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Proceedings of the XI International Congress of Genetics
The Hague, The Netherlands, September, 1963

Volume I

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PERGAMON PRESS LTD.

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Library of Congress Card No. 63-19264

Printep in the Netherlands by A.W. Sythoff - Leyden

PREFACE

This volume contains the abstracts of contributed papers, demonstrations and films
presented at the Xlth International Congress of Genetics. The editorial policy has
been to assemble the abstracts into sections not corresponding completely with the
titles of the symposia (see vol. II and III). Within each section we have endeavoured to
arrange the abstracts in a practical manner which may, however, vary from section
to section. Those abstracts delayed in some way are printed at the end of their respective
sections. The abstracts are numbered within each section; D refers to demonstrations
and F to films. In the alphabetical index of authors at the end of this volume references
are given to these numbers. Full addresses of the authors may be found by consulting
the membership list supplied at the Congress and printed at the back of volume III.

S. J. Geerts

CONTENTS

Sections and Section numbers.

1. Complex loci 1

2. Recombination 9

3. Molecular and microbial genetics 19

4. Gene action 37

5. Mutagenesis 55

6. Cytology 101

7. Cytogenetics 117

8. Cytotaxonomy and experimental taxonomy 131

9. Population genetics 143

10. Developmental genetics 169

11. Immunogenetics 189

12. Plasmatic inheritance 201

13. Plant genetics and breeding 209

14. Animal genetics and breeding 249

15. Human genetics 271

16. Human cytogenetics 299

17. Dermatoglyphics 315

18. Citation indexing 321

Author index 323

SECTION 1

COMPLEX LOCI

1.1. Genetic Fine Structure of the rosy Cistron in
Drosophila melanogaster. A. Chovnick,
A. Schalet, and R. P. Kernaghan (Storrs,
U.S.A.).

A highly efficient crossover selector system^1)
has been used to examine the fine structure of
a single cistron in Drosophila. This system per-
mits routine large-scale sampling approaching
that used in microbial studies, and permits
distinction between rare crossovers and spon-
taneous mutations. Such studies, involving a
large number of mutants of the rosy cistron,
permit the following general conclusions: (a)
The mechanism of recombination in higher
organisms, like that of microorganisms, does
permit crossing over within a cistron. (b) The
resolving power of such analysis of genetic
fine structure in Drosophila approaches that of
microbial systems.

of phenotypes exhibited by mutations in the
rosy region does not include complementary
recessive visibles and lethals or non-complemen-
tary lethals: 1. Lack of complementation among
all mutants at separable sites and failure to
detect mutants complementing rosy2 among 24
X-ray-induced rosy mutants selected over a
deficiency for the entire rosy region. 2. Absence
of rosy mutants which are lethal and unassociat-
ed with genetically detectable alterations involv-
ing closely neighboring loci among a sample of
13 rosy mutants with lethal effects localized to
the vicinity of the rosy region. 3. Absence of
complementary recessive mutations within the
rosy cistron among a sample of 1 1 lethals and
visibles selected within a small (0.5 map units)
chromosomal segment which included the rosy
region.

1. Chovnick et al., Amer. Nat. 96, 281, 1962.

1.2. On the Phenotypic Manifestation of Mutations
in the rosy Region of Drosophila melanogaster.

A. Schalet, R. P. Kernaghan, and
A. Chovnick (Storrs, U.S.A.).

According to the suggestion of Welshons
(1962) discontinuous mapping of complex loci
in Drosophila may be a function of the type of
mutant (recessive visibles) usually used. His
evidence shows that Drosophila results may be
reconciled with continuous mapping of mutant
sites in microorganisms when methods are
used to detect mutable sites for recessive lethals,
recessive visibles with atypical phenotypes and
possibly wild-type iso-alleles. While Welshons'
proposal may apply to many of the complex
loci in Drosophila, evidence from analysis of
the rosy region reveals that within a single
functional unit defined on the basis of a visible
and enzymatic phenotype, recessive visibles
give continuous recombination mapping of
mutable sites upon appropriate sampling. The
following considerations suggest that the range

1.3. Notch Pseudoalleles in Drosophila melanogaster.

W. J. Welshons, E. S. Von Halle, and
B. J. Scandlyn (Oak Ridge, U.S.A.).

The Notch locus is a complex one composed
of pseudoallele recessive visibles superimposed
upon an array of pseudoallele recessive lethal
Notch (N) mutants. The linear order from left
to right on the X chromosome of all pseudoalle-
les is fa -faao - A/40 - Nmc - spl - N103 - N>24 - NCo-
N%n-nd. The order of Ns" and nd is tentative but
both are to the right of yVCo. The genetic length
of the region has been estimated to be 0.11 units.

It was noted previously that no Ws had been
discovered in the region to the left of fano, but
it was presumed that they would eventually be
found there. Recent experiments performed
with Nen, spl, and fa yielded results which
placed Nel1 0.03 units to the left of fa, and in-
creased the genetic length of this complex locus
to 0.14 units.

Similar experiments utilizing TV39, fa and spl
localized N39 around fano. Since TV39 was known
to be cytologically deficient for salivary band
3C7, 85,600 chromosomes were tested from
N39lfano heterozygotes, but no recombinants
were found. It is probable that deficiency N39
includes the locus of fano.

Section 1 — Complex Loci

Obviously the region left of fano is capable of
producing recessive lethals as indicated by the
localization of NeU- Furthermore, the ./V39 ex-
periments indicate that the single salivary band
C37 cannot contain all the pseudoallele loci
comprising the functional Notch locus since
mutants like A/e" and NCo are localized at appre-
ciable distances to the left and right of the de-
ficiency.

as found by Green at the white locus, in which
event the resulting duplication in the present
case might be spectacle and the complementary
deficiency a lethal.

This investigation was supported by U.S.
Public Health Service Research Grant
GM-8889-02 from the Division of General
Medical Science.

1.4. An Apparent Non-equivalence in Crossover
Rates between Lozenge Alleles in Trans and
Cis Arrangement in Drosophila. Luolin S.
Browning and Edgar Altenburg (Houston,
U.S.A.).

The rate of recovered crossovers between
lzBS and lz46 in Drosophila was found to be
significantly greater from females heterozygous
for these two lozenge alleles in trans arrange-
ment as compared with cis as follows. Among
350,000 Fi from the trans arrangement (lzBS + /
+ lz46), 116 crossovers (46 lzBS lz46 and 70 + +)
were recovered, or about 1 crossover in 3000
offspring; among 436,500 Fi from the cis ar-
rangement (lzBS lz46 / + +) 75 crossovers
were recovered (75 being the combined number
of lzBS + and + lz46, both of lozenge phenotype
as contrasted to the spectacle phenotype of the
double mutant non-crossover class on the one
hand and the complementary normal on the
other), or about 1 crossover in 5820 offspring.
It is not considered very likely that this difference
in rates could be due entirely to the relative
viabilities of the crossover classes as compared
with the non-crossovers in the trans and cis
arrangements. Work earlier reported indicated
that the crossover rate between apricot (wa)
and white (w) was also greater for the trans than
the cis arrangement. In explanation of these
results, it is suggested that alleles might some-
how differ structurally. One series of lz-locus
experiments (involving a question as to the
identity of the alleles) gave an unexpected re-
sult in that the two lz alleles in trans arrange-
ment gave the apparent double mutant class
(10 phenotypic spectacles in 70,000 offspring)
but none of the complementary normal pheno-
type. Judd reported a result which we interpret
as similar to this, insofar as in the Fi from apri-
cot and buff in trans arrangement, the only
"exceptions" found were of white phenotype
(no normals). We have no explanation for this
kind of result except perhaps that it is due to
some type of non-homologous pairing such

1.5. A Study of Recessive Lethals on the Dot-like
Fourth Chromosome in Drosophila melano-
gaster. Benjamin Hochman (Knoxville,
U.S.A.).

A three-year study of 1352 fourth chromo-
somes, extracted from natural populations of
D. melanogaster, has uncovered 15 which are
lethal in the homozygous state. Allelism tests of
14 of the lethals (one was lost) demonstrate that
they occupy eleven different loci. One locus is
represented three times; a second locus twice;
and nine have a single representative each.
One of these nine is allelic to a lethal which has
arisen spontaneously in two different laboratory
stocks.

Crosses of the lethals to the six existing
chromosome 4 dominant visibles have thus far
localized one of them to the btu locus and a
second to that section of the microchromosome
delimited by the Minute-4 deficiency. None of the
lethals permits pseudodominance of any of the
seven non-allelic recessive visibles tested.

Several X-ray-induced lethal fourth chromo-
somes, generously provided by Drs. M. M. Green
and H. Gloor, have been examined for inter-
actions with the lethals of spontaneous origin.
Three heterozygous combinations which are
lethal have been observed to date.

If all of the spontaneous microchromosomal
lethals are "point" mutations, the number of
loci identified on chromosome 4 has been ap-
proximately doubled over the figure based here-
tofore solely on the known visible mutations.
The total number of "potentially-lethal" loci
to be expected on the microchromosome, as
well as the developmental period during which
each lethal acts, will be discussed.

This research was supported by Grant
RG 9845, United States Public Health Ser-
vice, National Institutes of Health.

Section 1 — Complex Loci

1.6. A "Mutable" Gene in Drosophila melanogaster.

William M. Hexter (Amherst, U.S.A.).

Attached-X females properly marked and
heterozygous for a specific allele of garnet
(g53d) and any other garnet allele (gx) were
singly mated by appropriately marked males,
and the female progeny were screened for the
presence of wild type (non-garnet-eyed) females.
In a total of 282,000 females, 15 were wild
type. In a similar test differing only in the homo-
zygosity of g53d, 0 wild type females were recover-
ed in 539,000 females. Of the 15 wild type fe-
males, 6 were non-diagnostic as to generating
mechanism; of the remaining 9, 6 could be inter-
preted as due to single crossover between pseu-
doallele loci. The remaining 3 are not so easily
explained. If the pseudoallele hypothesis were
correct it should be possible to demonstrate a
double mutant. Four of the presumptive 6
females were tested in a manner to reveal one
of the two garnet alleles should the double mu-
tant exist. The sum of these 4 experiments was
428,000 flies with 0 single garnet mutants re-
covered. An additional and independent test of
a presumptive double mutant was to place the
supposed double mutant chromosome (g53d gx)
in apposition to an appropriately marked g53d
free-X chromosome. Due to homozygosity of
g53d sucn a test would not be expected to yield
any wild type progeny. In fact, however, 22
wild types were recovered in 685,000 flies; of
these 22, 11 were associated with recombina-
tion and could be simply interpreted as a cross-
over between pseudoallele loci. The remaining
11 were non-recombinants for outside markers
and are not as easily explained. The conclusion
based on these results is that some, if not all,
of the wild types were due to mutability of
g53d when heterozygous with other garnet alleles.
Further genetic tests have not suggested, nor
ruled out, the possibility that g53d is a duplica-
tion.

segment of the forked locus) might be due to
duplication of the right forked sub-locus at a
pre-meiotic division, a suggestion which would
account not only for the relatively high rever-
sion rate of f3N (to f3N+), but also for the
restriction of the reversions in large measure
to diploid cells (oogonia and spermatogonia)
in irradiated material. Since a ring X would
get lost much more often than a rod as the
result of a duplication in a dividing chromosome,
the recovered reversion rate of f3N would ex-
pectedly be higher for a rod than a ring X, pro-
vided the reversions were due to duplications
(whether X-ray induced or spontaneous). In
accordance with the above suggestion, it has
been found that the spontaneous reversion rate
of f3N is much higher in a rod X than in a ring as
follows. Among 380,000 females heterozygous
for a rod and a ring (but homozygous for f3N)
there were 12 reversions recovered in the rod and
one in the ring. Insofar as these results would
indicate that the forked locus arose as the result
of a duplication, they would support the Lewis
theory of pseudoallelism in Drosophila.

Detailed data are in press in Proc. Nat. Acad.
Sci. Wash.

Supported by a grant from The National
Institutes of Health (CA-03 1 14).

This investigation was supported by U.S.
Public Health Service Research Grant GM
08889-02 from the Division of General Medical
Science.

1 .8. The Genetic Fine Structure of the Mutants
/."' and /.' in Drosophila melanogaster.
B. H. Judd (Austin, U.S.A.).

The zeste locus in Drosophila melanogaster is
located at 1.0 on the X chromosome. The mu-
tant zm (zeste-mottled) was found by Green as a
single male (sc zm) from the cross sc z ec ctj
wM x sc z ec ct. Later z1 (zeste-light) was found
by Becker as a single male in the sc zm stock.

Analysis of zm leads to the hypothesis that it
does not represent a change at the zeste locus,
but is the result of an asymmetrical exchange at
the white locus. The white locus is very closely
linked to zeste (1.5 on the X chromosome)
and is functionally related. It is postulated that
z1 arose as a similar asymmetrical exchange from

1.7. Evidence from Rod-ring Experiments for the
Duplicational Origin of f 3N Reversions in Dro-
sophila. Edgar Altenburg and Luolin S.
Browning (Houston, U.S.A.).

It was previously suggested that the rever-
sion of f3N (an allele located in the left sub-

The change in the white locus can be localized
within the two rightmost recombination sites
of the locus. The phenotypic expression of this
change leads to the conclusion that a portion
of the white locus has been duplicated. Recom-
bination experiments give support to this inter-
pretation.

The duplicated nature of the two mutants

Section 1 — Complex Loci

leads to further asymmetrical pairing and ex-
change within the locus when these chromo-
somes are used for crossover studies. A variety
of unusual recombination products presumably
resulting from asymmetrical exchanges have
been analyzed.

1.9. Biochemical Division of the White Locus in
Drosophila melanogaster. Hugh S. Forrest
(Austin, U.S.A.).

Despite the fact that the white locus in Droso-
phila melanogaster has been known for some
time to be divided into subloci separable by
crossing over and other genetic tests, there is
remarkably little knowledge of its fundamental
biochemical role. It is believed by some workers,
for example, that mutation at this locus results
in an "incomplete" or "non-functional" pig-
ment-synthesizing granule, resulting in loss of
synthetic ability for both the red and brown
pigments.

In a new attempt to investigate the biochemical
function of the locus, it has been shown that
all of the alleles at the locus so far tested can
be divided into two groups — those that accu-
mulate the compound xanthurenic acid, albeit
in smaller amounts than wild type, and those
that do not contain this compound at all. It is
reasonable to assume that xanthurenic acid
can be used as an index of the production of
3-hydroxykynurenine, an intermediate in the
biosynthesis of the brown pigments. Thus the
white locus may be separable into two functional
areas, one of which, in the mutant condition, is
unable to produce 3-hydroxykynurenine (meas-
ured as xanthurenic acid) and the other of which
can produce limited amounts of this material.
A theory relating this finding with the failure
or partial failure of mutants of the white locus to
synthesize the red eye pigments will be presented.

1.10. Complementation Groups at a Complex Locus
in the House Mouse. Dorothea Bennett and
L. C. Dunn (New York, U.S.A.).

The study of three newly arisen alleles has
provided the opportunity to compare the
effects of lethals of independent origin, and
especially to compare members of the same
complementation group with respect to second-
ary effects. Complementation groups are defined
as consisting of alleles from different sources
which are lethal in combination. Alleles in the
same complementation group are in general

indistinguishable; they produce similar syn-
dromes of abnormalities at similar times in
embryogeny, and have similar effects on male
transmission ratio and recombination. Combina-
tions of members of different complementation
groups are of course viable to some extent, but
the proportion of viable compounds found
varies greatly depending on the alleles concerned.
The three new alleles reported here were found
to belong to three different previously established
complementation group. Their characteristics
regarding embryonic effects, transmission ratio,
and recombination followed the general pat-
tern of being in each case indistinguishable from
other members of their group. However, one of
the new alleles was found to be clearly distin-
guishable from other members of its group on
the basis of degree of complementation with
another lethal allele. This suggests that alleles
in the same complementation group may not
have identical structures, and that quantitative
measurements of the degree of complementa-
tion amongst different alleles may be used as a
basis for assessing their similarity or dissimi-
larity.

1.11. Genetical Analysis of the R Chromonemal
Region of Mormoniella. P. W. Whiting and
Doris J. Bush (Philadelphia, U.S.A.).

Two eye-color factors, O and S, mutating
with relatively high frequency to oyster-white
and to scarlet respectively, make the "/?"
region of Mormoniella especially favorable for
genetical analysis. These colors serve as markers
for many other 7?-locus changes including semi-
lethals and lethals, steriles and near-steriles.
Complementation tests identify factor homolo-
gies. For factors such as lethals and male-steriles
which cannot be transmitted through haploid
males, diploid males heterozygous for these
deleterious factors are used. Two different
female-steriles have been found, masked in
lethal-bearing genes. Two different lethals,
complementary to each other and, therefore,
non-homologous, have proved non-complemen-
tary, "homologous", to a third lethal. It is sug-
gested that the last impairs some process essen-
tial to the normal functioning which is impaired
by each of the other two lethals. Formula of
each /Mocus gene is given in terms of its factor
states. By use of the gene formulae it is possible
to predict the phenotypes and breeding behavior
of the different compounds. It is postulated that
the complexity of the R region is not greater
than that of other chromonemal regions. Its
complexity has been explored and revealed by

Section 1 — Complex Loci

use of oyster and scarlet mutations, acting as
markers of each pleiotropic gene of the series.
Data in full are at present being published in
Genetics.

1.12. Partial Complementation of Six Multiple Alleles
in a Chlorophyll Mutation Locus in Barley.

Gerhard Holm (Lund, Sweden).

A locus in chromosome 1 contains at least 6
recessive vital viridis alleles which, when inter-
crossed, give Fi's exhibiting different degrees
of partial complementation judging from the
visually examined leaf colours. A spectrophoto-
metrical analysis of 8 of the Fi's verified this
interpretation and also showed that the degree
of complementation in an Fi apparently depends
upon the relative position in the locus of the
two alleles involved in the cross. The 6 alleles
probably belong to the same cistron and overlap
each other in a linear order. The less two alleles
overlap, the higher the complementation in
their Fi.

The pigment content in 5 of the alleles is the
same, 1.23 mg/g fresh weight, but in the 6th
(no. 4 in the linear order) it is significantly higher,
1.44 mg/g fr.w. Normal plants contain 2.00
mg/g fr.w.

The 6 multiple alleles appeared in a material
of 82 vital viridis mutations of which the resulting
76 most probably belong to single loci. 865 dial-
lelic crosses were made in the group and the
result indicates a very low frequency of multiple
allele loci among the vital viridis mutations.

1.13. Fine Structure of Genes Conditioning Resistance
to Erysiphe graminis hordei at the Mla
Locus in Hordeum vulgare. John G. Mose-

man (Beltsville, U.S.A.).

Several genes conditioning the resistant reac-
tion of barley Hordeum vulgare L. to infection
with the ascomycetous fungus Erysiphe graminis
DC. f. sp. hordei Em. Marchal have been found
at the Mla locus on chromosome 5. Genes Mla,
Mla2, and Mla3 at this locus were studied. When
genes Mla and Mla3 were homozygous they
conferred a similar degree of resistance, but
when heterozygous with a recessive gene condi-
tioning susceptibility they conferred a different
degree of resistance. Gene Mla2 conditioned a
lesser degree of resistance than genes Mla and
Mla3. The degree of resistance conferred when
the genes were MlaMla2, MlaMla3. or Mla2Mla3
also was determined. For each gene conditioning

the reaction of the host, a corresponding gene
has been found conditioning the pathogenicity
of the pathogen. Pathogenically different cultures
differentiated the genes at the MIa locus. The
relation of the pathogen genes corresponding to
host genes was determined by the pathogenicity
of haploid progeny cultures derived from crosses
between selected cultures. Pathogen gene Aa
corresponding to host gene Mla was inherited
independently of pathogen genes Aa2 and Aa3
corresponding to host genes Mla2 and Mla3,
respectively. Pathogen genes Aa2 and Aa3
were found to be at or near the same locus. The
utilization of degree of resistance conferred,
relation of corresponding pathogen genes, and
other host-pathogen relations in studying fine
structure of genes conditioning resistance to
E. graminis f. sp. hordei at the Mla locus in H.
vulgare were discussed.

1.14. Complementation, Recombination, and Biochemi-
cal Relationships within the Td locus in Neuro-
spora crassa. Ann Matthews LACY(Baltimore,
U.S.A.).

The present study of non-indole-utilizing
(NIU) tryptophan synthetase deficient mutants
indicates that the complementation and recom-
bination maps of the Td locus are not co-linear.
The clustering on the recombinational map bears
some relationship to the biochemical charac-
teristics of the mutants; however, the biochemi-
cal relationships between mutants grouped
together by complementation appears some-
what obscure.

The twenty NIU complementing mutants
tested are distributed among three of the four
complementing groups previously established
for NIU mutants (the fourth group is still re-
presented by Tdl only).

The ability to form cross-reacting material
(CRM) is not a prerequisite for complementa-
tion. Four CRM~ mutants are in the Tdl\
complementation group. These mutants, how-
ever, are not located near Tdl I on the recombina-
tion map, but are located in the vicinity of
Tdl, 7, and 24 (i.e. "profound region").

The ability to catalyze the formation of indole
from indole-glycerolphosphate is not limited to
members of the 7^71 complementation group,
but can be detected at varying levels in crude
extracts of at least 90% of the CRM+
NIU mutants tested (including Td3, 7, and 24).
CRM" mutants tested exhibit no detectable
activity in this reaction.

Of three temperature sensitive mutants stud-
ied (mutant phenotype at 25°C, not at 37°C),

Section 1 — Complex Loci

two are in different complementation groups and
exhibit different levels of ability to form indole;
the third neither complements nor forms indole.
The relationships between the NIU mutants
will be described in detail and their possible
significance in terms of gene structure and func-
tion will be discussed.

Supported by grants from the National
Science Foundation.

1.15. Further Analysis of Interallelic Complementa-
tion at the leu-2 Locus of Neurospora crassa.

S. R. Gross (Durham, U.S.A.).

A previous analysis of the complementation
behavior of a large number of leu-2 mutants of
Neurospora crassa yielded a complementation
map that was linear, overlapping and conti-
nuous/1) Evidence was presented that indicated
that the polypeptide whose structure was deter-
mined by the leu-2 gene was at least one of two
different polypeptide structural units of an
enzyme (3-carboxy-P-hydroxyisocaproic acid
isomerase, an enzyme which catalyzes the iso-
merization of (3-carboxy-(3-hydroxyisocaproate
and cc-hydroxy-(3-carboxyisocaproate, interme-
diates in leucine biosynthesis. An analysis of the
complementation interaction between leu-2 and
leu-3 alleles as well as enzymological analyses
indicated that both the leu-2 and leu-3 loci were
involved in the determination of the structure of
P-carboxy-[3-hydroxyisocaproic acid isomerase.
The evidence obtained was interpreted as indicat-
ing that complementation between leu-2 mu-
tants resulted from a protein-protein interac-
tion in the formation of a polymer consisting of
a chains coded by the leu-2 gene and p chains
coded by the leu-3 gene, and that at least in the
case of enzymes synthesized as a result of com-
plementation, the isomerase consisted of at
least two a and two p polypeptide chains. A
recent study of the physical chemistry of the
isomerase obtained from several complementing
pairs of leu-2 mutants has revealed that the
molecular weight of the normal enzyme as well
as that of the heteroallelic enzymes are the same,
ca. 80,000. The enzymes obtained by complemen-
tation are remarkably similar to the normal
enzyme with respect to the functional area of the
protein. Several physical properties of the
heteroallelic enzymes differ markedly from that
of the isomerase enzyme. These differences,
however, are probably confined to areas not
directly involved in substrate binding.

1.16. Homology Tests on X-ray-induced Recessive
Lethal Mutations in the ad-3 Region of
Neurospora crassa. F. J. de Serres (Oak Ridge,
U.S.A.).

Forward-mutation experiments on a balanced
heterokaryon^1) have shown that 76 per cent
of the ad-3 mutations are recessive lethal (ad-3RL)
on adenine-supplemented medium and that 40
per cent of these were ad-3 A ad-3BRJj double
mutants. Atwood and Mukai(2) have shown that
such recessive lethal mutations can be analyzed
by homology tests. Such tests on the ad-3RIj
mutations show that they form an inclusive
complex; all combinations show homology
except some of the ad-3ARlj +ad-3BRL combina-
tions. The apparent homology of certain ad-3ARL
+ ad-3BRJj combinations is of particular interest
since the trikaryon involving two such mutations
in combination with a viable double mutant
(from a cross of two viable mutants ad-3Av
x ad-3Bv = ad-3Av ad-3Bv) is capable of
growth on minimal medium. The behavior of
these trikaryons shows that there is an intact
essential region (designated region X) present in
the ad-3Av ad-3Bv double mutant that is non-
functional in any of the ad-3ARJj or ad-3BRL
mutations showing homology. The simplest
assumption is that region X is located between
the ad-3 A and ad-3B loci, and present data are
consistent with this hypothesis. The interaction
patterns of such ad-3RL mutations in homology
tests offer a unique opportunity for the analysis
of the genetic composition of the ad-3 region,
and the results of these tests will be described
in terms of a complementation map.

1. Gross, Proc. Nat. Acad. Sci. 48, 922, 1962.

1. De Serres and Osterbind, Genetics 47, 793-
796, 1962.

2. PN AS 39, 1027-35, 1953.

1.17. Complementation and Genetic Mapping of pan-2
Mutants Induced in a Reversion of a Primary
pan-2 Mutant. Mary Case (New Haven,
U.S.A.).

Twenty-nine secondary pan-2 (pantothenic
acid-requiring) mutants have been obtained
following X-irradiation of a reversion induced
by X-rays in a complementing primary pan-2
mutant B5. Thirteen of the 29 mutants comple-
ment with at least one primary mutant, and all
13 can be positioned on a linear complementation
map of the locus. The behavior of the secondary
mutants establishes the existence of an additional
complementation unit at the pan-2 locus. All

Section 1 — Complex Loci

mutants were crossed to B5 to determine whether
the mutants are identical to B5. In addition,
crosses were made to five other pan-2 alleles
located at various points on the genetic map of
the locus. Two of the mutants map at the same
site as B5; however, one of these mutants
is a non-complementing type while the other
exhibits a different complementation pattern
from B5. Fourteen of the mutants are located
near B5 in the proximal region of the genetic
map. Ten are located near the distal end of
the map, while the three others are located at
different sites near the middle of the map.
The relationship between complementation and
genetic maps of primary and secondary mu-
tants will be discussed further.

Supported in part under a contract, AT
(30-l)-872, with the U.S. Atomic Energy Com-

mission.

1.18. Allelic Recombination and Complementation in
Adenine-requiring Mutants of Schizosaccha-
romyces pombe. C. Ramirez, J. Friis and U.
Leupold (Bern, Switzerland).

Allelic recombination and complementation
have been studied in several groups of mutants
blocked in adenine biosynthesis, including two
groups accumulating a red pigment (ads and
adi) and one group blocked in an early reaction
preceding the pigment blocks (adi).

In the ad-? group, 152 u.v.-induced allelic
mutants have been demonstrated to represent
mutations at 33 different but closely linked sites,
recombination frequencies ranging from 1 to
77° prototrophic recombinants per 106 as-
cospores. The mutant distribution is character-
ized by "hot spots", one particular site having
contributed as many as 26 per cent of the mu-
tants. Complementation has not been observed
in any of the pairwise mutant combinations
tested.

In the ad\ group, however, complementation
leading to a more or less prototrophic phenotype
has been found in many of the diploid combina-
tions tested. The complementation pattern of
138 mutants of u.v.-induced, diethylsulfate-
induced and spontaneous origin may be re-
presented by a linear complementation map in
which eight types of complementation behaviour

define five different "complementation units".
The complementation and recombination maps
show a considerable degree of co-linearity.

A more complex complementation pattern has
been found in allelic combinations of 158 u.v.-
induced mutants of the constitution ad§. The
results of complementation tests involving
21 different types of complementation behaviour
may be represented in a complementation map
in which both ends are closed to circles. With a
few notable exceptions, the complementation
and recombination maps show again a sig-
nificant degree of co-linearity.

1.19. Studies on the Genetic Fine Structure of the
ad7 and adg loci of Schizosaccharomyces pom-
be. H. Gutz (Berlin, Germany).

Leupold has done a very extensive intragenic
recombination analysis in the adi and ad& loci
of Schiz. pombe. The ad& locus shows the
phenomenon of allelic complementation. In
addition to the intragenic recombination analysis
Leupold has constructed a complementation
map of this locus. All mutants analysed by
Leupold have been induced by ultraviolet irradia-
tion. The ad- and adz mutants are not randomly
distributed in the maps. There are a few "hot
spots", and the mutation sites appear to be
concentrated in several parts of the map.

Taking the maps of Leupold as a basis, I
have analysed the intragenic distribution and
the complementation pattern of mutants which
were induced by nitrous acid and X-rays. In
both loci (adi and ad&) the intragenic distribu-
tion of the nitrous acid-induced mutants is quite
different from that of the u. v. -mutants. They
show some pronounced "hot spots". Above
half of the sites induced by nitrous acid are not
present on the u.v.-map. From the non-random
distribution of the mutants it is concluded that
a direct deamination of the DNA-bases is
not to be considered as mutation mechanism.
All X-ray mutants map on single sites, and no
intragenic deletions have been found.

According to the results of Leupold, the
complementation map of the ade locus is non-
linear. Seventy-nine per cent of the nitrous
acid-induced mutants show complementation.
Among these mutants at least 6 complemen-
tation types were found which are not present
in the u. v. -mutants.

SECTION 2

RECOMBINATION

2.1. Factors Affecting Distributive Pairing between
Nonhomologues in Drosophila melanogaster.
Rhoda F. Grell (Oak Ridge, U.S.A.).

Meiotic associations that lead to the regular
separation of nonhomologous chromosomes
occur with high frequencies in Drosophila me-
lanogaster females of particular genotypes.
The relation of the size of the nonhomologues
to the regularity of their association has been
investigated by measuring the nondisjunction
frequencies between a free fourth chromosome
and a series of X-duplications in the progeny of
females of the genotype y2/j>2/Dp(l ;f),y+; T(3; 4)
86D, m//T(3 ; 4)86D, red; ciD mated to xy,
yB/Y males. The duplications were obtained and
characterized by Cooper and Krivshenko and
vary in size from ~ 0.3. to 3.0 times the meta-
phase length of chromosome four. The percent of
nondisjunction between chromosome four and
the duplication, given in ascending order of
duplication size, are, / ~ 0.3 = 4.49 ± 0.19, /
0.5 = 0.39 ± 0.07, / 0.7 = 0.12 ± 0.06, 0.9 =
0.08 ± 0.03, 1.0 = 0.13 ± 0.05, 1.1 = 0.02 ±
0.02, 1.1 = 0.08 ± 0.03/ 1.4 = 0.38 ± 0.08,
1.6 = 0.39 ± 0.08, 2.0 = 0.42 ± 0.07, /
2.5 = 2.03 ± 0.39, 3.0 = 1.89 ± 0.24 /. The
lowest frequencies of nondisjunction (~ 0.08 per
cent) occur with those duplications closest in size
to chromosome four (0.7 - 1.1) whereas the
highest frequencies occur with the smallest
(~ 0.3) and largest (2.5 and 3.0) duplications.
The results indicate size alone is not responsi-
ble for pairing efficiency since the progressive
change in duplication size is not associated
with correlated changes in nondisjunction fre-
quency. Instead, the duplications fall into five
discrete classes (designated by slashes in the
sequence above) within which size variation
causes no apparent related change in segregation
behavior. This suggests the possibility of sites
that participate in nonhomologous associations
as has been postulated by Gershenson and
Cooper for homologous associations.

2.2. Nonrandom Assortment of Compound Chromo-
somes with Nonhomologous chromosomes in
Oocytes of Drosophila melanogaster. E. H.
Grell (Oak Ridge, U.S.A.).

The requirements for nonrandom assoi cment
of nonhomologous chromosomes was studied
by R. F. Grell (1962). For noncompound
chromosomes it was determined that a chromo-
some must not have been a crossover. (The
term compound chromosome is intended to
include chromosomes formed by the joining of
two homologous chromosome arms to one
centromere.) If two nonhomologous chromo-
somes are noncrossovers they may pair and pass to
opposite poles of the first meiotic division spindle.
The pairing in which nonhomogolous elements
may participate has been termed distributive
pairing to distinguish it from exchange pairing
which occurs prior to crossing over and only
involves homologous chromosomal regions.

The studies reported here deal with distributive
pairing of nonhomologous chromosomes when
at least one member of the pair is a compound
chromosome. A familiar compound chromosome
is the attached X. The two arms are attached in
reverse order on either side of a centromere.
Crossing over occurs between the two arms
with almost the same frequency as for normal
free X chromosomes. Yet, from females of the
genotype xx/0; SMI, Cy/T(2;3)A, 90 per cent
of the recoverable gametes have the attached
X or SMI, Cy and only 10 per cent have both or
neither chromosome. Despite the fact that more
than 95 per cent of the tetrads contain an ex-
change, the attached X shows a very highly
nonrandom assortment with a second chromo-
some. Experiments that compare compound X's
with an exchange and those without an exchange
indicate that in contrast to free X's, exchange
has no influence on the extent of nonhomologous
pairing. All compound chromosomes tested
(various compound X's and the attached 4's)
have shown nonrandom assortment with non-
homologous chromosomes.

2.3. On the Nature of the Event that leads to Recom-
bination of Pseudoalleles of the m-dy locus in
Drosophila. Allan B. Burdick, Robert A.
Shleser, Evelyn Barbour Bendbow, and
Rosanne Abbadessa (Lafayette, U.S.A.).

The recombination map of the m-dy locus

Section 2 — Recombination

shows an unambiguous sequence of five se-
parable mutant elements extending over about
0.07 map units. The sequence is v . . . m61e,
«759a, m, dy, dyeok . . . g with several other ele-
ments being inseparable from one or more
members of the sequence. A number of insertio-
nal-type crossover products (double and triple
crossovers with respect to the outside markers)
have been obtained from free-X recombina-
tion tests. These have involved nine different
elements of the locus, and their explanation
poses a problem as to the nature of the event that
leads to recombination of the elements of the
locus. Although the cis-trans test of the most
distal elements (m61e and dy60k) indicates that
they occupy separate functional sites, comple-
mentation tests among the various elements
indicate that they could all belong to the same
cistron. The m6le dy60k m-phase double mutant
has been tested in //a/;s-phase with all of the
internal elements of the locus and has never
yielded a wild-type recombinant — that is, no
double crossing over takes place within the
locus — although m61e dy60k has broken down
several times to yield dy60k types. Attached-X
recombinations of m61e and m yielded 31 events
of which 14 were reciprocal. One of the m61e
m m-phase double mutants has been /ra/w-phase
tested to m61e, /?/59a, and m and has not given
any wild-type recombinants. Our present opinion
is that recombinations within this locus are of
the same classical reciprocal kind that have
been postulated for interlocus recombinations,
and that the insertional-type crossovers we have
obtained can be explained on a classical basis.

Supported by a grant from the National
Science Foundation.

2.4. Genetic Ultrafine Structure of the T4rII Region.
Irwin Tessman (Lafayette, U.S.A.).

Recombination frequencies as low as 10 6
per cent have been measured in the rll region of
phage T4. At this sensitivity, hot spots in the
rll region have been resolved into two or more
genetic sites, separable by recombination. No
lower limit to recombination frequencies is
apparent. Therefore, lack of recombination
cannot guarantee identity of genetic sites.

The largest recombination frequencies in the
rll region are greater than 1 per cent and the
lowest less than 10-6 per cent. The ratio, 106,
is greater than the number of nucleotides in the
entire phage. Therefore, in this range there

must be extreme deviations from even rough
additivity of recombination frequencies. Within
the range of very low recombination frequencies,
marked deviations from additivity are, in fact
found.

2.5. Effect of Negative Interference on Genetic Map
Concepts and Its Consequences for Intra-
allelic Recombination Studies. W. D. Hanson
(Raleigh, U.S.A.).

Experimental studies involving intra-allelic
recombination have yielded results which are not
entirely compatible with classical recombination
concepts. Negative interference noted in intra-
allelic recombination studies suggested that
given a genetic recombination at a point one
could expect additional points of recombina-
tion or a "cluster" of points of recombinations
(one or more) within some interval. The products
of meiotic division were described in a proba-
bility space so that the probability of chromo-
some types could be formulated. The products
of meiosis for linked loci were formulated for
classical recombination studies and for intra-
allelic recombination studies assuming that
cluster recombinations could occur in some inter-
val. The results involved the probability of an
"odd cluster", length of cluster interval, and
genetic map distance between linked loci. Cluster
recombinations within a relatively short interval
should not upset classical recombination meas-
ures. The frequency of chromosome types
(with respect to marker genes) with selection for
intra-allelic recombinations reflected a bias
identified with cluster effects. Relative reverse
mutation rates could also be a factor; however,
for crosses involving a locus bounded by two
markers a contradiction existed for linear order
of alleles vs. relative mutation rates. A positive
bias in map distance involving a non-selected
marker was associated with cluster recombina-
tions which was a maximum for a closely linked
marker and decreased with increased map dis-
tance. Published data supported cluster recom-
bination concepts, as compared to mutation,
with a positive bias in recombination being a
consistent feature in these data.

2.6. Polarized Intragenic Recombination in Neuro-
spora crassa. Noreen E. Murray (Stanford,
U.S.A.).

Methionine-independent progeny from crosses
between many pairs of combinations involving

10

Section 2 — Recombination

nineteen alleles at the me-2 locus were classified
with respect to the markers present on both sides
of the me-2 gene. One of the two classes of
methionine prototrophs having markers re-
combined occurred in excess over the other;
when the markers entered the cross in the op-
posite phase, a similar excess was found in the
reciprocal class. The linear order of me-2 alleles
determined from these asymmetries was consis-
tent with a map based on prototroph frequencies.

Pronounced asymmetries were also observed
in the numbers of the two parentally-marked
classes of prototrophs; when the markers en-
tered the cross in the opposite phase these asym-
metries were reversed. The asymmetries between
the two parentally-marked classes were correlated
in direction with the asymmetries between the
two classes having markers recombined.

The results may be interpreted in terms of
multiple exchanges within small, discontinuously
distributed, regions of effective pairing — the
asymmetries would result from a reduction of
coincident exchanges in the region proximal to
the selected interval.

The coincident exchange frequency in the
proximal region is influenced by the position of
the more proximal of the two me-2 alleles, but is
independent of the recombination frequency
between the two alleles. The reduced coincidence
in the proximal region could be explained if the
me-2 region is situated immediately distal to
some discontinuity which imposes a nonran-
domness of exchanges by reducing the chance
that the effectively paired region could be proxi-
mal to, yet extend into, the me-2 region.

Supported by Public Health Service research
grant El 462.

2.7. Conversion and Crossing-over as Recombination
Mechanisms in Ascobolus immersus.
W. Gajewski, A. Kruszewska, A. Makare-
wicz, A. Paszewski, S. Surzycki and H. Bie-
lawska (Warsaw, Poland).

Crosses between mutants with white ascos-
pores from one series give wild type, dark re-
combinants due predominantly to conversion.
The frequencies of conversion-type asci are
roughly proportional to the distances between
the sites involved which enables one to map them.
When mutants from one series are crossed with
wild type strain the frequencies of 2:0 asci
increase in percentage from one end of the series
to the other approaching the value of recombina-
tion between the extreme sites of the series.

This is interpreted that the frequency of 6:2
asci in crosses between two white mutants
represents only that part of copy choices which
begin or end in between two sites. It seems that
the beginning of the switches is random along
the analysed unit whereas the return to the origi-
nal matrix is rather nonrandom.

One mutant, namely 186, crossed with wild
strain shows very high frequencies (10- 12 per cent)
of 6:2 and 2:6 asci of conversion type. Besides,
less frequent asci 5 : 3, 3 : 5, 7 : 1 andl : 7 also appear.
They are not due to aberrant chromosome
segregation or mutation. The asci 5:3 and
3 : 5 result probably from copy errors of half-
chromatids. The 7:1 and 1:7 asci may be the
result of two copy errors it two successive re-
plications. The tetrad analysis of visible asco-
spore mutants in Ascobolus immersus on suffi-
ciently large scale reveals the predominant role
of conversion as a mechanism of recombina-
tion within a small piece of genetic material .

2.8. Effect of Crossing-over on the Process of Sponta-
neous Mutation. G. E. Magni (Pavia, Italy).

It has been recently observed that the rates of
spontaneous mutations occurring during meiotic
division are higher than those occurring during
vegetative reproduction (mitosis) in the same
type of cells (yeast). W

Further experiments have shown that most
spontaneous back-mutations at locus hii-i
during meiosis, in either one of the two identical
alleles carried by diploid yeast cells, are con-
nected with crossing-over in the specific locus.

Data regarding the amount of negative inter-
ference in the adjacent chromosome regions and
the polarization of the intragenic crossing-over
will be presented.

The hypothesis of unequal crossing-over
within the locus leading to back mutant chroma-
tids will be discussed.

1. Magni and Von Borstel, Genetics 47, 1097,
1962.

2.9. Further Investigations on Somatic Gene Conver-
sion in | the Tomato. Rudolf Hagemann
(Gatersleben, Germany).

In heterozygous sulf+sulf plants a green
yellow variegation occurs (sulf means any mutan
allele of the chlorophyll deficient sulfurea series
either of the sulpUT3- or of the sulf™« group)

11

Section 2 — Recombination

The genetical analysis demonstrated that the
sulf+ allele, stable in homozygous condition,
becomes unstable in presence of a sulf allele.
This type of allele-induced instability in somatic
cells is termed somatic gene conversion/1)
Further experiments have shown:

(1) A two-element-system (as found in maize)
is not involved.

(2) In hybrids between sulf homozygotes and
taxa of the subgenus Eulycopersicon the pet-
centage of variegation is only determined by the
"conversion activity" of the special sulf allele
present. The environmental conditions (tempera-
ture, nutrition, illumination) do not signifi-
cantly alter the percentage of variegation. Con-
version-table sulf+ alleles have not yet been
found.

(3) In hybrids between sulf homozygotes
(Lycopersicon esculent um) and L. hirsutum
(subgenus Eriopersicori) or Solatium pennellii
variegation occurred very seldom, thus indicat-
ing an influence of the genotypic milieu.

(4) The somatic conversion is confined to the
sulf locus. But as to the different alleles, conver-
sion is not highly directed. The sulf allele, con-
verted from sulf+, need not be identical with the
sulf allele already present; e.g. in a sulf+ sulfpuTa-
90 per cent piant the Sulf+ allele is converted
to sulfva-z more frequently than to sulfpuTa,
and even the sulfvu™ alleles arisen may differ
in their conversion activity from s«//pura"90

per cent.

performed. In the first type the marker asco that
gives rise to white ascospores was used. The
asci were dissected and second division segrega-
tions scored. In the second type biochemical
markers that were separated by various distances
were used and recombinants scored.

The protein synthesis inhibitor, chloramphe-
nicol which inhibits rejoining and also inhi-
bits growth of Neurospora, did not affect re-
combination. We interpret this to mean that
protein synthesis is not involved in crossing-
over.

The only inhibitor that gave consistently
higher recombination values when added at the
time of meiosis was 5-fluorodeoxyuridine
(FUdR) a compound that, in some systems, has
been shown to be a specific inhibitor of DNA
synthesis.

Bromodeoxyuridine (BUdR) a compound that
is structurally similar to FUdR but is a thymi-
dine analog that becomes incorporated into the
DNA did not have any affect on recombination
nor did uridine which is usually added with
FUdR to prevent effects on RNA.

The simplest explanation for these experi-
ments is that genetic recombination, which
entails the exchange of chromatids, is specific
for DNA and that a functional mechanism for
DNA synthesis is required when the bonds re-
joining the two pieces are synthesized. This is
unlike the rejoining or repair of X-ray induced
breaks that are non-polarized and are inhibited
by chloramphenicol.

1. Z. Vererbungsl. 89, 587, 1958.

2.10. Preliminary Experiments on the Chemistry of
Crossing-Over. Sheldon Wolff and Frede-
rick J. de Serres (Oak Ridge, U.S.A.).

Experiments with metabolic inhibitors have
indicated that protein synthesis is necessary for
the rejoining of radiation-induced chromosome
breaks. Because of these experiments Wolff has
postulated that the bonds formed at rejoining
are in protein. Although crossing-over may in
some ways be likened to breakage and rejoining
there are some differences. We, therefore,
treated Neurospora crassa with specific metabolic
inhibitors at the time of crossing-over to see if
recombination is affected when certain cellular
synthetic processes are blocked. In particular,
the experiments were designed to see whether or
not protein synthesis inhibitors which can in-
hibit the rejoining of radiation-induced breaks
can affect crossing-over.

Several experiments of two different types were

2.11. Tetrad Analysis of Crosses of Neurospora
crassa Grown on Media Containing Various
Concentrations of 5-bromouracil and Cytidine
Sulfate. S. F. H. Threlkeld (Hamilton,
Canada).

Crosses of two Neurospora crassa strains A>
arg-3 (30300), cr, tryp-2 (75001), ylo (Y30539y)
and a, pyr-1 (H263), pdx-1 (37803) were made on
various media, by conidiating 5-day-old cul-
tures of the protoperithecial parent, a pyr-1
pdx-1, with conidia from the other strain.

Standard Neurospora reproductive medium
was supplemented in the following ways to give
four types of media: (i) cytidine sulfate 100mg/l.,
5-bromouracil 0 mg/1.; (ii) cytidine sulfate
100 mg/1., 5-bromouracil 100 mg/1.; (iii) cytidine
sulfate 45 mg/1., 5-bromouracil 100 mg/1.;
(iv) cytidine sulfate 45 mg/1., 5-bromouracil
0 mg/1.

Crosses grown on media (i) and (ii) showed no
significant differences with respect to ascus
patterns or recombination frequencies. Com-

12

Section 2 — Recombinanon

pared with the crosses grown on media (i) and
(ii), crosses grown on media (iii) showed a
highly significant increase in asymmetrical te-
trads, and in the recombination frequency be-
tween the marker ylo and the respective centro-
mere. Crosses grown on media (iv) were infertile.
These results will be discussed.

2.12. Changes of the Recombination Frequency by
Means of Different Concentrations of Ca and
Mg in Barley. Milos Ondrej (Prague,
Czechoslovakia).

Fi generation of hybrids between cultivar
Stupicky plnozrny and the new-breeding Solary
38-50 were cultivated in sand cultures and
watered with these kinds of nutrient solution:

1. with normal content of Ca and Mg (N)

2. with 100 < decreased content ( — )

3. with 2 X increased content (+)

The last batch of plants were cultivated in field
conditions (C). The content of the nutrient
medium influenced all the quantitative charac-
ters of the analysed plants.

In F2 generation the recombination frequen-
cies for rough-smooth and long-short rachila
hairs were tested.

The lowest recombination frequency was
found in the progeny of plants C, higher in N,
more higher in (-f) and highest in ( — ) nutrient
solution.

The results are in controversy with those
authors which used EDTA to influence the re-
combination values. It is possible to explain it in
two different ways:

1. The meiotic cells were not influenced by
the decreasing or increasing content of Ca and
Mg. They were influenced only by the general
physiological changes of the whole organism.

2. EDTA acts on crossing over another
way other than direct binding of ions Ca and Mg
from chromosomes and their environment.

2.13. Effect of Temperature on Recombination in
Sphaerocarpus. W. O. Abel (Heidelberg,
Germany).

The effect of temperature on recombination
in Sphaerocarpus was tested in 3- and 4-factor
crosses. The position of the loci used in the
crosses enabled an analysis of recombination
almost in the whole now known left arm of the
linkage group I. The sporophytes grew during
the (for the recombination) important stages of
development in temperatures of 10, 18, 26 or

33°C. The temperature of 33°C had to be re-
duced for physiological reasons to 19°C during
the night. The temperatures of 10, 26 and 33°C
caused for all chromosome sections higher re-
combination frequencies than the temperature
of 18°C. We found therefore a minimum at
18°C for the recombination frequency. This
minimum was very striking and statistically
significant in the chromosome section near the
centromere. The double crossing over frequency
was higher in the experiments at 10, 26 and 33°C
than at 18°C. It is very likely that the increase
of the double crossing over frequency was
caused through a decrease of crossover inter-
ference. This demonstrated the alteration of the
second division segregation for the locus
"crassa" from 91 per cent at 18°C to 69 per
cent at 33°C.

The observation of a minimum for the re-
combination dependent on temperature, the
reaction of crossover interference and the high
sensitiveness to temperature of the chromosome
region near the centromere are comparable
with the findings of Plough and other authors
in Drosoplu'la.

2.14. The Genetic Effect of Mitomycin C. Robin
Holliday (Hertford, Great Britain).

All mutagenic agents which have so far been
tested are able to induce somatic recombina-
tion in diploid strains of fungi. Apart from their
specific mutagenic properties, such agents may
have a more general metabolic effect, in particu-
lar the temporary inhibition of DNA synthesis
relative to RNA and protein synthesis. In order
to determine which of these properties is respon-
sible for the induction of recombination, a
heterozygous diploid of Ustilago maydis and a
heteroallelic diploid of Saccharomyces cerevisiae
have been treated with the antibiotic mitomy-
cin C, which is known to be a specific inhibitor
of DNA synthesis. It was found that in Ustilago
mitomycin is somewhat more efficient than ul-
traviolet light in stimulating reciprocal mitotic
crossing over; whereas in Saccharomyces it is
less efficient than ultraviolet light in stimulating
nonreciprocal recombination or gene conver-
sion. Parallel experiments with haploid strains
of Ustilago have shown that mitomycin has
no mutagenic activity. It is proposed that agents
which induce recombination but not mutation
be termed recombinagens. Experiments are now
in progress which will attempt to determine
whether recombination can occur during mito-
mycin treatment, i.e. in the absence of genetic

13

Section 2 — Recombination

replication, or wncther it occurs only subsequent
to the treatment.

2.15. Centromeres and Sites of Affinity Located on
Saccharomyces Chromosomes. Carl C. Lin-

DEGREN, YUH LlN HWANG, J. K. BHATTACHAR-

jee and Gertrude Lindegren (Carbondale,
U.S.A.).

The analysis of data from more than 5000
yeast tetrads has revealed eleven independently
segregating centromeres and five sites of affinity,
confirming the data previously reported by
Lindegren et al. (1962) concerning Chromo-
somes I to X. The centromeres (C), their markers
and the number of linked genes, in parentheses,
are:

Ci, ad-1 (1); Cn, ga-1 (8); Cm, le-2 (5);
Civ, tr-1 (1); Cv, ur-3 (14);

Cvi, hi-2 (4); Cvn, le-1 (3); Cvm, ar-4 (1);
Cix, hi-6(2);Cx, met-3 (1).

The new eleventh centromere-marker, met- 14,
is about 10 strains from Centromere XI. A
cluster of 4 sites of affinity linked to 6 genes lies
near Centromere V; a fifth, linked to 2 genes,
lies near Centromere VIII. Four segments
containing 11 genes are not yet linked to cen-
tromeres. The homogeneity of data between
crosses has been examined and gene-to-centro-
mere distances have been calculated to a high
order of accuracy.

2.16. Comparison of Sexual Recombination in Escheri-
chia coli with Gene Recombination in Strep-
tomyces violaceoruber. S. G. Bradley (Min-
neapolis, U.S.A.).

E. coli cells and S. violaceoruber hyphae
contain several to many sets of genetic informa-
tion. In neither organism is the germ plasm
separated from the cytoplasm by a well-defined
nuclear membrane. Cellular or hyphal fusion
therefore is tantamount to nuclear fusion. The
resulting heterogenomic state is maintained
during several cell divisions in E. coli and indefi-
nitely in the mycelia of S. violaceoruber.
Homogenomy is established in S. violaceoruber
by serial plating of spores. In both microbes,
parental types are recovered frequently from
recombinant clones. Many recombinant pheno-
types may be derived from a single heterogeno-
mic clone. Different sublines of a heterogenomic
clone often show disparate patterns of segrega-
tion for one or more characters. Recombinant
clones may appear homogeneous for some ex-

pressions but heterogeneous for others. These
results are best explained in terms of repeated
opportunity for recombination during vegetative
growth. The recombinational process in both
microbes is essentially similar to parasexuality
as described in Aspergillus by Pontecorvo. S.
violaceoruber differs from E. coli in that the
mycelial growth habit of the former stabilizes
the heterogenomic state whereas the relatively
regular cell division of the latter establishes
homogeneity rather quickly. If pedigrees based
on single cells and serial spore analyses are
compared, this difference disappears. Another
difference is that S. violaceoruber seems to
possess two linkage groups capable of indepen-
dent segregation whereas E. coli has a single
linkage group.

2.17. Mitotic Recombination within the pabal Re-
gion of Aspergillus nidulans. Alexandra
Putrament (Warsaw, Poland).

The results of analysis of 393 mitotic recom-
binants within the pabal region of Aspergillus
nidulans can be summarized as follows:

Chromatid segregation following intragenic
mitotic recombination is not random. Wild-
type (selected) strands segregate preferentially
with non-recombinant ones.

Reciprocal, non-reciprocal and probably
inexactly reciprocal exchanges can occur in
intragenic mitotic recombination.

In intragenic mitotic recombination within
one effective pairing segment exchanges can
occur between three or even four chromatids.

Negative interference observed in intragenic
mitotic recombination in Aspergillus nidulans
is higher than that observed in meiotic recombi-
nation within the same gene (pabal). In fact
it resembles rather the pattern of intragenic
meiotic recombination observed in Neurospora
crassa.

Polarity of recombination observed in the
present work has the same tendency as that ob-
served in meiotic recombination within the
pabal region; it is even more pronounced.

2.18. Mitotic Recombination in Translocation Hetero-
zygotes of Aspergillus nidulans. Etta Kafer
(Montreal, Canada).

A variety of rare segregants can be isolated
from vegetative diploid cells of the normally
haploid fungus A. nidulans. These segregants
are of a few specific types and most of them

14

Section 2 — Recombination

appear to be produced either by mitotic crossing-
over or by mitotic non-disjunction. Unusual
types of segregants have been isolated from cer-
tain diploids. All results obtained are consistent
with the hypothesis that these diploids contain
chromosomal aberrations, most probably trans-
locations. Such aberrations cause consistent
complete linkage of the markers of the two
linkage groups involved in the haploids resulting
from repeated steps of mitotic non-disjunction.
This is expected if near-haploids with unbalanced
chromosome complements, that is dis omic and
nullisomic for translocated segments, are inviable.
The frequency of haploids is therefore reduced
(evident, e.g. relative to the frequency of diploid
segregants resulting from more than one event
of mitotic recombination).

Similarly, mitotic crossing-over in chromo-
some arms heterozygous for translocated seg-
ments regularly leads to the formation of unu-
sual segregants. Suitably marked diploids were
produced heterozygous for either or both of the
translocations T(VI-VII) and T(I-VII), as well as
normal and translocation homozygotes. Mitotic
crossovers selected in one of the involved arms
were found to be unstable and presumably unbal-
anced. They produce regularly, by further steps of
mitotic recombination, better growing diploid
and haploid sectors on complete medium.
Segregation patterns specific for each transloca-
tion were observed and segregants of each type
were found to show very characteristic pheno-
types. Information on the position of the
breakage points as well as the relative size of the
translocated segments was obtained.

2.19. Somatic Recombination in Coprinus lagopus.

K. M. Swiezynski (Warsaw, Poland).

In a vegetative mycelium, arising from a
compatible or incompatible di-mon mating,
frequently new nuclei arise, containing markers
originating from two different nuclei.

Dikaryotic mycelia containing such nuclei
fruit easily. In the progeny markers often appear
in the proportion 2:1. The 1 : 1 proportion of
alleles is also frequent.

In common AB heterokaryons, containing
two types of nuclei with complementary growth
requirements, occasionally prototrophic nuclei
arise and may be isolated. Mycelia containing
such nuclei mate with compatible strains. In
the progeny of the fruibodies obtained from
such matings again the alleles often appear in
the proportion 2 : 1 or 1 : 1.

The simplest explanation is that in hetero-
karyons nuclei may fuse and become diploid.
Such nuclei are unstable and turning back to

the haploid state in vegetative mycelia give rise
to somatic recombinants. Hypothetical diploid
nuclei, containing different alleles of the A mating
type locus, are apparently very unstable and
were never isolated. On the other hand diploid
nuclei homozygous for the A locus are compara-
tively stable and are able to form dikaryons
with compatible haploid nuclei.

The results will be published in detail in the
journal Genet ica Polonica.

2.20. Somatic Recombination in Arabidopsis.

Y. Hirono and G. P. Redei (Columbia, U.S.A.).

Somatic segregation in heterozygotes may be
brought about by: deletion (breakage), sorting
out of extranuclear elements, non-disjunction
(reduction), mutation (gene conversion), trans-
location and crossing over. In higher organisms,
the distinction among these possibilities is
difficult partly because the lack of special cell
markers and mainly because of the determinate
differentiation of the cells. In Drosophila con-
vincing positive evidence is available for somatic
crossing over, though without breeding test but
with supporting cytological observation of
somatic pairing. Until now, only in certain fungi
has it been possible to demonstrate the occur-
rence of mitotic recombination with progeny
test, though the responsible chromosomal
mechanism is not amenable to cytological anal-
ysis. In Arabidopsis plants (crucifer) heterozy-
gous for three good linked markers (ch1: chloro-
phyll-b free; gi2: late-vigorous; and pa: dark
green, dwarf) somatic segregation was induced
by X-raysand the seed obtained from the pheno-
typically different two sectors has been subjected
to further genetic analysis. Though the irradia-
tion caused multiple hereditary alterations, in
the case of one plant the experimental data can
be interpreted only by genetic exchange at the
four-strand stage. Observations on somatic
metaphases of wild type plants indicate that
juxtaposition of the homologous chromosomes
occasionally occur in Arabidopsis. Full details
will be reported in Genetics.

Contribution from the Missouri Agricul-
tural Experiment Station. Journal Series No.
2543. Approved by the Director.

2.21. Mitotic Recombination in Yeast. B. S. Cox

(Liverpool, Great Britain).

A diploid yeast is described with which it has
been possible to apply three of the selective

15

Section 2 — Recombination

techniques described by Pontecorvo for de-
tecting mitotic recombination, namely the use of
<a) a morphological gene and (b) a recessive
resistance gene in heterozygous combination
with their wild-type alleles, and also the use of
recombination between "heteroallelic" mutants
in the same cistron to produce a heterozygote of
wild phenotype. Roman's (1956) finding, that
selection using the last system results in the
detection of exclusively non-reciprocal events
best explained by a copy-choice theory of re-
combination, is confirmed, but it is also found
that some of the recombinants so selected can-
not be explained so simply and may provide
evidence of either four-strand or inexact recip-
rocal recombination at mitosis.

When homozygous recombinants from hetero-
zygous loci are selected, for in this diploid they
are found to fall into two classes: 90 per cent or
more are found to be homozygous at all the
linked marked loci, both proximal and distal to
the "selected" locus; the remainder are non-
recombinant at any other marked locus, either
proximal or distal.

It is proposed that the second class arise by a
recombination process similar to that, already
described, which is observed in selecting for
recombination between heteroalleles. The other,
majority, class may be the result of chromatid
non-disjunction rather than mitotic crossing-
over. That marker genes unlinked to the selected
genes remain heterozygous in all these recom-
binants indicates that this non-disjunction is
not general, that is, that the recombination is
not due to the occurrence of the first division of
meiosis.

2.22. U.V.-induction of Somatic Recombination in
Yeast. David Wilkie (London, Great Britain).

In diploid yeast cells the change from the
heterozygous Ad2 ad2 (white colony, proto-
trophic) to the homozygous recessive ad2 ad2
(red, adenine-requiring) takes place spontane-
ously with a low frequency and is usually detected
as a red sector in a white colony. The change can
be induced by u. v. -irradiation and irradiated
cells when plated out give rise to colonies classi-
fiable as follows:

(1) all white, showing no recombination;

(2) half-sectored, or otherwise showing sec-
toring from the centre of the colony,
indicating segregation at the first mitotic
division;

(3) all red. The relative frequency of this class
increases with dose suggesting the recip-
rocal white cell had a lethal defect;

(4) a. showing a small red sector or sectors
mainly at the periphery,
b. mottled red and white.

(4) a and b are interpreted as showing an
unstable condition of partial induction which is
apparently transmissable. In other words, it
appears that the u.v.-induction is not an all-or-
none event and cannot be attributed directly to a
photochemical change. Further evidence of the
complexity of the recombination process is seen
in the results of preliminary experiments which
show that u.v.-induction is dependent both on
the intensity of the irradiation and on the
temperature at which it is administered. It is
suggested a comparison along these lines be
made with heteroallelic systems where recombi-
nation appears to involve a different mechanism.

2.23. Extrabasidial Nucleic Recombinations in a Basi-
diomycete Coprinus radiatus. Nicole Prud'
homme (Gif-sur-Yvette France).

It is possible to make triheterokaryons of
Coprinus from three monokaryotic strains. In
these heterokaryons new nuclei have been
demonstrated, formed through an exchange of
genetic material between two of the original
nuclei. The study of these nuclei leads to the
following facts:

(1) High frequency of aneuploid nuclei
(single or multidisomic).

(2) Possible recombinations of the genes
located on non-homologous chromosomes of
the original nuclei.

(3) Apparently total linkage between genes
located on homologous chromosomes, even if
these genes are 35 c.o. units apart from each
other.

(4) More frequent appearance of some nu-
clear types than others.

These data suggest a mechanism of parasexual
recombination analogous to that described in
various Ascomycetes, associated with possible
selection of some nuclear types.

2.24. The Purple Suppressor System in Coprinus lago-
pus. D. H. Morgan (Hull, Great Britain).

It has been established that certain suppressor
mutations in Coprinus may be functionally alle-
lic with one another, as judged by a complemen-
tation test, but at different chromosomal loca-
tions as judged by recombination data.(1)
Further work on the purple-suppressor system is
now reported.

16

Section 2 — Recombination

When different cultures, in which spontaneous
suppression of the mutant gene purple has taken
place, are crossed with wild type, the proportion
of unsuppressed purples appearing in the prog-
eny varies (between suppressors) from 0 per
cent to about 19 per cent. The scatter of values is
continuous rather than discrete. Identification
of the expected reciprocal recombinant class
has been attempted, but unequivocal evidence
for its occurrence and for the 1 : 1 segregation of
suppressors has not been obtained. Evidence
from the resolution of dicaryons formed between
suppressed and unsuppressed cultures shows
however that suppression is not due to cyto-
plasmic factors. Suppressed cultures are quite

stable and recombinant suppressed cultures,
from crosses with wild type, have exactly the
same characteristics as their parents with
respect to segregation of purples and comple-
mentation reactions. Infertility is frequent in
crosses involving suppressed purples. It is pos-
sible that the spontaneous suppression of purple
involves chromosomal changes, in which case
non-complementation between suppressors
which have different characteristic purple segre-
gations would be less surprising.

1. D. Lewis, 1960; D. H. Morgan, 1961.

17

SECTION 3

MOLECULAR AND MICROBIAL GENETICS

3.1. Further Studies on Incorporation of Homologous
DNA into the Mouse Germ Cells in vivo.
Chai Hyun Yoon (Chestnut Hill U.S.A.).

DNA in the thymus glands was tagged with
thymidine-H3, and the extracted thymus-
DNA-H3 was injected into the gonads of mature
male mice. The germ cells of recipient mice
were studied with both squash and section met
thods for the evidence of thymus-DNA-H3
incorporation in vivo. Quantitative data will be
presented.

3.2. Evidence for the Source of Messenger RNA and
the Control of Its Production in the Regulation
of Gene Activity. Alfred Marshak (Phila-
delphia, U.S.A.).

Ribonucleic acid from cytoplasm (c-RNA) is
so constituted that on treatment with weak
alkali all of its constituents are released as
nucleotides. In contrast, ribonucleic acid
from nuclei of the same or similar cells (n-RNA)
when similarly treated yields adenine, guanine,
and cytosine and adenosine in amounts equal to
the nucleotide released. The release of these
bases is correlated with the presence of material
extractable in lipid solvents which renders the
ribosidic bonds susceptible to alkali hydrolysis.
When both components of n-RNA are accounted
for, it is found to have base ratios resembling
those of DNA. The component of n-RNA not
associated with lipid has ratios similar to those of
c-RNA. Previously it was shown that n-RNA is
the precursor of ribosomal RNA. The present
experiments indicate that it contributes to the
ribosomes that portion of its nucleotides which
are not associated with lipids. The n-RNA thus
has the properties required of a source of mes-
senger RNA. It is proposed that the association
of lipid or lipoprotein with the n-RNA provides
a mechanism for the control of gene activity
through regulation of the release of messenger
RNA from n-RNA or by other means.

3.3. The Structure of Adapter RNA. Geoffrey
Zubay (Upton, U.S.A.).

Adapter RNA is the carrier of amino acids to

the template for protein synthesis. Part of its
function is to arrange the amino acids in a
specific order prior to peptide synthesis. It does
this by the following means: each adaptor
combines with one — and only one — amino
acid; and each adaptor is presumed to have a
trinucleotide sequence which hydrogen-bonds
to a complementary site on the messenger
RNA template. Physico-chemical measure-
ments indicate that adapter RNA is a single
polynucleotide chain about 67 nucleotides long.
It has been proposed that the polynucleotide
chain has a bend near the middle, with the two
halves of the chain interacting in double helical
fashion. to In a more recent and detailed model,
it has been suggested that the only nucleotides
not in the double helix configuration are the
three in the bend, and the two on the nucleoside
end which combines with the amino acid.('2)
This structure will be discussed with regard to
(1) stereochemical problems of protein syn-
thesis^23) and (2) chemical methods for demon-
strating the location and composition of the
coding nucleotides. (4)

Research carried out at Brookhaven National
Laboratory under the auspices of the U.S.
Atomic Energy Commission.

1. G. Brown and G. Zubay, /. Mol. Biol. 2,
287, 1960.

2. G. Zubay and J. Bergeron, Symp. A-II-I,
8th Intern. Microbiology Congr., Montreal,
1962, in press.

3. G. Zubay, Science, in press.

4. G. Zubay, Biophys. Biochem. Res. Commtin.,
in press.

3.4. The Genetic Implication of the Methylated Bases
in Transfer RNA. Erwin Fleissner, P. R.
Srjnivasan and Ernest Borek (New York,
U.S.A.).

Transfer RNA is characterized by the unique
presence of a small number of methylated
purines and pyrimidines. The origin of these
methyl derivatives has been obscure until re-
cently. We have shown that the methylated bases

19

Section 3 — Molecular and Microbial Genetics

are not incorporated a priori into the primary
structure of s-RNA but are acquired by the
methylation of the polynucleotide chain by a
multiple enzyme system - RNA methylase—
which uses s-adenosyl methionine as the methyl
donor.d)

Studies of the RNA methylases from different
species revealed that the enzyme systems are
endowed with great species specificity.

The s-RNA from a given source while fully
methylated with respect to its homologous
methylating enzymes can accept excess methyl
groups from an enzyme from a different species.
These findings tend to eliminate the possibility
that the methylated bases are involved in the
coding of amino acids. If it is assumed that the
amino acid code is universal, the methylating
enzymes of all species would be expected to be
uniform. Yet we found that a presumably fully
encoded s-RNA of one specieo can accept methyl
groups from a heterologous enzyme system.

The species variation of the RNA methylases
has also been adumbrated by the analytical data
on the distribution of the methylated bases of
s-RNA from various sources.

However, the biological function of the spe-
cies variation of the enzyme and, indeed, of the
methylation itself awaits elucidation.

1. E. Fleissner and E. Borek, Proc. Nat. Acad.
ofSci., U.S.A. 48, 1199, 1962.

3.5. Synthesis of Specific RNA on Different Sites of
the Phage T2 Chromosome in vivo and in
vitro. R. B. Kcesin, M. F. Shemjakin, G. M.
Gorlenko, I. A. Bass and A. A. Prozorow
(U.S.S.R.).

1. Different types of phage-specific mRNAs
are synthesized during early and later stages of
the intracellular development of the phage T2 in
E. coli B. The phenomenon is in accordance
with the synthesis of different proteins during
those stages (early enzymes and phage particle
protein). Experiments on the formation of
complexes of Cl4-mRNA and phage DNA show
that the mRNAs, synthesized on different stages,
correspond to different regions of phage DNA.

2. Experiments with chloramphenicol indicate
lhat the regulation of the synthesis of different
specific mRNAs depends on the synthesis of
proteins.

3. In vitro experiments showed that phage
DNA, added as a primer to an extract from
uninfected E. coli cells, directed only the synthesis
of a type of mRNA, which is similar to that

synthesized in vivo during the early stages of
infection. The same mRNA was obtained when
native deoxyribonucleoprotein from T2 phage,
DNA from phage-infected cells or renatured
T2-DNA were used as primer.

4. Also in systems containing T2-DNA as a
primer and partially purified enzymes from un-
infected E. coli B or B. prodigiosan} cells only
early mRNA was synthesized. In the in vivo
experiments with RNA-polymerase it was pos-
sible to obtain the synthesis of specific mRNAs
formed on different regions of the phage chro-
mosome (DNA) in the infected cells. The isola-
tion and purification of DNA does not change
the order of reading of the genetic information.

3.6. The Active Complex in Protein Synthesis. R.

Haselkorn, V. Fried, and J. Dahlberg
(Chicago, U.S.A.).

RNA from turnip yellow mosaic virus has
been used to characterize the complex responsible
for protein synthesis in cell-free extracts from
Escherichia coli. Three types of experiment
were performed, all utilizing zone centrifugation
through sucrose gradients. In the first type,
viral RNA and coli ribosomes are mixed, frac-
tionated on the gradient, and the RNA located.
Secondly, the RNA ribosome mixture is frac-
tionated, and the individual fractions are as-
sayed for their ability to incorporate amino
acids. Finally, the RNA-ribosome mixture is
allowed to incorporate amino acids, then frac-
tionated, and both the RNA and newly made
protein located. All three experiments give es-
sentially the same result, namely, that in the
cell-free system 5-10 per cent of the ribosomes
participate in protein synthesis and the active
complex consists of one 70S ribosome and one
molecule of viral RNA.

3.7. A Model System for Studying the Genetic Deter-
minants of Protein Configuration. Charles J.
Epstein and Robert F. Goldberger (Bethes-
da, U.S.A.).

It is generally accepted that the genetic infor-
mation contained in linear DNA molecules is
transferred, via messenger RNA, to linear
polypeptide chains. However, the process by
which these chains are converted to "native"
proteins is poorly understood. The reduction and
reoxidation of ribonuclease by Anfinsen, White
and co-workers provided a model system for
studying this process, and led to the conclusion

20

Section 3 — Molecular and Microbial Genetics

that all information necessary for the secondary
and tertiary structure of this enzyme is contained
within the primary amino acid sequence. More
recently, similar results have been obtained
with other proteins, indicating that this conclu-
sion may be generally applicable. Because of
the relatively long time required for the refolding
process in vitro, in contrast to the rapid synthesis
of native proteins in vivo, a catalytic system was
sought and found in rat liver extracts. This
system, consisting of soluble microsomal and
supernatant factors, greatly accelerates the reac-
tivation of reduced ribonuclease.

In order to study the effects of amino acid
alterations on the configuration, as opposed to
the "active center", of proteins, chemically
modified, but still active, enzymes have been
prepared. The effects of the modifications on
folding of the extended molecules (following
reduction) are now being studied.

Much of this work was done in conjunction
with Dr. Christian B. Anfinsen.

3.8. The Evolution of the Fibrinogen Molecule'

Russell F. Doolittle and Birger Blomback
(Stockholm, Sweden).

Fibrinogen is a protein molecule which is
apparently found in the blood plasmas of all
vertebrate species. During its conversion to
fibrin by thrombin, certain segments of the
parent molecule are proteolytically released.
These peptides, usually termed the fibrinopep-
tides A and B, range from 13 to 21 amino acids
in length, depending on the species from which
they are obtained. Comparisons of the amino
acid sequences of fibrinopeptides from a variety
of vertebrates has permitted us to follow a
flow of amino acid substitutions through these
regions of the fibrinogen molecule. Complete
sequences are now available for the A and B
fibrinopeptides from ox, human, sheep, and pig.
In addition, partial amino acid sequences of
the peptides from several other mammals and
the lamprey eel (Petromyzon marinus) are re-
ported. The differences in sequence are greater
than expected. Thus, the B fibrinopeptides of
sheep and ox differ in ten of twenty residues.
On the other hand, certain positions and seg-
ments of the peptides appear to have been
maintained throughout all the species examined.
Only one fibrinopeptide has been isolated from
lamprey eel fibrinogen so far. Its partial amino
acid sequence suggests that it has characteristics
of both the A and B mammalian fibrinopeptides.

For example, it contains tyrosine-O-sulfate, an
unusual amino acid found in the B fibrinopep-
tides of several mammals, but its C terminal
portion corresponds to the C terminal sequence
of the mammalian A fibrinopeptides. Although
our observations are restricted to those amino
acid substitutions which have survived, as op-
posed to all those that may have occurred, it
is possible to draw some conclusions about the
plausibility of suggested amino acid codes.

3.9. Control of DNA Replication in an Organism
with Synchronous Mitosis. E. Guttes and
S. Guttes (Providence, U.S.A.).

The nuclei of the plasmodial slime mold,
Physarum polycephalum, divide in synchrony
every 12-14 hr. The replication of DNA occurs
during a period of 3-4 hr immediately following
mitosis. The role of the nuclei in the initiation
of this process was investigated by examining
their competence for DNA replication at various
stages of the replication cycle. For this purpose,
the normal correlation between the nuclear and
the cytoplasmic replication cycle was disrupted
by implanting nuclei representing one stage of the
cellular cycle into Plasmodia representing an-
other. We found that hostplasmodia at a stage
of the cell cycle at which they are able to sus-
tain DNA biosynthesis and replication, cannot
initiate DNA replication in implanted nuclei,
unless these have undergone mitosis just prior
to the implantation. This failure is not due to a
lack of functional integration between the host
Plasmodium and the implanted nuclei, nor to
unspecific damage to the latter as a result of the
implantation procedure. DNA feeding with
subsequent exposure to tritiated thymidine re-
sulted in strong DNAse labile cytoplasmic la-
belling at all times of the intermitotic period. It is
concluded that only at mitosis the nuclei acquire
competence for DNA replication and that this
event is a critically controlling factor in the
timing of DNA replication. The structural
alteration of the nuclei in acquiring of compe-
tence is not reflected in their in vitro behaviour.
In an experiment involving exposure of fixed
nuclei to a suitable DNA-polymerase prepara-
tion*1) we found that nuclei in ethanol-fixed
smear preparations were at no time of the repli-
cation cycle able to serve as primers, whereas
nuclei which were previously fixed with denatur-
ing ethanol-acetic acid(2> were able to serve as
primers. An investigation with the electron
microscope^3) has shown that in vivo the onset of
DNA replication coincides precisely with the
time at which prenucleolar material is beginning

21

Section 3 — Molecular and Microbial Genetics

to be dissociated from the chromosomes after
mitosis. We assume that the acquisition of
competence at mitosis for DNA replication
may result from a dissociation between the
DNA double helix and a previously stabilizing
(prenucleolar?) protein.

1. Unpublished results of R. C. von Borstel,
D. M. Prescott, and F. J. Bollum; Bollum,
personal communication.

2. Unpublished result of E. Guttes and F. J.
Bollum.

3. In collaboration with R. A. Ellis, Brown
University, R. I.

3.10. Incorporation of H3 Thymidine by K12 (A)
Induced by Streptonigrin. Charles M. Radding
(Michigan, U.S.A.).

K12(A) is induced by streptonigrin (SN), an an-
tibiotic and anti-tumor agent^1)- Like Mitomycin
C, SN initiated a rapid breakdown in vivo of the
DNA of E. coli to acid soluble products: 35
per cent of H3 labelled DNA was rendered acid
soluble in 2 hr. Vegetative multiplication of
phage was also initiated, and, in spite of the
rapid degredation of the bulk of cellular DNA,
all cells could be induced to produce about
50 phage per cell.

The net incorporation of H3 thymidine (in a
1 min pulse) into acid insoluble material was
90 per cent inhibited in 20 min by SN in lysogenic
and non-lysogenic cells. Only in lysogenic cells
was a recovery observed in the ability to incor-
porate H3 thymidine. The peak of this recovery
coincided approximately with the appearance
of mature intracellular phage. Consistent
with incorporation of H3 thymidine into DNA,
both before and after treatment with SN, was
the sensitivity of the acid insoluble radioactivity
to heating in acid, and stability to heating in
alkali. Further studies will attempt to determine
if the incorporation of H3 thymidine by lysogenic
cells induced by SN is equivalent to the specific
synthesis of A DNA. Preliminary study of
defective A lysogens indicates that they are
separable into those which can or cannot in-
corporate H3 thymidine after treatment with
SN.

3.1 1. Replication of T2 DNA. F. R. Frankel and
L. Ingraham (New York, U.S.A.).

Bacterial cells infected with bacteriophage T2
contain phage particles and intermediate DNA
structures. Chloramphenicol (CM) arrests the
production of phage but permits continued
synthesis of DNA. On removal of the inhibitor,
most of the accumulated DNA is utilized in the
formation of complete phage particles. We have
observed that immediately upon addition of
CM to an infected culture, not only does phage
synthesis cease, but molecules of DNA like
those contained in phage particles are no longer
produced. Instead, about 25 per cent of the
total DNA of these cells is recovered as unusual
DNA structures, in many respects similar to,
but apparently larger than, phage DNA.(1) This
material is not a precursor of phage DNA,
since on removal of CM (1) it does not diminish
in amount, and (2) radiophosphorus enters
phage DNA without passing through these
structures. If fluorodeoxyuridine is added to a
CM-treated culture, total DNA synthesis
abruptly stops. However, the amount of these
unusual DNA structures continues to increase.
Therefore, this material may be derived from the
actual phage DNA precursor, which itself
resists isolation, and perhaps it reflects the
unusual structure of the latter. In fact, subjecting
the extracts to gentle shear further increases the
yield of these unusual molecules. We are examin-
ing the hypothesis that phage precursor DNA is
composed in large part of exceptionally large,
fragile DNA molecules.

1. Proc. Nat. Acad. Sci. 49, 1963.

1. M. Levine, Bacteriological Proceedings 1963,
in press.

3.12. Inhibitory Effect of Chloramphenicol on Mating
Fragmentation of T4 Phage DNA. Andrzej
W. Kozinski (Philadelphia, U.S.A.).

In our previous paper we have shown that
parental T4 DNA undergoes an extensive frag-
mentation in the vegetative pool. As a result,
final progeny molecules contain only small
fragments of parental DNA as semi-conservative
sub-units^1 2). In the present studies, we have
shown that chloramphenicol inhibits fragmenta-
tion of parental DNA in the vegetative pool,
allowing at the same time strictly semi-conser-
vative replication and apparently unimpaired
net synthesis of progeny DNA. We conclude
that molecular matings in the DNA vegetative
pool require concomitant synthesis of protein.

22

Section 3 — Molecular and Microbial Genetics

presumably facilitating the recombination be-
tween DNA molecules.

1. A. W. Kozinski, Virology, 13, 124, 1961.

2. A. W. Kozinski and P. B. Kozinski, Virology,
1963 (in press).

3.13. The Arrangements of Nucleotide Sequences
in T2 and T5 Bacteriophage DNA Molecules.

C. A. Thomas, Jr. and I. Rubenstein (Balti-
more, U.S.A.).

The genetic map of T2 and T4 bacteriophage is
circular but the DNA molecule that is liberated
by phenol extraction is a linear duplex of poly-
nucleotide chains. If the genetic map is related
to the physical structure of the DNA molecule
the problem arises as to how a linear molecule
can give rise to a circular map. An explanation
can be made on the basis that the bacteriophage
liberate molecules which have nucleotide se-
quences which are circular permutations of each
other. Thus, markers which are most distant on
one molecule are closest together on another.
To test this hypothesis, the middles of T2 and
T5 DNA molecules were mechanically deleted
and the absence of certain nucleotide sequences
was tested by "renaturation" or "reannealing"
experiments using columns containing dena-
tured DNA immobilized in agar beads. The re-
sults indicate that when the middles are deleted
from the T5 DNA molecule, some special se-
quences are removed; whereas, when the middles
are deleted from the T2 DNA molecule, no
special group of sequences are removed. This
would indicate that the T2 molecule had a
random beginning point while the T5 molecule
has a special beginning point. These results would
predict that T5 should have a single linear ge-
netic map.

3.14. The Action of Streptonigrin on Genetic Re-
combination between Bacteriophages. Myron
Levine and Marcene Borthwick (Ann
Arbor, U.S.A.).

The antibiotic streptonigrin preferentially af-
fects DNA metabolism in bacteria and is an
excellent inducer of phage production in indu-
cible lysogenic bacteria^1). Streptonigrin also
has a striking effect on phage recombination.
Short treatments (as short as 2 min exposures
to lOrg/ml of the antibiotic) of mixedly in-
fected E. coli cells results in 2-3-fold increases in
the frequency of recombination between T4rII

mutations. Of special interest is the finding of a
critical time during which infected complexes
must be treated in order to obtain this effect on
recombination. Increases in recombination fre-
quencies are observed following treatment of
infected cells early in the latent period, within
the first 10 min. Treatments at later times in the
latent period result in normal recombination
frequencies. The data suggest a change in sus-
ceptibility to the action of streptonigrin on
genetic recombination at about the 8th to 10th
minute of the infection. Similar effects have been
observed with phage P22 of Salmonella typhi-
murium.

1. M. Levine and M. Borthwick, Bacteriologi-
cal Proceedings, 1963.

3.15. Correlated Variation of Recombination Potential
and DNA Content among T4 Phage Particles.

Gisela Mosig (Cold Spring Harbor, U.S.A.).

A population of T4 phages is heterogeneous
with respect to the following properties which
appear to be correlated:

(a) potential for genetic recombinants.

(b) buoyant density in CsCl

(c) size of their DNA molecules.

Phages from prematurely lysed cultures show
slightly greater buoyant density in CsCl, and,
when used as parents in crosses, yield a lower
fraction of recombinants for given markers
than phages from spontaneously lysed cultures.

DNA isolated from unselected phage particles
falls into three classes differing in molecular weight
(recognized by column chromatography, sedi-
mentation rate, and susceptibility to hydrody-
namic shear). If unit length is assigned to the
majority (class I DNA), class II DNA measures
0.6, and class III DNA varies between 0.05 and
0.2. The DNA from these classes does not
differ in base composition.

Phages of different buoyant density differ in
DNA content. Those of average density (the
majority) contain a single molecule of class I
DNA. Less dense phages yield many recombi-
nants and contain DNA of class II. Dense phages
yield few recombinants and contain DNA of
classes I and III, or of class III only. A small
fraction of very low density phages also contain
DNA of class III only.

The following working hypothesis is proposed:
The dense phage particles yield few recombinants
because they are diploid over a wide range of
their genome and the diploid state interferes with
heterologous pairing.

23

Section 3 — Molecular and Microbial Genetics

3.16. Recombination of Purified DNA of Bacterio-
phage OX 174 in Spheroplasts. H. S. Jansz,
C. van Rotterdam and J. A. Cohen (Rijswijk
(Z.H.), The Netherlands).

The DNA-spheropIast system^1) for the deter-
mination of the biological activity of purified
DNA of bacteriophage OX 174 was employed in
a biochemical approach to the elucidation of
the mechanism of gene action. In order to study
genetic recombination in this system phage and
bacterial mutants were derived from OX 174
wild type (Oo) and its host E. coli C (Co) ac-
cording to the method of D. Pfeifer/2)

0o is designated genetically as hl+h2+ indicat-
ing that it is able to be adsorbed to and to infect
Co but not the Oo resistant bacterial strains CI
and C2. Ol (hi h2+) is able to infect Co and
CI but not C2. 02 (hi h2) is able to infect Co,
CI and C2. 03 (hl+h2) is able to infect Co but
not CI and C2. In a cross 03 x 01 recombi-
nants 02 were obtained (recombination fre-
quency 10 A). The DNA's isolated (using phenol
extraction) from purified batches of Ol and 03
were infectious to spheroplasts of E. coli
K12, producing approximately 107 phages/ml.
Mixed infection of spheroplasts of E. coli
K12 with DNA's from Ol and 03 produced
approximately 103 recombinants 02. The effects
of u.v. -irradiation and chain scission by DNAse
of one of the parental DNA's on infectivity and
ability to form recombinants were studied.
U.V. irradiation causing a fall in infectivity of
DNA down to 5 per cent of its original value
did not weaken its efficiency in giving rise to
recombinants. However, chain scission of
03-DNA by DNAse corresponding to an aver-
age of 3 hits per molecule destroys its recombining
faculty.

1. Cf. G. D. Guthrie and R. L. Sinsheimer,
J. Mol. Biol. 2, 297, 1960.

2. Z.fiir Vererbungslehre 92, 3, 1961.

3.17. The Response of Mutants of the Bacteriophage
f 2 to a Bacterial Suppressor Gene. Norton D.
Zinder (New York City, U.S.A.).

Garen and Siddiqi have isolated a suppressor
gene for certain alkaline phosphatase negative
mutants of the bacterium Escherichia coli. This
same suppressor gene can also suppress specific
mutants of the "r2" type of the bacteriophage
T4 (Benzer and Champe). The assumption is
that this suppressor gene can work on mutants
in many different cistrons.

Following nitrous acid treatment of the bac-
teriophage f2, we have obtained mutants which
respond to this same suppressor gene. These
phage mutants are being studied for their
effects upon the bacteria without the suppressor
gene. If, as supposed, mutations which respond
to this suppressor can occur in all cistrons, by
determining at what stage in their life cycle these
mutants are blocked we can infer the genetic
functions of the phage. The determinations in-
clude the production of new enzymes, infective
RNA, phage coat protein and phage particles.
In addition, RNA from the mutants is added to
E. coli extracts and the nature of the protein
products it stimulates determined.

3.18. Studies on Plaque Mutants of the RNA Phage
fr. F. Kaudewitz and P. Knolle (Berlin-
Dahlen, Germany).

Phage fr is related to the RNA phages descri-
bed by Loeb and Zinder. f1) Its physical charac-
teristics have been described by Hoffmann-
Berling.(2' The occurrence of plaque-size mutants
has been reported in phage suspensions treated
with nitrous acid (Kaudewitz and Knolle. (3)
Turbid mutants have been obtained by the same
method. These mutants are stable upon cloning.
They are inactivated by wild type fr antiserum,
their burst size is smaller than that of the wild
type phage. The infection of cells of Escherichia
coli K-12 strains by these mutants is inhibited
by RNase as is the infection by phage fr. The
analysis of recombination experiments obtained
from mixed infections involving a number of
different mutants of this type will be presented.
The data will be published in full in the Zeit-
schrift fur Vererbungslehre.

1. T. Loeb and N. Zinder, Proc. Natl. Acad.
Sci. 47, 282, 1961.

2. H. Hoffmann-Berling, Z. Naturforschg.,'m
press.

3. F. Kaudewitz and P. Knolle, Nature, in
press.

3.19. Host-range Mutations of the Bacteriophage
0X-174 and the Specificity of Its Host E. coli.

G. A. van Arkel and J. H. van de Pol
(Utrecht, The Netherlands).

The host-range phenotype provides a useful
tool in genetic studies with bacteriophage 0X-
174, which, because of the single-strandedness

24

Section 3 — Molecular and Microbial Genetics

of its DNA, has been of vivid interest to both
biochemists and geneticists. We therefore
studied some properties of host-phage rela-
tionships with this phage.

Plating wild type E. coli CI 22 with an excess
of wild type OX- 174 will result in the selection of
phage-resistant bacteria (designated by C/0).
Among them three types of C/0 mutants could
be distinguished, on the strength of differences
in the frequence with which plaques of host-
range phage mutants (Oh) were found after
plating C/O cells with wild-type phage.

The largest class, some 80 per cent of all
C/0 isolates, comprises the mutants for which
no host-range particles (i.e. less than 10_u)
were observed in wild-type lysates, not even
after heavy mutagenic treatment with nitrous
acid. These bacterial strains (C/OR) are consid-
ered to be resistant to all first step host-range
mutants. On a second type of C/O bacteria
("normal", C/ON) small, turbid host-range
mutant plaques (Ohn) were found with a fre-
quency of 3 x 10~9. From Ohn, but not directly
from wild-type phage, mutants yielding clear
plaques could be isolated (0hc). The host-
range patterns of independently isolated strains
of 0hn and Ohc are identical for all C/O in-
dicator strains. A third, extremely rare, type of
C/O mutants ("sensitive", C/OS) was charac-
terized by a higher frequency (4 x 10~T) of large,
clear plaques of host-range mutants (Ohs)
found after plating with wild-type phage. C/0S
also served as a host for 0hn and Ohc. On the
other hand, C/ON was resistant to Ohs.

Results obtained, using Shigella paradysen-
teriae Y6R as the host bacterium, partly paral-
leled those with E. coli.

Our findings will be discussed in relation to the
mechanism of phenotypic expression of bacte-
rial resistance and host-range properties of the
phage.

3.20. Studies of Proteinless Mutants of Tobacco
Mosaic Virus. A. Siegel and M. Zaitlin (Tuc-
son, U.S.A.).

Two mutants of tobacco mosaic virus have
been isolated which are unable to induce the
formation of complete virus particles upon in-
fection. Strain PM 1 induces the formation of no
detectable virus-like protein. Strain PM2 induces
the formation of a non-functional, low molecular
weight protein serologically related to tobacco
mosaic virus protein. This virus-like protein,
although it aggregates into rods under slightly
acidic conditions, will not reconstitute with
virus nucleic acid to form complete virus parti-

cles. The PM2 protein differs from parent strain
protein in one of the first forty amino acids,
starting from the acetyl-N end. Infectious ma-
terial present in phenol extracts of PM infected
leaves can be reconstituted with virus protein to
form highly infectious particles. It has also been
found that infectious nucleic acid can be sep-
arated from the bulk of the leaf RNA on
methylated albumin columns.

3.21. Hereditary Changes induced by the Host in
Bacteriophage Lambda. L. Fischer-Fantuzzi
and E. Calef (Naples, Italy).

A particular strain of K12 sensitive to lambda
(called U 173) upon infection with the hdi
mutant of bacteriophage lambda yields wild
type progeny phage or lysogenic derivatives
which in turn will release wild type phage.

Both vegetative and induced phage appear to
be mixed, wild type and mutant.

The wild type and the mutant obtained from
the growth on strain U 173 breed true.

This hereditary modification is associated with
the appearance of phages of non standard
density.

3.22. The Genetic Nature of the Exclusion of Bacte-
riophage T2 by T4 with Mixed Infection on
E. coli B. B. de Groot (Leyden, The Nether-
lands).

Cross products of bacteriophage T4 and T2
show the efficiency of plating (e.o.p.) of T4 on
certain E. coli K strains; they have the same
glucose-substitution of HMC as T4, and the
frequency of T4 markers is about 90 per cent.
This phenomenon, the exclusion of T2 by T4*
has been ascribed to one gene ("Bar") by Strei-
singer and Weigle.^1) The genetic nature of
exclusion was investigated in the following ways:

(1) Single bursts of T4 T2 crosses were ana-
lyzed completely. The majority of the progeny
particles showed the T4 host range. A few of
them showed either the u.v. sensitivity of T2,
or did not exclude the markers of T2, or appear-
ed to plate with a low e.o.p. on E. coli K strains,
like T2.

(2) Simultaneous infection of E. coli B with
heavily u.v. -irradiated T4 and unirradiated T2
resulted in marker rescue of the e.o.p. of T4,
when plating the infective centres on E. coli
K (A h). An isolated marker-rescue product did
not exclude T2 markers on crossing. Both these
experiments suggest that the "Bar"-properties.

25

Section 3 — Molecular and Microbial Genetics

result from more than one factor and cannot
be ascribed to one gene with a pleiotropic
effect.

1. P.N.A.S. 1956.

3.23. Mutants of Prophage PI which Affect Host-
induced Modification. S. W. Glover (London,
Great Britain).

Bacteriophage A plates with an efficiency of
1 .0 on Escherichia co/i strain C 600 (K), and with
an efficiency of 10 4 on K made lysogenic for
phage PI. This phenomenon is called restriction.

The A phage which comes through K(P1) plates
with an efficiency of 1.0 on both K and K(P1)
and is designated A (PI). This is called modifi-
cation. After one cycle of growth in K the
modified phage A(P1) loses its modification.

By several different methods to be described
derivatives of K(P1) have been isolated in which
the carried PI prophage is abnormal. So far two
types of abnormal PI have been characterized.
One type has lost the ability to restrict A but is
still able to modify it. The other type has lost
both the restricting and modifying functions of
PI. The results of genetic crosses between these
abnormal PI phages will be discussed.

3 .24. Different DNA Systems undergoing Host-
induced Modification in Bacteria. J. Schell
and S. W. Glover (London, Great Britain).

In the system of host-induced modification by
bacteria carrying prophage PI, A DNA is
restricted and modified (see previous abstract,
S. W. Glover).

Data will be presented and discussed concern-
ing the restriction and modification of DNA from
other sources, including:

(a) Chromosomal DNA in conjugation.

(b) Episomal DNA in Flac infection.

(c) Colicin factors in colicinogeny transfer.

(d) Phages other than A.

The nature of the abnormal PI prophage
described in the previous abstract (S. W. Glover)
has been investigated with these systems.

3.25. Affinity of Coliphage P2 for Prophage Sites.
Erich W. Six (Iowa City, U.S.A.).

Affinity of P2 for its prophage sites (I, II, etc.)

on the chromosome of Escherichia coli strain C
was studied. Usually strain C derivatives with
P2 at site I liberate phage with strong preference
for this site, whereas phage from donors car-
rying P2 at site II show no strong site preferences.

The site occupied in the donor cannot be
solely responsible for the site affinity of the pha-
ges: P2 may retain its low site I affinity even
while occupying this site in the donor, and the
site I affinity of phages from donors carrying
P2 at site II or other sites -/- I varies.

P2 from donors producing phage with strong
preferance for site I occasionally looses this prefer-
ence when lysogenizing another cell. Donors
producing phage with low site I affinity also pro-
duce some phages with strong site I preference.

All P2-like phages, including P2 Hy dis and
PK, show some affinity for site I, even if liber-
ated from donors other than E. coli strain C.
But P2-related phage P4 shows no affinity for
site I.

Existence of P2 in at least two states: one
with high preference for a particular chromoso-
mal site, one without particular site preference,
resembles the behavior of fertility factor F.
Since only relative affinities were measured,
loss of site I preference may indicate either
decrease of affinity for this site or increase in
affinity for other sites.

3.26. On the Induction of Lysogenic Bacteria by
Halogenated Pyrimidines. E. Geissler (Berlin,
Germany).

The paper summarizes results of experiments
on the induction of Escherichia coli K12 (lamb-
da) by halogenated pyrimidines. An induction
results, if the lysogenic bacteria are incubated in
SS-medium (containing Bacto vitamin-free
casamino acids) supplemented with 0.5 ug/ml
of either 5-fluorouracil (FU) or 5-fiuorodeoxyu-
ridine (FUdR).

The inducing activity of FUdR is greatly
enhanced by 200 ug/ml 5-bromouracil (BU) —
which itself does not induce — even if an ap-
parently sublethal concentration of FUdR
(0.05 ug/ml) is used. BU is ineffective in combina-
tion with a further reduced concentration of
FUdR (0.005 ug/ml), or if its own concentration
is lowered (20 ug/ml).

The inducing activity of FU seems to be non-
specific with regard to the later stages of nu-
cleic acid synthesis: The effect can be reversed
by 200 ug/ml BU, 0.5 ug/ml uracil, 25 ug/ml
deoxycytidine (CdR) or 200 ug/ml thymidine.
These substances revert the bacteriocidic ac-
tion of FU too, but a nearly normal multiplica-

26

Section 3 — Molecular and Microbial Genetics

tion-rate was achieved only by addition of CdR.
In modified SS-medium (containing normal
Bacto casamino acids instead of the vitamin-
free preparation) FU does not induce but still
shows its bacteriocidic activity. Investigations
with FU in SS-medium supplemented with
vitamins are in progress. Preliminary results
show that several vitamins are apparently able
to reduce or to abolish the inducing action of
FU without interfering with its bacteriocidic
activity.

These results suggest that the halogenated
pyrimidines used exert their inducing action
by a rather unspecific disturbance of (nucleic
acid) metabolism of the lysogenic cells so
treated. There are no reasons for assuming a
direct action of these agents on the deoxyri-
bonucleic acid of the prophages or on DNA
synthesis. Other experiments on the inducing
effect of ultraviolet- and X-rays on BU-labelled
lysogenic cells indicate that ultraviolet rays too
do not induce by a direct action on DNA.

this prophage, photoreversal of induction is
less for a given dose of u.v. than in wild-type
lambda. But if doses giving the same surviving
fraction of lysogenic bacteria are compared,
the u.v. -sensitive prophage is at least as suscep-
tible to photorepair as wild-type lambda.
Acridines and methyl purines do not enhance
u.v. induction of u.v. -sensitive lambda. These
results can be attributed to the inability of the
u.v. -sensitive phage to undergo dark repair of
u.v. damage to the nucleic acid region that
controls production, or acceptance, of repressor.
When lysogenic bacteria are forced to incor-
porate 5-bromouracil, their prophages become
more sensitive to u.v. induction. Acridines and
methyl purines enhance u.v. induction in such
bacteria, but there is no photoreversal of induc-
tion. Additional data indicate that the photo- and
dark repair systems act on the same u.v. dama-
ged sites in nucleic acid, but act independently.

The paper will be published in Acta Biologica
et Medica Germanica (Berlin).

3.27. Genetic and Environmental Factors affecting
Prophage Induction. Margaret Lieb (Cali-
fornia, U.S.A.).

A prophage has one or more genes whose
product(s) apparently repress the function of
other genes involved in phage replication.
Although the repressor may be a protein, DNA
and/or RNA are probably the sites affected by
many treatments that induce phage develop-
ment in lysogenic bacteria. In some lysogenic
bacteria, the phage system(s) blocked by the
hypothetical repressors are extremely sensitive
to induction (derepression) by u.v. light. Methyl
purines and acridines, which enhance mutation
in u.v. -irradiated bacteria, also enhance induc-
tion of lambda prophage replication. These
substances combine with DNA and presumably
distort the molecules in previously denatured
regions. In this way, they either prevent reversal
of u.v. damage by a dark repair system in the
bacterium, or otherwise increase the probability
that an absorbed u.v. quantum will result in
mutation or induction. These substances do not
enhance prophage induction by agents other
than u.v. and have little effect on killing of non-
lysogenic bacteria.

We have also studied a lambda prophage that
is unusually sensitive to induction by u.v. For

3 78. Transfer of Information determining Lysogeni-
zation in Bacteria by Transforming DNA.
B. Gyorffy (Budapest, Hungary).

Immunity to subsequent superinfection with
closely related phages to that carried as prophage
is a general property of lysogenic bacteria. This
immunity could be transferred by the DNA
isolated from the lysogenic strain 41/63-3/ of
Rhizobium meliloti into the sensitive strain 41,
on which a brief preliminary report was already
published/1) Further experiments were per-
formed, involving bacterial DNA and phage
DNA labelled with 32P and/or 14C, and transfor-
mations with DNA isolated from temperate
phages, respectively, to gain more insight into
the process of transfer of the capacity to estab-
lish the lysogenic complex in the recipient bac-
teria after transformation. The results obtained
presenti reliable evidence to separate the
transforming efficiency of the bacterial DNA
and prophage DNA, respectively, involved in the
transformation of immunity to superinfection
by the DNA isolated from the lysogenic strain/2)

Studies on this line also are in progress with
Bacillus subtilis, where DNA from the lysogenic
strain 3NT is used to transform the recipient
strain 168, and the results on transformation

1. K. Szende, T. Sik, F. Ordogh and B. Gyorf-
fy, Biochim. Biophys. Acta 47, 215-217, 1961.

2. T. Sik, B. Gyorffy, K. Szende and M. Cza-
ko, to be published in Acta Microbiol. Hung.,
1963.

27

Section 3 — Molecular and Microbial Genetics

of the information determining lysogenization
will be presented. (3)

The possible interpretation of the process of
transformation of lysogenization and immunity
in line with current ideas of the specific repressor
control mechanism will be discussed.

3. B. Gyorffy, K. Hatnay and I. Kallay, to
be published in Acta Microbiol. Hung., 1963.

3.29. Bacteriophage Transformation. G. Veldhuisen,

R. A. OOSTERBAAN, P. H. POUWELS, H. S.

Jansz and J. A. Cohen (Rijswijk (Z.H.),
The Netherlands).

Transformation of bacteriophage T4 with
purified DNA has been demonstrated earlier/1)
Spheroplasts of Escherichia coli B were mixed
with a purified DNA preparation obtained from
T4rII+ phages and infected with urea-disrupted
particles of a T4rII-mutant. By plating on E. coli
K 112-12 (A h) # 3 and on E. coli B the yield of
transformants and total phage yield were deter-
mined respectively; the rll+/rll ratio (transfor-
mation frequency) varied between 10_6to 10 T for
native DNA using a concentration of approx.
lOug/ml.

We wish to report some further results:

(1) More reproducible results were obtained
using spheroplasts prepared from cells that
were grown to exhaustion in minimal medium
plus 0.04 per cent glucose.

(2) Heating of the DNA preparation (5 min
at 100° C) in 0.01 m phosphate buffer pH7
increases the transformation frequency and
also the total number of transformants by
about 5-10 times.

(3) The increase in biological activity after
heating follows the increase in optical density
at 260 mu measured after rapid cooling of
samples that had been heated at various
temperatures.

(4) DNA molecules fragmented mechanically
to a weight average molecular weight of
about 275,000 were still biologically active.
Degradation by means of limited amounts
of DNA-ase gave similar results.

(5) The transformation of T4rII mutant is
equally effective with DNA prepared from
T2, T4 and T6. Among the transformants,
obtained using T4rII+-DNA and disrupted
T6rII, some are able to grow on B/6 suggest-
ing that other T4 markers may be inherited.

3.30. Electronmicroscopic Studies of Cells and DNA
Molecules during the Genetic Transformation
of Bacteria. Alexander Tomasz and Wal-
ther Stoeckenius (New York, U.S.A.).

Competence: The ability of certain bacterial
cells to absorb highly polymerized biologically
active DNA molecules in the process of genetic
transformation must necessarily involve the
adsorption of the molecules to the cell surface
and their eventual penetration through the
bacterial surface structures. Attempts are being
made to study this process in the electron
microscope. Conditions will be described under
which the method of Kleinschmidt(1) can be
adapted to visualize DNA strands in association
with competent bacteria.

The particular question asked is whether there
is any localization on the bacterial surface for
the adsorption and entry of DNA molecules.
Thin sections of pneumococcal cells^ revealed
the existence in these cells of unusual membrane-
like structures which were often attached to the
developing septum of dividing cells. In view of
the suggested correlation between the division
state of the cells and their competence13' this
observation directed our attention to the septum
of dividing cells as possible sites for the entry of
DNA molecules into the cell.

1. Cf. Veldhuisen et al., Biochim. Biophvs.
Acta 61, 630, 1962.

1. Z.f. Naturf. 16b, 730, 1961.

2. Publication under preparation — Drs. J. Ja-
mieson and E. Ottolenghi.

3. Hotchkiss, Proc. Nat. Acad. Sci. U.S. 40,
49, 1959.

3.31. Some Factors affecting the Frequency of
Streptomycin Resistant Transformants through-
out the Evolution of Competency. M. Ko

houtova, H. Kopecka, and J. Konicek.
(Prague, Czechoslovakia).

Two different transformation media for the
transformation of the streptomycin sensitive
strain of Pneumococcus (PMS1IB) to the strepto-
mycin resistant one were used. (1) Neopepton-
heart infusion broth enriched with some ingre-
dients and (2) the so-called NS-medium (H. E.
Taylor). With both media we obtained similar
results as in the type-transformation experi-
ments previously performed in our laboratory,
concerning the presence of salts with monovalent
cations and their support in a good transfor-
mation yield.

The competent recipient culture was diluted
into the appropriate transformation medium

28

Section 3 — Molecular and Microbial Genetics

with donor DNA. To the medium KC1 salt was
added to the final concentration of 0.075,
0.15, 0.2 and 0.3 m. The number of transform-
ants rose 6, 60, 120 and 20 times respectively
in comparison to the control without the added
salt. The optimal concentrations of 0.15 or
0.2 m KC1 were later used in the transformation
reactions. It was found that the salt showed its
effect only in the time of evolution of competency
when the rising tendency of competency in the
recipient culture was noticed and not during the
falling one.

When small inocula of the recipient culture
were used and sometimes two waves of compe-
tency were obtained, the presence of KC1 salt
was still more effective in the rising period of
the second wave of competency. While in the
first rising period the difference between the
control without the added salt and the medium
with the salt was 2 to 5 times higher, the yield of
streptomycin resistant transformants in the
second rising period was even 10 to 15 times
higher.

This effect of salts with monovalent cations
is not to be confused with the effect of divalent
cations (Fox and Hotchkiss).

If we used in the transformation medium
different bovine serum albumin preparations
(BSA) in which various amounts of residual con-
tents of deoxyribonuclease could be determined,
we also found different answers to the presence
of KC1 salt added to the medium. Higher
amounts of DNAse in BSA (or in the cultiva-
tion medium) require the presence of KC1 salt
for a good yield of streptomycin resistance
transformants (1-10 per cent) while with a very
low content of deoxyribonuclease the presence
of KC1 salt is inhibitory.

Series of our results may indicate that the
presence of small amounts of DNAse in the
transformation medium may play its active
role in the transformation reaction when properly
counterbalanced.

3.32. Genetic Transformation Among Living Pneu-
mococci in the Mouse. Elena Ottolenghi and
Colin M. Macleod (New York, U.S.A.).

Experiments were performed to determine
whether deoxyribonucleate-mediated transfor-
mation could take place spontaneously among
genetically distinguishable pneumococci growing
side by side in the same living host, the mouse.
It was found that when living, unencapsulated,
nonpathogenic, streptomycin-resistant pneumo-
cocci and living, streptomycin-sensitive pneumo-
cocci which synthesized only a small amount

of type III polysaccharide capsule were injected
sequentially into the peritoneal cavity of mice,
recombinants could be recovered from about
30 per cent (26/83) of the mice. The recombinants
were streptomycin-resistant, synthesized type III
polysaccharide in almost the full amount, and
were more pathogenic for mice than either pa-
rental type. The recombination was most likely a
result of deoxyribonucleate-mediated transfor-
mation since it could be completely prevented
by exogenous deoxyribonuclease. It may be,
therefore, that transformation can serve as a
mechanism of recombination in nature for some
microorganisms such as pneumococci.

This data will be submitted for publication in
the Proceedings of the National Academy of
Sciences of the United States in 1963.

3.33. Linkage of Genetic Factors affecting Similar
Function and Derived from Different Bacterial
Species. Arnold W. Ravin and Joscelyn de
Sa (Rochester, U.S.A.).

Certain strains of streptococcus and pneumo-
coccus are known to be capable of transforming
each other by means of their respective deoxy-
ribonucleic acids (DNAs). In pneumococcus,
moreover, various spontaneous mutations that
confer different levels of resistance to strepto-
mycin (str-r mutations) have been shown to be
genetically and physically linked. In DNA-
mediated transformations of pneumococcus,
for example, str-r mutations conferring high
levels of resistance generally replace (i.e. are
allelic with) str-r mutations conferring low
levels of resistance. In order to determine
whether str-r mutations arising spontaneously
in streptococcus and pneumococcus were also
genetically linked, therefore, a streptococcal
mutant resistant to a low streptomycin concen-
tration was exposed to DNA from a pneumococ-
cal mutant capable of resisting a high antibiotic
level. Conversely, pneumococcal mutants capa-
ble of resisting only low concentrations of the
antibiotic were exposed to DNA from a strep-
tococcal mutant capable of resisting high strep-
tomycin concentrations. Transformants pro-
duced in such interspecific reactions were isolated,
measured for level of streptomycin-resistance,
and DNA prepared from them for test on strep-
tomycin-sensitive strains. The latter test was
undertaken to determine whether the genome
of the transformed bacterium continued to
harbor its original str-r mutation and, if so,
whether the original mutation was linked to the

29

Section 3 — Molecular and Microbial Genetics

introduced transforming marker, or whether
the original mutation had been replaced by the
introduced marker. In inter-specific transfor-
mations involving two low-level and one high-
level pneumococcal mutations, one low-level
and one high-level streptococcal mutations,
the results demonstrate that mutations affecting
a similar function (streptomycin-resistance)
but arising in different bacterial species are
genetically linked.

3.34. Biochemical and Genetic Studies of Integra-
tion and Recombination in the B. subtilis
Transformation. Walter F. Bodmer and A. T.
Ganesan (Palo Alto, U.S.A.).

DNA was isolated from a multiple marked
strain of B. subtilis grown on N15 H2 medium
with added H3 thymidine. Competent cells
were prepared in an N14 H1 medium containing

p32

Using the labelled DNA as donor, transforma-
tion was terminated at 30 min with deoxyribonu-
clease. The DNA purified from the donor-
recipient complex was fractionated pycnogra-
phically. Donor and recipient DNA differed
in buoyant density and were traceable by differ-
ential counting of P32 and H3. Donor atoms were
found in density strata corresponding to native
donor but also in native recipient, hybrid, and
"denatured" donor DNA. These DNA fractions
were assayed for their genotypic content by
transformation. Donor and recombinant geno-
types were found in all of these strata but the
last, which is biologically inactive.

The association of donor atoms (i.e. H3) with
a recipient stratum persisted on refractionation
of the DNA, and was also found when transfor-
mation was terminated after only 10 min. The
number of donor atoms found in the other strata
decreases when the cells were allowed to grow
after transformation. However, the H3 count
in the recipient DNA stratum remains approxi-
mately constant. The continuous distribution of
H33 label over strata of varying densities implies
the integration of donor DNA segments, which
were considerably smaller than the average size
molecule present in our preparations of trans-
forming DNA.

3. 35. DNA - mediated Transformations to Improved
Enzymatic Activities. Stephen Zamenhof and
Lilo H. Heldenmuth (New York, U.S.A.).

A mutation resulting in an improvement in

enzymatic activity (and, sometimes, in survival
or in adaptive value) over and above that of a
wild strain, may be caused by a change in regu-
latory mechanisms or an "improvement" in the
structural gene determining the enzyme. At
least the latter should be a change in DNA
and might be transferable by DNA-mediated
transformation.

In the present study several spontaneous
mutants of Bacillus subtilis were isolated having
various enzymatic activities improved over
those of the wild strain. The DNA of some of
these mutants was able to transform the wild
strains in a single step to strains with improved
enzymatic activities. The transformable im-
provements studied to date were: (1) In utiliza-
tion of citrate as the only carbon source; (2) In
production of paraminobenzoic acid (PABA);
(3) In production of phenylalanine. Other im-
provements will also be discussed. Sulfanilamide-
resistant strains of B. subtilis, first isolated by
S. Greer in this laboratory, did not require sul-
fanilamide for growth but were better PABA-
producers than the wild-type; both features are
transformed concomitantly, in both directions.
Some of the (3-thienylalanine-resistant mutants
were better phenylalanine-producers; both fea-
tures are also transformed concomitantly. The
improvements that could not be transferred by a
single step transformation were those which
accumulated in different strains as a result
of more than one mutation, e.g. slime produc-
tion, or utilization of pyruvate as the only car-
bon source.

3.36. Participation of RNA in the Transfer of
Genetic Information of a Virus of the DNA-
type. S. Gershenson (Kiev, U.S.S.R.).

Injection into healthy larvae or pupae of
Bombyx mori of RNA isolated from those in-
fected with nuclear polyhedrosis virus causes a
high frequency of the disease, whereas RNA
from healthy B. mori is non-infectious. The
technique used for isolating the infectious
RNA (extraction with phenol, high speed cen-
trifugation) makes it improbable that the infec-
tivity of the preparations is due to an accidental
admixture of the virus. This possibility is further
disproved by the fact that these preparations
are infectious for larvae of Pier is brassicae wich
are completely immune to the virus itself. The
virus developing in P. brassicae infected with
RNA from virus-diseased B.mori is infectious
for B.mori but not for P. brassicae. Infectivity
of the RNA preparations from virus-diseased
B.mori is destroyed by RN-ase but not by DN-

30

Section 3 — Molecular and Microbial Genetics

ase. Dilution of the preparations leads to an
approximately exponential decrease of infecti-
vity. Two alternative explanations are suggested:
transfer of genetic information of the virus by
means of RNA synthetized by infected host
cells, or formation in these cells of an infectious
RNA-DNA complex. Resistance of the infective
principle to DN-ase and its susceptibility to RN-
ase seem to speak in favour of the first explana-
tion.

3.37. DNA Transformation for Oxytetracycline
Resistance in B. subtilis. M.Polsinelli (Pavia,
Italy).

A strain of B.subtilis resistant to about 300y/ml
of oxytetracycline has been produced through
multistep selection and the DNA prepared from
it used for transforming a sensitive strain.

Transformation was carried out with such
DNA and selection of transformed bacteria
performed on media containing a minimal
inhibiting concentration of oxytetracycline.
The degree of resistance of transformed clones
was individually tested. The population of the
transformed bacteria shows a trimodal distri-
bution of resistance suggesting that at least two
genes are involved in the process.

Transformation by means of DNA taken from
clones belonging to the first and second peak
of the distribution shows that such clones are
transmitting a single genetic factor for oxy-
tetracycline resistance, while DNA from clones
of the third peak transforms sensitive bacteria
in a way similar to that of the original resistant
strain. Further results will also be discussed.

3.38. Multi-site Mutations and Deletions. Minna B.
Rotheim and Arnold W. Ravin (New York,
U.S.A.).

By means of DNA-mediated transformations
in pneumococcus 13 spontaneous mutations to
streptomycin resistance have been mapped at
only three sites, which may be designated A, B
and C. Mutations occurring at she C replace,
but never combine with, those at sites A and B.
For this reason mutations at site C are considered
to be multisite mutations; such mutations have
generally been considered to be deletions. Marker
str-r41 has been mapped at site C. In one
culture of str-r41 a new mutant appeared having
a hundred-fold lower level of resistance. Experi-
mental data indicate that the lower level of
resistance is not due to the action of a suppressor
on a still present str-r41 mutation, but due to

a new mutation. This latter mutation has been
called str-r42, and in transformation reactions
may be replaced by, but never combines with,
marker str-r41. Moreover, by recombining with
site A and B mutations, the str-r42 mutation is
shown to occupy a new site (D). Therefore, mar-
ker str-r42 lies within the region covered by
the mutation str-r41, suggesting that marker
str-41 may partially revert to leave a small
mutated segment which confers a lower level of
streptomycin resistance. Defining deletions oper-
ationally as mutations incapable of back muta-
tion within the same segment of genetic material
that was involved in the original mutational
event, these results establish str-r41 as a multisite
mutation that is not a deletion.

3.39. Transformation of Genetic Traits Associated
with Sporulation in Bacillus subtilis. John
Spizizen, Bernard Reilly, and B. Dahl
(Minneapolis, U.S.A.).

Studies in Bacillus species strongly indicate an
association of a number of enzyme systems
formed in the post-logarithmic growth period
with the sporulation process. Thus the forma-
tion of proteolytic and cell-wall lytic enzymes
appears to be associated with sporulation. A
genetic approach to this problem has been
initiated with the finding of a linkage group con-
taining genes which control the synthesis of a
proteolytic enzyme, a wall-lytic factor or en-
zyme, and sporulation. These studies were
made with transformable Bacillus subtilis,
strain 168. Asporogenous mutants unable to
synthesize protease and the lytic factor could
be transformed with deoxyribonucleic acid
(DNA) isolated from sporogenous strains with
the production of sporogenous organisms con-
taining also genes for protease and lytic factor.

Partially asporogenous mutants have also
been found which contain the genes for protease
and lytic factor. The asporogenous defect in
such mutants appears to have arisen from a
separate locus, so that sporogenous recombinants
could be obtained with crosses of the two as-
porogenous types. Evidence will also be pre-
sented which strongly suggests that certain par-
tially asporogenous mutants are heterogenotic
for some regions of the sporulation genome.

3.40. Genetic Studies on Sporogenesis of Bacillus
subtilis. I. Takahashi (Ottawa, Canada).

By the use of temperate bacteriophage PBS 1,

31

Section 3 — Molecular and Microbial Genetics

it was possible to demonstrate transduction of
sporogenesis in an asporogenous strain (Sp~l)
of Bacillus subtitisM) It was also reported that
the locus sp-1 was linked to prototrophy and to
streptomycin and erythromycin resistance loci
at different frequencies. Examination of sporo-
genous (sp+) recombinants obtained from a
number of other asporogenous (sp~) strains by
transduction with SB19-ery (prot, sp+, str-r and
ery-r) as donor, revealed that three distinct sp
loci were linked to amino acid markers (serine,
phenylalanine and tryptophan-phenylalanine)
at relatively high frequencies. By reciprocal
transduction performed with fourteen sp~
strains, nine genes which govern spore formation
in this organism were recognizable. The finding
that certain sp loci are linked to amino acid
markers or to antibiotic markers and the re-
sults of the reciprocal transduction experiments
suggest that these sp~ characters are under the
control of chromosomal determinants. In addi-
tion, an anomalous recombination pattern was
observed with strain Sp_H12-3. This strain was
never transduced to sp+ even when a wild type
donor was used, although this strain could act
effectively as donor for other sp' strains and was
transducible for prototrophy and erythromycin
resistance. The mechanism responsible for this
anomalous recombination will be discusesd.

18th hour the growth stops and in the next
24 hours neither autolysis nor further growth was
to be observed. From the filtrate of such cultures
it is possible to isolate a factor (probably
polypeptide) which inhibits the growth of the
producing strain. The inhibiting effect can be
antagonized with proteins or with denaturated
proteins.

The mutant C-46 (Am+) and the B. subtilis
wild types grow well on a synthetic medium in
which the sole nitrogen source in (NH4)2S04.
In these cultures autolysis does not take place.

The activity of ALA-dehydrogenase, which —
according to literature — probably plays a role
in the ammonia assimilation has been deter-
mined in the lysozym-lysat of cells.

Inthelysate of (Am-) cells active (100 per cent)
ALA-dehydrogenase, in the lysate of wild type
reduced (10 per cent) ALA-dehydrogenase have
been found. It is not possible to find ALA-
dehydrogenase in the lysozym-lysat of C-46
(Am+) cells.

The results indicate that in the case of B. sub-
tilis the ammonia is not assimilated by means
of ALA-dehydrogenase. Presumably the re-
pression of the process of ammonia assimilation
is simultaneous with the induction of ALA-
dehydrogenase.

1. B.B. Res. Comm. 5, 171, 1961.

3.42. The Initial Events in Transduction of Galactose
Fermentation in Salmonella typhimurium Cell.

J. Hubacek (Prague, Czechoslovakia).

3.41. Inheritable Changes in the Nitrogen Assimila-
tion of Bacillus subtilis. Istvan Turtoczky
and Imre Fedorcsak (Budapest, Hungary).

The growing and the nitrogen assimilation
of B. subtilis C-46 (Am") and C-46 (Am+)
as well as B. subtilis wild types have been
studied in aerated synthetic medium containing
different nitrogen sources.

The mutant C-46 (Am-) is not able to the assim-
ilation of ammonia. It requires glutamic acid
to its growth. In a medium in which the sole
nitrogen source is glutamic acid, autolyis takes
place regularly in the 18th hour. In the filtrate
of autolysat it has not been possible to find the
presence of infective phage particles. The auto-
lysis does not take place if (NHi)2S04 or
NaHCC»3 is also present in the medium besides
glutamic acid; or if 20-30 vol. per cent CO2 is
mixed to the air used for aeration. The autolysis
does not occur if the nitrogen source in the
medium is only glutamic acid but if the Mg++
concentration of the medium is reduced from
1.0 mMmg/l.toO.l mMmg/liter. In this case in the

The frequency of the transduced cells is reduced
by the transfer of the Salmonella typhimurium
recipient cells in the early period after the adsorp-
tion of the transducing phage from a complete
to a minimal medium and subsequent cultivation
for 90 min at 37°C. The potentially transduced
cells are most sensitive to this treatment at the
onset of transduction process (3 min after the
adsorption of the phage) and then after cultiva-
tion in compit-te medium the sensitivity gradually
decreases. After 90 min cultivation in complete
medium the frequency of transduction is practi-
cally not influenced by the transfer of the cells to
minimal medium.

In order to time the period of the incorporation
of the transducing element its elimination in the
non-integrated form has been followed using
acriflavine. It was shown that the incorporation
requires the multiplication of the cells and takes
place during the period from the onset of growth
up to 110-120 min of cultivation (2-3 divisions).
The sensitive phase of transduced cells to the
minimal medium precedes this incorporation
event. We assume, therefore, that the reduced

32

Section 3 — Molecular and Microbial Genetics

frequency of the transduced cells in the minimal
medium is due to the recovery of the cell from
the transducing element when it is in the non-
integrated form.

Further experiments have shown that the
reduced frequency of transduction can be achiev-
ed not only by transferring the cells from com-
plete to minimal medium, but also by chilling the
cells in complete medium from 37 C to 0°C.
When the cells were treated in such a way, 10~N20
per cent of the amino acid pool was lost while
some amino acids were removed quantitatively.
Biochemical analysis of this fact leads to eluci-
dation of the importance of amino acid pool in
the genetic expression of a new marker.

3.43. The Biochemistry of Dissociation of Bacillus
brevis GB. V. N. Stoletov, S. V. Shestacov,
V. M. Glazer, V. D. Filippov (Moscow,
U.S.S.R.).

The comparative biochemical examination of
the dissociating forms of Bacillus brevis GB has
been carried out. The chemical constitution of
cells, the content of some significant metabolites,
some enzymic activities, and the occurrence of
some metabolic processes have been investigated
in rough R-form, smooth S-form, and in two
variants of mat flat M-form of B. brevis. R-form
and variants of M-form are known to synthesize
the gramicidin C in contrast with S-form and
second M-form variant.

All studied variations of B. brevis have the
same nucleotide composition and DNA content.
Gramicidin-producing variants have been shown
to content the higher amounts of RNA. Flat
forms differ from R- and S-forms in their free
amino-acid and polypeptide content and compo-
sition, and the content of free nucleotides and
some phosphorus-containing fractions.

The role of cell membrane in metabolic changes
in the time of dissociating transition are dis-
cussed with respect to the study of respiratory
characteristics of intact cells and protoplasts of
various dissociating forms of B. brevis. Specific
differences in lipide and polysacharide compo-
sition have been found for three forms of disso-
ciation.

The activity of some amino-acid metabolism
enzymes of dissociating forms has been investi-
gated in respect to their capability to enzymic
induction, repression, and feed-back inhibition.

Obtained results are interpreted as being due
to existence of correlation between certain
biochemical and cultural features in various
forms of B. brevis. The data of biochemical ana-
lysis of dissociants are regarded from the point

of view of detection of regularity in metabolic
changes in the time of bacterial dissociation. The
latter is assumed to be a form of displaying the
special adaptive variability of microorganisms.

3.44. Genetic Analysis of Streptomycin Dependence
in Proteus mirabilis. Helmut Bohme (Gaters-
leben, Kreis Aschersleben, Germany).

By selection in the presence of streptomycin
several different streptomycin dependent (str-d)
mutants can be obtained from the same wild type
strain of P. mirabilis. Most of these str-d mutants
require for growth in minimal medium besides
streptomycin the aminoacids methionine, cys-
teine and arginine. All str-d mutants can be
transduced to nondependence by phages grown
in wild type (str-s) cells. Transduction with pha-
ges propagated in str-r mutants results exclusively
in str-r transductants; str-s, str-r and str-d thus
being allelic. Since in transductional crosses
between nine different str-d mutants no wild type
recombinants were obtained, the str-d mutations
seem to be multi-site mutations. By selection on
streptomycin-free nutrient agar streptomycin
nondependent revertants can be obtained from
str-d mutants. In most of the str-d strains two
types of revertants result: auxotrophic and proto-
trophic revertants. The numerical relation be-
tween these two types as well as the growth
factor (isoleucine and valine, in other cases
arginine) required by the auxotrophic revertants,
are specific for each str-d mutant. The auxotro-
phic revertants arise as result of a mutation in a
suppressor locus; it can be shown by transduction
with phages grown in these revertants that they
still contain the str-d allele. The suppressor could
not be transferred by transduction into str-d
strains. The auxotrophic revertants can be trans-
duced to prototrophy using phages grown in
wildtype bacteria. The resulting prototrophic
transductants further contain the suppressed
str-d allele. Thus in spite of the always simulta-
neous origin of the suppressor and the growth
factor requirement, both can be separated by
transduction.

3.45. A Genetic Basis for Changing Virulence in Two
Plant Pathogenic Fungi. E. W. Buxton (Har-
penden. Great Britain).

Mutants of physiologic races of Fusarium
oxysporum and Verticillium albo-atrum resistant
to acriflavine or actidione were used to show
how heterokaryosis and asexual recombination

33

Section 3 — Molecular and Microbial Genetics

produce biotypes with increased virulence and
wider host range. Heterokaryosis alone, without
mitotic recombination, immediately increases
virulence when two mildly virulent strains ana-
stomose. The nuclear ratio in inoculum of
heterokaryons containing nuclei of labelled
pathogenic and non-pathogenic strains deter-
mines the extent of wilt in the host, the rate the
rhizosphere is colonized and the ability to survive
in soil. Haploid nuclei bearing genes conditioning
virulence are selectively increased in hetero-
karyons during host infection and their rate of
multiplication in the heterokaryon is directly
related to the development of wilt symptoms.
Similarly, material released from host roots into
the rhizosphere selectively increase nuclei con-
ditioning virulence, but a few nuclei from non-
pathogenic strains also enter the host as com-
ponents of a heterokaryon. In soil, a few nuclei
conditioning virulence survive in heterokaryons
largely composed of nuclei from purely sapro-
phytic strains. Heterokaryotic chlamydospores,
the resting bodies of F. oxysporum, have been
isolated from soil.

The control of both the parasitic phase and
survival in soil through adjustments of the nu-
clear ratio in heterokaryons provides the wilt
fungi with a rapidly adapting mechanism that
helps explain some of their variability.

ing, typical wild type colonies characterized by
abundant cleistothecial formation. Ascospores of
this isolate were exposed to ultraviolet light for a
period of time such that 2.5 per cent of the asco-
sporessurvived the treatment. Among the sur-
viving colonies, three strains were isolated which
were capable of producing conidia abundantly
over the entire colony surface at both 25° and
37°C. One of the conidiating mutants was selected
for the isolation of conidial color, auxotrophic
and antimicrobial agent resistant mutant strains.
Since the conidial mutant retained the ability to
form cleistothecia, the markers carried by the
conidial strain have been mapped based on ran-
dom ascospore analyses. Additional conidial
mutants have been isolated from the original
non-conidial parental strain. Each conidial strain
has been marked genetically and intercrossed
with other conidial mutant strains in an attempt
to locate the gene or genes which control conidi-
ation in this strain. Since the conidial strains
differ from one another morphologically, it is
probable that more than one locus is involved or
that there exist several alleles of the same gene.

3.48. Sexual Polarity in Streptomyces. G. Sermonti,

I. Spada-Sermonti and S. Casciano (Rome,
Italy).

3.46. Utilization of a Dikaryotic Organism (Coprinus
radiatus) for the Study of Lethals. Madeleine
Gans (Gif-sur-Yvette, France).

A method is described which enables us to
discover, preserve and study from a genetical
point of view the compensable and non-com-
pensable lethal mutations (according to Atwood)
induced in a dikaryotic mycelium.

Preliminary results show that from about
twenty lethals induced by nitrous acid, two only
are compensable by the growth medium.

3.47. Genetic Control of Conidiation in Aspergillus
rugulosus. R. W. Tuveson and D. O. Coy
(Chicago, U.S.A.).

A group of intersterile strains (R~) has been
found in Streptomyces coelicolor A3(2). They are
fully fertile with all the other strains, which in
turn are fertile among themselves. In summary:

R+ x R+ = fertile

R+xR' = fertile

R" x R~ = sterile
In R+ x R^ crosses, the contribution to the
progeny of markers of the R+ strain is, as a rule,
reduced as compared with that of the R" markers,
although in rare cases the reverse is observed.

The transfer (or integration) of the R+ markers
of the two linkage groups appears to be corre-
lated in the different zygotes. When defects are
observed on the R"" side, the deficiencies in the
two linkage groups also appear to be correlated.

A strain of Aspergillus rugulosus isolated from
sand in the Indiana Dunes State Park produces
abundant cleistothecia when grown on complex
and minimal medium at 37°C. Under these con-
ditions, conidial production is limited. When this
isolate is incubated at 25°C for extended periods
(14 days or more), conidia are produced abun-
dantly at the colony periphery. These conidia
when transferred to complex or minimal medium
and incubated at 37°C give rise to non-conidiat-

3.49. Directed Mutation of the Mating Type Alleles
as an Explanation of Homothallism in Yeast.
Donald C. Hawthorne (Seattle, U.S.A.).

Hybrids of homothallic Saccharomyces cheva-
lieri and heterothallic Saccharomyces cerevisiae
yield asci in which two of the spores give rise to
heterothallic cultures, i.e. they remain haploid
and show a mating reaction when mixed with

34

Section 3 — Molecular and Microbial Genetics

test strains. The other two spores form homo-
thallic cultures which readily diploidize and then
are capable of sporulation. The 2: 2 ratios in the
asci reflect the segregation of a single gene, D,
for diploidization or homothallism. Gene D
segregates independently of the mating type locus.
Although gene D is seemingly epistatic to the
mating type alleles, aandx, it in reality acts by
causing their mutation. Under the influence of
gene D, either mating type gene mutates to the
other allele at rates which range from one mu-
tation per two cell divisions to one per thirty cell
divisions. The haploid cells fuse as soon as the
mutant allele is expressed and the zygotes give
rise to stable diploids which are heterozygous for
mating type. The heterozygous condition of the
mating type alleles blocks any further action of
the gene D.

3.50. Establishment of Respiratory Capacity during
Zygote Formation between Complementing
Respiratory Deficient Mutants of Yeast.

H.Jakob and A. SELS(Gif-sur-Yvette, France).

Two classes of respiratory deficient mutants are
known: genie mutants called segregational
"petites" and cytoplasmic mutants called vege-
tative "petites", both lacking several respiratory
enzymes especially cytochrome-oxidase. The
cross of two haploid complementing strains (ex.
g. cytoplasmic petite genie one, or between two
non allelic genie mutants) gives rise to diploid
cells able to synthesize the complete respiratory
system.

We have studied the kinetics of the establish-
ment of respiratory capacity in such crosses in-
volving p5, p7 and p~ neutral mutants, during
the zygote formation, and immediately after it.
Every cross shows characteristic kinetics which
can be followed by the overall respiratory rate
and/or by cytochrome-oxidase activity of the
extracts. In certain crosses a greatly enhanced
respiratory activity is brought about immediately
after the zygote formation.

These experiments will lead to more precise
knowledge about the interactions between the
different genes or between these latter and the
cytoplasmic factor.

3.51 . The Tetrade Analysis of Some Bakers' Yeasts.

L. Sedlarova (Bratislava, Czechoslavakia.

In some hybrids and production strains of the
bakers' yeasts a genetic analysis has been made
of cell and giant colony shape, fermentation

characters and the production of biomase. The
results of this analysis will be discussed, together
with the process of spore copulation.

3.52. Directed Hereditary Changes of Fermentative
Properties of Yeast and Indirect Selection. K. V.

Kossikov and O. G. Raievskaia (Moscow,
U.S.S.R.).

Mutational change of fermentative properties
of yeast Saccluiromyces globosus induced by
specific substrate (sucrose) can be markedly
enhanced by changing the cultivation conditions.
The greatest effect is obtained by increasing the
sucrose concentration in the media (up to a
certain limit).

The joint results show that the hereditary
changes of fermentative properties in yeast (in
the present case the appearance of their ability
to ferment sucrose) are associated with their
metabolic activity; this fact does not allow to
connect these results with the selection of spon-
taneous mutations.

In order to confirm the absence of spontaneous
mutations of the character that is being studied
in the above-mentioned experiments on directed
changes, tests with indirect selection are being
carried out.

The results obtained show the ineffectiveness in
the present case of indirect selection. The results
will be discussed with due regard of the additional
experimental data.

3.53. Regulatory Mutations Concerning Threonine
and Methionine Biosynthetic Enzymes in Sac-
charomyces cerevisiae. H. de Robichon-
Szulmajster and W. Sly (Gif-sur-Yvette,
France).

In S. cerevisiae, methionine and threonine
share a common synthetic pathway which bran-
ches from homoserine. The common steps in-
clude:

(A) (B)

Aspartate v-^ Aspartyl-phosphate ^ Aspartic

(C)
semi-aldehyde «-» Homoserine

In addition to the control they can exert on
enzymes of their own branch (after homoserine),
each end product control its individual rate of
synthesis, by repression and feedback inhibition
of different enzymes in the common pathway.
Thus, enzyme A is inhibited and repressed by
threonine(l) and enzyme C is inhibited and re-
pressed by methionine (2). Structural genes for
these two enzymes are unliked(3).

An attempt has been made to find regulation

35

Section 3 — Molecular and Microbial Genetics

mutants, using resistance to amino acid analo-
gues for selection. Haploid mutants, resistant to
the methionine analogue, ethionine, have been
selected. They fall into three classes, recessive,
semidominants and dominants. Most of them are
methionine (or methionine-precursors) excretors.

In one of the semi-dominant haploids, in
which resistance to ethionine appears to be
digenic, aspartokinase (enzyme A) is no longer
repressed by exogenous threonine, but regulation
of homoserine dehydrogenase (enzyme C) is un-
changed. The inhibitibility of aspartokinase is
not modified in this mutant.

These observations were unexpected since
ethionine is not an analogue of threonine which
is important in the repression of this enzyme.
Since non-repressibility by threonine, which re-
presents an apparent regulation mutation, was
found in one of these mutants, the other members
of the three classes of mutants are being examined
enzymatically and genetically, in hopes of iden-
tifying and mapping the characters which fulfil
the criteria of regulation mutations.

1. De Robichon-Szulmajster and Corrivaux,
Biochem. Biophys. Acta (1963).

2. Karassevitch and de Robichon-Szulmaj-
ster, Biochem. Biophys. Acta (1963).

3. Mortimer and Hawthorne; de Robichon-
Szulmajster, unpublished.

analysed. Different new combinations were
found.

Several other yeasts were used later.

Using diploid yeasts all the transformed cells
were heterozygous for the newly acquired abili-
ties. Heterothallic haploid yeasts treated with
DNA derived from homothallic diploid yeasts
yielded diploid homozygous cells.

A hypothesis is presented which can explain
these facts.

The mendelian laws still hold, the foreign
DNA is localized exclusively in the nucleus. In
our opinion the different hereditary characters,
dominant or recessive, are transferred independ-
ently from each other.

Attempts to fractionate the DNA preparations
by means of an Ecteola column so that only one
or a few genes were present in each fraction were
unsuccessful.

Very confusing was a phenomenon which is
called pseudo-transformation, a temporary
change of characters, which decreases after sub-
sequent transferring into the same broth.

Our previous hypothesis was that the pseudo-
transformation was induced by the RNA still
present in the DNA preparations. Harris showed
that also enzymes still were present. This RNA
or the enzymes are adsorbed at the cell walls.
Thorough washing, however, did not always
remove the pseudo-transformation. It seems that
sometimes the substances have reached inside the
cells.

3.54. Transformation in Yeast. W. F. F. Oppenoorth
(Delft, The Netherlands).

DNA has been extracted from bacteria with-
out loss of its genetic characters. The transfor-
mation of yeast also was possible but to a much
less extent. When an experiment was successful
only few transformed cells were present.

The transfer of fermenting abilities was in-
vestigated. A yeast, a hybrid obtained by Winge
(303-9), not capable of fermenting disaccharides
was treated with DNA extracted from Sacch.
chevalieri, able to ferment sucrose and raffinose
1/3. The DNA was added into malt extract and
inocculated with the acceptor yeast. After some
days the yeast was full grown and transferred
into different sugar Durham tubes. After several
transfers cells were isolated with the micro-
manipulator and the fermenting abilities were

3.55. On the Possibility of Carrying Out the Func-
tional Test for A I lei ism in the Ascomycete Asco-
bolus immersus. Jean Mousseau (Gif-sur-
Yvette et, France).

In some crosses between independent mutants
with colourless spores there appear asci con-
taining 6 mononuclear spores and 1 binuclear
spore. Genetic analysis of such asci shows that
in some of them the binuclear spore is hetero-
caryotic possessing a nucleus from each parental
type. These heterocaryotic spores are of wild
type phenotype (brown red colour). This shows
that complementation takes place between mu-
tations of the parental strains and that the mutant
allele is recessive with regard to the wild type
allele. These double spores therefore make it
possible to carry out a functional test for allelism.

36

SECTION 4

GENE ACTION

4.1. Studies on the Function of Histones and Similar
Basic Proteins. I. Leslie (Belfast, Great Britain.

Chemical similarities have recently been de-
tected between the histones associated with
DNA and the basic proteins associated with
RNA in ribosomes. However, the function of
this class of protein is not yet known. From their
chemical and physico-chemical investigations,
Stedman and his collaborators have proposed
that histones act as gene-regulators.

Latent-nuclease activity in ribosomes is known
to be associated with basic proteins or proteins
having compositional similarities to histones.
The hypothesis that "histone-nucleases" control
gene-expression in metazoan cells (Leslie, 1961)
is being investigated with the aid of biochemical
and tissue culture techniques.

Fractionation on carboxymethylcellulose and
electrophoresis on starch-gel has been applied to
the basic proteins of nuclei and ribosomes. With
two procedures for detecting nuclease activity, it
has been found that ribosomal RNA is most
readily degraded by proteins of the lysine-rich
type. Degradation by the arginine-rich protein is
negligible.

Pulse-labelling and chase experiments with a
human cell strain (HLM) are being used to
follow the synthesis of the different types of
RNA and basic proteins, when the cultures are
treated with either fluorodeoxyuridine, which
selectively inhibits DNA formation, and actino-
mycin C, which partially inhibits RNA forma-
tion. The results will be described.

phage and superinfecting phage respectively. The
prophage is not induced, the few prophage
markers found in the progeny are rescued by
recombination. The same appears to be true
when a "virulent" A is used as the superinfecting
phage. In superinfection of cells lysogenic for A
with another A and 434 hy, the superinfecting A
is not multiplied: the recovery of the corres-
ponding type in the progeny is below the multi-
plicity of infection. These particles derive their
protein coat from the unrepressed 434 functions.
These results suggest either that the control of
replication does not entirely depend on the re-
pression of synthesis of "'early proteins", or that
the functions involved in replication are not
interchangeable between A and 434 hy. If this is
the case, the genetic loci for these functions
should map in a small region which includes
cistron ci (Kaiser and Jacob, 1957) but does not
extend as far to the left as the site of the defective
mutation d22 (since healthy A can be rescued
from A d-22 by recombination with 434 hy).

1. Kaiser, A. D. and Jacob, F. Virology, 4,

509, 1957.

This research has been supported by Euratom-
U.L.B.-contract 016-61-10 ABIB, by the Air
Force Office of Scientific Research, OAR
62-17 (through the European Office Aerospace
Research, United States Air Force) and by
the Belgian Fonds National de la Recherche
Scientifique.

4.2. Data on the Control of Genetic Replication in
Temperate Bacteriophages. R. Thomas (Brus-
sels, Belgium).

When a lysogenic bacterium is superinfected
with a temperate bacteriophage which is closely
related to the prophage but has a different pat-
tern of immunity, the superinfecting phage
multiplies normally. The question can be asked
whether functions supplied by the superinfecting
phage will induce the prophage.

Suitably marked derivatives of A and 434 hy
(Kaiser and Jacob, 1957) (1) were used as pro-

4.3. Regulatory Function of Sorbose on Sugar Meta-
bolism in Neurospora. Walter Klingmuller
(Berlin, Germany).

Growth of Neurospora on filter sterilized su-
crose medium is blocked by sorbose. (1) By
treatment with nitrous acid mutants have been
produced that are not blocked. These mutants
also grow on fructose/sorbose and on sorbose
alone, the wild type does not. Growth on those
media is in dense, small colonies, the growth rate
somewhat retarded as compared to other media.

Growth on sorbose alone may be due to im-
purities in this sugar. Growth on fructose/sorbose

37

Section 4 — Gene Action

and on sucrose/sorbose point to a regulatory
mechanism related to the (3-galactosidase system
in E. coli. The mutants are supposed to be con-
stitutive for a sucrose splitting enzyme, which in
the wild type can be repressed by sorbose.

A genetic factor for colonial growth has been
crossed into the mutant strains, to check for
correlations between the repressor function of
sorbose and its ability to induce colonial growth.

1. De Serres, Kolmark and Brockman, Nature
193, 556, 1962.

4.4.Genetic Studies on ur Mutants of Coprinusradia-
tus (Fr. ex. Bolt). Georges Prevost (Gif-sur-

Yvette, France).

The species studied is a basidiomycete having
dikaryotic phase. This work has been under-
taken in order to understand physiological me-
chanisms underlging the regulatory systems which
occur in dikaryotic organisms.

Four groups of alleles controlling the bio-
synthesis of uracil have been found: ur 1. (Chro-
mosome VI), ur 4. (Chr. IV), ur 7. (Chr. VII) and
ur 13. (Chr. III). On the other hand a gene r,
linked to ur 7 (max. 6 UCO) permits the utili-
zation of orotic acid as a source of uracil by
strains ur 1. and ur 4. but not by strains ur 7. and
ur 13. The gene /- is recessive, which means that
the dikaryon (//;■ /. r + ur. 1. r+) cannot utilize
orotic acid. The r gene does not control a per-
mease system but it permits the participation of
orotic acid in the pathway of uracil biosynthesis.
The very high sensitivity of the wild type strains
to the action of 5-fluorouracil (inhibition of
growth by concentration of 10~7 m of 5-FU) has
led to the utilization of this product for selection
of possibly derepressed or/and desinhibited mu-
tants. Monogenic mutants resisting to 5-FU
(10~4m) have been obtained. These mutants can
be classified in several allelic groups and are re-
cessive or semi-recessive. Their relationship to
mutants ur~ as well as the mechanisms of resist-
ance to 5-FU have been analysed.

4.5. Further Studies with Methionine Mutants of
Salmonella typhimurium LT2. D. A. Smith
and J. D. Childs (Birmingham, Great Britain).

The results of growth response, cross-feeding
and transduction experiments with 166 mutants
suggest that at least 6 genes (met A, B, C, E, F
and G) are involved in the synthesis of methionine

via cystathionine and homocysteine. Met A mu-
tants are unable to carry out one, and metB
mutants the other, of two successive steps in the
synthesis of cystathionine, metC mutants cannot
synthesize homocysteine and metE, F and G
mutants possess metabolic blocks between homo-
cysteine and methionine. MetE mutants have an
alternative requirement for vitamin B12. All
metC mutants appear to be heat sensitive.

Colicine mediated genetic transfer results in-
dicate the sequence metB — (metF — metE) — met A
— metC for 5 of the 6 genes. Only metB and metF
are close enough to be transduced by the same
phage particle.

Intragenic abortive and complete transduction
tests between all the mutants of each gene are
being carried out. Mutants of each of the met A,
Fand G genes all fall into single complementation
groups. Those of the metB gene fall into at least
three and those of metF gene into two groups.
The occurrence of suitable met A and F deletion
mutants permit some mapping of sites of mutat-
ion within these genes.

4.6. The Gene-Enzyme-Complex Involved In L-arabi-
nose Metabolism. Ellis Englesberg, Nancy
Lee and Richard Cribbs (Pittsburgh, U.S.A.).

Forty-seven L-arabinose nonutilizing mutants
sites of Escherichia coli, strain B/r, have been or-
dered between the markers threonine (thr) and
leucine (leu) by transduction experiments with
phage Plbt employing three factor crosses. The
mutant sites fall into four different gene loci, as
defined by both genetic and functional criteria
and are arranged in the order thr — D, A, B,
C— leu.

Genes A and B are the structural genes for l-
arabinose isomerase L-ribulokinase, respectively.
Gene D is probably the structural gene for l-
ribulose-5-phosphate-4-emimerase. Gene C is
characterized by mutants which are deficient in
all three enzymes. Recent experiments (Helling,
Weinberg, and Englesberg, unpublished), con-
ducted with K12, using temporary merozygotes,
have shown that C mutants complement both
A and B mutants thereby eliminating the possi-
bility, previously considered, that C is an operator
locus. Mutations in the B gene have a dual effect
causing alterations in the structure of the kinase
and increased or decreased inducible levels of
isomerase and epimerase. The mutant sites
affecting high and low inducible levels of these
two enzymes are distributed in a random fashion.
The amount of inducible L-ribulokinase CRM
formed by some B gene mutants is greater or less
than the amount of L-ribulokinase (as deter-

38

Section 4 — Gene Action

mined antigenically) produced by the parent
strain. These changed inducible levels of isome-
rase, epimerase and kinase CRM vary coordi-
nate^. It is proposed that coding in the B gene
besides determining the structure of L-ribuloki-
nase may set the rate of production of the mes-
senger for kinase, isomerase, and epimerase.

4.7. Complementation of Arabinose Negative Mutants
of Escherichia coli. R. B. Helling (Pittsburgh,
U.S.A.).

L-arabinose non-utilizing mutants of E. coli,
strain B/r, fall into four groups. A mutant in
group A, B, or D lacks one of the first three
enzymes involved in arabinose breakdown, while
gene C mutants lack all three enzymes. ^

In order to study the complementation pattern
of these mutants using sexual merozygotes, the
mutant sites were transduced from E. coli strain
B/r (which mates poorly) to E. coli strain K-12,
which had the arabinose genes of strain B/r.
Induction of arabinose enzymes was measured in
merozygotes obtained from a cross involving
two different arabinose negative mutants, under
conditions where neither parent could produce
the enzyme. Four complementing groups were
found which corresponded to the four known
genes. Each group complemented all other
groups; thus C gene mutants are not similar to
known operator or regulator mutants, and gene
C produces a cytoplasmic product necessary for
the induction of the arabinose enzymes, possibly
a permease.

zyme map within two separate segments of gene
G. (»)

Enzyme production is coordinately repressed
in the presence of L-histidine ^ or 1,2,4-tria-
zole-3-alanine (TRA). Both compounds elicit
pyrophosphate exchange reactions, activities
which are not repressed by L-histidine. (5) TRA
allows synthesis of afunctional proteins in histi-
dine auxotrophs and is recovered from protein
hydrolyzates. (6) Mutations at (a) gene(s) outside
of the histidine region simultaneously confer
TRA-resistance and a relative intensitivity to
repression by L-histidine. (7)

The site of repression is part of, or adjacent to,
gene G. Deletion of this "operator" region elim-
inates production of theenzymes by the histidine
genes. For example, mutant lusG-203 fails to
recombine only with the seven most terminal
sites of the G gene. Yet no histidine enzymes are
produced. Translocation of the remaining seven
structural genes into an episome, or further
deletion of genetic material to the "right" of the
histidine genes, allows return of coordinate gene
function which is, however, now unaffected by
the presence of L-histidine. W

1. See Lee and Englesberg, Proc. Nat. Acad.
Sci., U.S. 48, 1962.

1 . Ames and Hartman, In : The Molecular Basis
of Neoplasia, p. 322, 1962.

2. R. G. Martin, J. Biol. Chem. in press, 1963.

3. D. E. Sheppard and Hartman, Fed. Proc, in
press, 1963.

4. Ames and B. Garry, Proc. Nat. Acad. Sci. 45,
1453, 1959.

5. Ames and B. Garry, unpublished.

6. A. P. Levin and Hartman, Bact. Proc, in
press, 1963.

7. J. R. Roth, Ames and Hartman, unpublished.

8. Ames, Hartman, and F. Jacob, J. Mol. Biol.,
in press, 1963.

4.8. Genetic Control of L-Histidine Biosynthesis in
Salmonella. Philip E. Hartman and Bruce N.
Ames (Bethesda, U.S.A.).

Eight closely linked genes specify the structures
of eight proteins involved in L-histidine biosyn-
thesis. The gene order and the relative biochem-
ical steps are: £(2?), F(A), ,4(3), 7/(5?), 5(6 and
8), C(7), D(9), and G(l). A coding ratio of three
has been calculated for the complete genetic
region, about 1.3 x 104 nucleotide pairs long. W

L-histidine and thiazole alanine feedback in-
hibit reaction 1 by altering the conformation of
the protein. (2) The sites of mutation in histidine-
excreting mutants with feedback-resistant en-

4.9. Restoration of Operon Activity by Suppressors.

J. R. Beckwith (London, Great Britain).

The genes clustered in the Lac region of the
E. coli chromosome are under the control of a
single operator region adjacent to the gene for
(3-galactosidase. 0° mutants, located in this
region, prevent the synthesis of all three enzy-
matic activities associated with the operon, |3-
galactosidase (z), permease (y), and galactoside
transacetylase. It has been proposed that these
mutations prevent the transcription of messenger-
RNA from the operon. In this paper the iso-
lation and characterization of suppressors of an
0° mutant is reported. By the use of melibiose,
an cc-galactoside which requires permease but

39

Section 4— Gene Action

not (3-galactosidase for its metabolism, it was
possible to select for suppressed mutants of the
type z~y+ in addition to z+y+ revertants selected
on lactose. The chromosomal location of the
suppressors for many of these strains has been
determined. They fall into four classes: (1) intra-
operon suppressors, and extra-operon suppres-
sors which map near (2) the methionine, (3) the
serine and (4) the tryptophan markers on the E.
coli chromosome. None of the extra-operon
suppressors involves translocations of the defect-
ive operon to another part of the chromosome.
These results are difficult to interpret by the
hypothesis that the original 0 mutant is blocked
at the level of gene transcription. They are better
explained by a model in which the mutation has
resulted in an inhibition of translation of mes-
senger-RNA into protein. Dominance tests with
these suppressors provide further indication of
their character.

4.10. On the Possibility of a Specific Repression at
the Genetic Level. M. Luzzati, L. Clavilier
and G. Pere (Gif-sur-Yvette, France).

At present, no case of specific repression of a
genetic event is known. Such repression as has
been described — i.e. the repression of the form-
ation of a set of biosynthetic enzymes by the
final product of the pathway — has always been
concerned with the phenotype of the cell.

We have discovered a phenomenon (1) which
concerns a specific inhibition at a genetic level.
We have studied gene conversion of the adl
locus in S. cerevisiae. The ad~h mutation leads to
a simultaneous requirement for adenine and
histidine.

In a diploid heteroallelic for ad3 the frequency
of prototrophic revertants due to gene conversion
is of the order of 10_4/day/cell. L-histidine(but
not adenine) prevents some of 95 per cent of the
gene conversion. The conversion of four diffe-
rent heteroallelic combinations within the same
locus are also inhibited by L-histidine to the same
extent. However, L-histidine has no effect on the
gene conversion of another locus th2.

Various reconstitution experiments have per-
mitted us to exclude explanations based on a
selection mechanism.

Inhibition is not due to an effect of histidine on
the expression of ad3+ gene in a ad3~ cytoplasm,
for histidine has no effect on the back mutation
of either a haploid or a homoallelic diploid for
the same locus.

If the ad3 gene is a type of operator mutant,
one could imagine that the cytoplasmic repressor
formed in the presence of histidine might, by

reacting at the gene level, inhibit gene conversion,
which is known to be a mitotic nonreciprocal
recombination.

1. L. Clavilier, M. Luzzati, P. P. Slonimski,
Compt. Rend. Soc. Biol. 154, 1970, 1960.

4.11. Genetic Control of 5-Fluorouracil Resistance in
Saccharomyces cerevisiae. Francois Lacroute
(Gif-sur-Yvette, France).

Growth of the wild type is inhibited by a
10~6m concentration of 5 FU. Many resistant
mutants have been obtained with thresholds
varying from 3 X 10~5m to more than 10_3m.

Sixteen independent mutants have been
analysed. They show single gene segregation;
they belong to four different and unlinked loci
and are dominant or semi-dominant.

Allelic relationships of these 5 FU resistance
genes with the four loci for uracil dependence
already known in S. cerevisiae and the mecha-
nisms of resistance will be discussed.

The release in the cultures of resistant mutants
of a substance promoting the growth of uracil
requiring mutants suggests that feed-back in-
hibition or repression has been relieved in these
resistant strains.

4.12. Gene Control of Differential Cell Function.

William K. Baker (Chicago, U.S.A.).

The pattern of pigmentation in variegated eyes
of Drosophila produced by position effect forms
a useful system for studying the ontogenetic
differentiation of cellular function. A comparison
of the size and distribution of twin sectors
produced in the eyes by induced somatic crossing
over in D. virilis and D. melanogaster with the
size and distribution of pigment patterns caused
by position effect indicates that the latter patterns
also have a cell-lineage basis.

From the size of certain pigmentation sectors
in the variegated eyes, it is concluded that pig-
ment potentialities of a given sector of the eye
can be determined in early larval development,
many days before pigment formation commences
in the eye. Evidence is marshalled that this early
determination — in spite of the clonal sectors it
makes — is not based on any type of somatic
mutation in the strict sense. Rather it is a regu-
lation of gene action whose level is rather stably
inherited within the somatic cells that will
comprise one of the sectors of the compound eye.

40

Section 4 — Gene action

The validity of these concepts will be illustrated
by studies on position effects causing white varie-
gation in D. melanogaster and peach variegation
in D. virilis.

4.13. Non-specific Genetic Control of Variegation in
Drosophila melanogaster. Janice B. Spofford
(Chicago, U.S.A.).

The Su- V locus (IIIL, 41.4) has already been
described; one allele enhances, the other sup-
presses variegation of white, facet and diminutive
induced by Dp (1 : 3) wm 264.58a, a short inser-
tion from the X into the proximal heterochro-
matin of IIIL. Neither allele at the locus is do-
minant. The extent of variegation depends on both
the individual's own Su- V genotype and his
mother's. This reports a study of the effects of
the Su-V locus on position-effect variegation due
to other rearrangements, all X chromosome
inversions: scs (scute-8), v3p (yellow-3 of Patter-
son), wm4 (white-mottled-4), and rst3 (roughest-3).
For scs, the number of scutellar bristles was
counted; for v3p, the amount of black pigment
in the core of the bristle was arbitrarily scored,
the scutella being mounted in euparol and ex-
amined immediately at 120x; for vvm4, the total
drosopterin per head was measured by densito-
meter after paper chromatography; and for rst3,
the proportion of eye which was rough was
estimated. In all cases the results agree with those
already described above for white mottling due
to the duplication. Possible modes of action of
the suppressor locus will be discussed.

The cell phenotype is not yet fixed at the time
of the replication. The expression is irreversible,
however, after the completion of cell different-
iation. The alternative functions of the im allele
can be induced or repressed by various physical
and chemical agents (temperature, irradiation,
metabolites of the cysteine pathway, etc.). The
transmission of this gene through the "germ
line" is clearly Mendelian and linkage has been
established. Spontaneous mosaicism in hetero-
zygotes was not observed. Seeds harvested from
white or green fruits are genetically identical and
repeat the mosaicism. Apparently the im alter-
ation is not due to mutation in a ,, structural"
gene but rather to a stable change of a "regu-
latory" element. A detailed report will be pub-
lished in Genetics.

Supported by National Science Foundation.

4.15. The Mode of Transmission of Exogenotic
Fragment in Bacterial Crosses. E. Calef and
L. Fischer-Fantuzzi (Naples, Italy).

Structural organization in heterogenote of K12
can be explored by interrupted mating.

The analysis of results of some crosses between
Hfr heterogenote and haploid F- show that
markers on the exogenotic piece are transferred
with the same timing as those of the endogenote.
Such mode of transmission is suggestive of an
organization involving some kind of attachment
between the exo- and the endogenote.

4.14. A Genetic Oscillatory Mechanism in Arabidopsis.

G. P. Redei (Columbia, U.S.A.).

In homozygous condition the recessive X-ray
induced im gene periodically and repeatedly
causes the turning on and off of chloroplastid
differentiation, and consequently of leaf pigment
synthesis. Due to this oscillating function all
parts of the mutant plants exhibit sectors. The
individual cells are either all white or normal
green without any intermediates. The frequency
of the switch may be very high. The pattern of
variegation indicates non-randomness. It appears
that the altered cells transmit their temporary
character to their several mitotic offspring.
Though there is some resemblance to certain
mutable systems observed in other organisms,
there are several obvious differences. The change
of state of the cell lines dividing parallel to the
axis of differentiation is fairly well synchronized.

4.16. Thermosensitive Mutations affecting the Re-
plicating Capacity of the Sex-factor of E. coli.
F. Cuzin and F. Jacob (Paris, France).

It has recently been suggested that in bacteria
a self-replicating unit, or replicon, such as a
chromosome or an episome, determines its own
replication system. (1> This hypothesis predicts
the existence of mutations affecting specifically
the replication of a given element such as an
episome or a chromosome. From bacteria car-
rying an F-Lac+, mutants have been isolated in
which the episome replicates normally at 30° but
not at 40°. At 40°, the episome is diluted out by
the bacterial multiplication. It is not destroyed,
however, since, when bacteria grown at 40° are
shifted down to 30°, the multiplication of the
episome in those cells, which will carry it, is
resumed. This thermosensitivity is genetically

41

Section 4 — Gene action

controlled by the episome as shown by transfer
experiments.

When the thermosenisitve F-Lac episome,
however, is integrated in the chromosome as
Hfr, it replicates normally at 40°, suggesting that
in this case F is no longer reproduced by the F
system of replication but by the chromosomal
one. In bacteria carrying both a thermosensitive
F-Lac, and a normal F-Gal factor, the replication
of the F-Lac is normal at high temperature, a
result which suggests that the thermosensitive
factor is diffusible.

1. F. Jacob and S. Brenner, C.R. Acad. Sci.
257, 298-300 1963.

4.17. The Mechanism of Chromosome Mobilization
by F-lac in Escherichia coli K12. John Scaife
and Julian D. Gross (London, Great Britain).

Male cells of E. coli K12 harbour the episomic
sex factor, F. During conjugation these are able
to donate genetic material to female cells, which
do not carry the F-factor. F-episomes incorporat-
ing genes from the bacterial chromosome have
been isolated, e.g. F-lac.

The chromosome of E. coli is circular; in
conjugation, however, it is transferred as a linear
structure. The opening of the circular chromo-
some and its subsequent transfer are associated
with an interaction between the sex factor and
the chromosome.

In the case of a cell harbouring F-lac this inter-
action occurs in the vicinity of the lac region of
the chromosome, which suggests that the two
structures synapse and interact within the region
for which they are genetically homologous. It is
proposed that the interaction is, in fact, a genetic
exchange in the homologous region, causing the
insertion of the F-lac into the chromosome, which
then acquires the ability to be transferred as a
linear structure. A prediction of this model is
that lac will be transferred as the first chromo-
somal marker.

We have made crosses using male cells bearing
different lac markers on the chromosome and the
F-lac factor. Analysis of the progeny from crosses
of this type has yielded results which are con-
sistent with this prediction from the model for
chromosome mobilization outlined above.

4.18. High Frequency Resistance Transfer System in
Escherichia coli. Tsutomu Watanabe (Tokyo,
Japan).

Cells of Escherichia coli K-12. which have just

received R factors (episomic resistance factors),
are able to transfer them in unusually high fre-
quencies unlike usual R+ cells. HFRT popula-
tions which contain high ratios of such competent
donor cells can be obtained following the pro-
cedure for obtaining HFCT (or HFC) of Ozeki
and Stocker (1960). We have recently found that
HFRT contains a large number of cells with
higher drug resistance than usual R+ cells. The
R factors of the clones which grew on higher
drug concentrations were found to have mutated
resistance markers which confer higher drug
resistance. Jacob et al. (1960) have shown that
partial polyploids carrying multiple copies of
FMac synthesize unusually large amounts of
(3-galactosidase. In view of their finding, our
results may be interpreted as due to a gene
dosage effect by the unusually vigorous repli-
cation of R factors in HFRT cells. Transfer of R
factors is very frequent because of the many
copies of R factors in cytoplasm and also of the
increased production of RTF-specific mating
substance. The gene dosage effect is also exerted
on the biochemical mechanisms of drug resis-
tance and HFRT cells are thus phenotypically
more resistant than usual R+ cells. HFRT cells
are able to divide for several divisions on higher
drug concentrations until the R factors are
reduced to "stable autonomous state" or in-
tegrated state. Accordingly, the probability of
mutation of R factors per cell is higher in HFRT
cells than in usual R+ cells.

4.19. Linkage of Colicinogenic Factors with an F
Agent and with Nutritional Markers in the
Chromosome and in an Episome of Escherichia
coli. Pierre Fredericq (Liege, Belgium).

Some wild strains E. coli producing colicins
V or B have been found to carry their colicino-
genic factors closely linked to an F agent of
sexual polarity. Almost every recombinant ob-
tained in crosses with F- derivatives of E.coli
K12are F+ or F- according to whether it receives
or not the colicinogenic factor. In such crosses, an
unusual type of recombinant has been obtained,
where the F agent has been integrated in the
chromosome. Genetical analysis of that recom-
binant revealed that a B colicinogenic factor is
still linked with the F agent and integrated with
it in the chromosome. In that Hfr strain the
leading markers is mal and the most distal str.
There is no transfer of the Hfr and colicinogenic
properties when selection is made for transfer
of the proximal marker met. There is however a
linked transfer of both properties when selection

42

Section 4 — Gene Action

is made for more distal markers. The rate of
transfer of both properties increases from 0.01
when selection is made for transfer of pro to 0.70
when selection is made for transfer of str, the
most distal marker.

Linkage of colicinogenic factors with nutritio-
nal markers has also been observed in an F1
episome. In a cross of a wild strain E. coli,
carrying V and B colicinogenic factors linked to
an F agent, with a deletion mutant tryABDC,
Tl-r of E.coli B, some recombinants, selected
for transfer of try+, had a very peculiar behaviour
towards phage Tl, being partially susceptible.
They were found to be partial diploids, carrying
the wild try region, including cysB and 77,
linked to the F agent and to the V and B colici-
nogenic factors in an F1 episome. These recom-
binants have been further crossed with different
cysB or try mutants of E.coli B or K12. The new
recombinants appear to carry, like the male
parents, an episome in which F is linked to the
V and B colicinogenic factors and to the entire
cys try region. Preliminary transduction experi-
ments by phage Pike grown on such a diploid
strain reveal that genetic material from either
the chromosome or the episome can be trans-
ferred. In many transductants the introduced
fragment is not integrated and remains in the
episomic state, giving rise to stable diploids.
Among them a few received all the known
markers from the episome, including the colici-
nogenic factors. From the data, the order (F),
cysB, tryA, tryB, tryD, tryC, Tl, (colV), (colB)
can be inferred.

The research reported in this document has
been made possible through the support and
sponsorship of the U.S. Department of Army,
through its European Research Office, Univer-
sity of Liege, Belgium.

4.20. Controlling Episomes. G. W. P. Dawson
(Dublin, Great Britain).

Instability at a number of loci in Salmonella
typhiimtrium have been studied and their in-
stability appears to arise by an attachment of
genetic particles to the locus. To point the simi-
larities between these particles and controlling
elements in higher organisms and episomes in
bacteria they are called controlling episomes. At
some loci controlling episomes suppress gene
expression as well as causing instability. Different
loci have different avidities for particular con-
trolling episomes. Different strains have different
frequencies of controlling episomes. Controlling

episomes can transpose from one locus to another

4.21. Controlling Episomes at the su-leuA locus of
Salmonella typhimurium. P. F. Smith-Keary
(Dublin, Great Britain.

Slow growing reversions of the leucine re-
quiring strain leu-151 of Salmonella typhimurium
are due to mutations at one of a number of sites
within a linked suppressor locus su-leuA; many
of these reversions are highly unstable. This in-
stability is caused by a particular type of episome
called a controlling episome which becomes trans-
posed from some unknown location to a site
within su-leuA where it induces a high frequency
of mutation. The instability is retained at this
site, through successive back and forward
mutations of su-leuA, until the controlling epi-
some becomes transposed to another location.

Some strains of leu-151 revert to the unstable
types at a much higher frequency than other
strains. This is attributed to these strains having
a higher frequency of controlling episomes
available for transposition to su-leuA; in these
strains the frequency of unstable reversions can
be greatly reduced by treatment with acridine
orange. The frequency of stable reversions is the
same in all strains of leu-151 and is unaffected
by acridine orange treatment.

A controlling episome located within su-leuA
can be transposed to a locus modifying proline
requirement, resulting in instability for proline
independence and concomitant stability at
su-leuA .

4.22. Transposed Inducer Function in E. coli. K. C.

Atwood (Urbana, U.S.A.).

From a K-12 stock carrying F-lac, a mutant
was screened that requires an inducer of B-
galactosidase for growth on medium containing
glycerol as sole carbon source. Inducing levels of
the nonutilizable inducers, methyl-thiogalacto-
side or isopropyl-thiogalactoside satisfy the re-
quirement. The inducer requirement is also pre-
sent with amino acid mixtures or succinate as
carbon sources, but not with any hexose or
pentose that can be used by the parent strain.
Neither induced nor uninduced cells of the
mutant produce B-galactosidase. The mutant is
interpreted as a transposition of controlling
elements such that the system normally in control
of B-galactosidase synthesis now controls a
different enzyme, apparently an enzyme involved

43

Section 4 — Gene Action

in the synthesis of hexoses and pentoses from
smaller compounds.

4.23. Discrimination among Copying, Plasmid, and
Episome Models for Conversion-type Inheri-
tance in Maize. E. H. Coe, Jr. (Columbia,
U.S.A.).

Specific models for conversion-type inheritance
or paramutation can be based on gene-extrinsic
or gene-intrinsic mechanisms. The basic obser-
vations in regular conversion-type systems (fail-
ure of normal segregation of parental phenotypes
in certain hybrids contrasted with normal se-
gregation in others) can be conceived to depend
upon (1) an independent but gene-maintained
material, (2) a gene-associated but transferrable
material, or (3) a gene-intrinsic feature that may
be forcibly copied. These conceptions are res-
pectively parallel to (1) the properties of plasmids
in many organisms, (2) the properties of episomes
in bacteria, and (3) special crossing-over models.
Criteria by which these systems might be distin-
guished in eukaryotes include time of conversion,
concurrence of chromosomal deletion with loss
of the conversion effect, infectivity, synchrony in
replication of the chromosome and the unortho-
dox system, association of the system with
particular alleles, and the mechanics of origina-
tion of the phenomenon. Because the regular
conversion-type event at the B locus in maize is
(a) completed late in the life cycle, (b) eliminated
by X-irradiation concurrently with linked
chromosomal markers, (c) apparently not in-
fective, (d) replicated synchronously with the
chromosome, (e) unrelated to allele function, and
(f) originating from a gene-independent system,
a dual model (a gene-associated but transfer-
rable material) is invoked. Specific predictions of
the dual model are being tested. A full report is
in preparation.

4.24. Control of Activity at a Mutable Locus in Maize.

Peter A. Peterson (Ames, U.S.A.).

Control systems as formulated from a study
of mutable loci in maize are characterized by the
following features; the definable elements that
comprise the system vary in time and rate of
action upon a locus, undergo transposition to
various sites in the genome, and affect the qual-
itative expression of a locus.

The mutable locus described here (ai colorless,
to A\ color on chromosome 3) consists of two
elements — / (Inhibitor) that controls the action

of the gene in question when intimately associated
with it and En (Enhancer) that controls the action
of / by removing or inactivating /. The expression
of the / pattern is dependent on the presence of
En, however En cannot be recognized unless
there is an / controlled locus. Each of these
elements is identified by a pattern of mutability
as determined by the time (manifest in size of
mutant area) and frequency (manifest in number
of mutant areas) of mutation events.

Recent experiments show that these elements
can also control the qualitative expression of the
locus. This is manifest in the wide assortment of
gene expression derived from certain of the
mutable alleles that have reduced levels of gene
action. The phenotypes in these cases range from
very light pales to dark pales that are heritable
and remain stable at each level in future gene-
rations. Experiments also indicate that additional
differences among the elements may be expressed
in their differential rate of change to other pattern
types or to colorless forms.

These results show that a particular gene locus-
can be affected both quantitatively and qualitas-
tively by the controlling elements in the genome.

4.25. Transposition of Mutability among Components
of a Compound Allele of the A1 Locus in Maize*

M. G. Neuffer (Columbia, U.S.A.).

An unstable condition has arisen at the site
of the (3 component of the compound Ab allele
(a (3) on chromosome 3 in maize. The mutability
is expressed as the loss of (3 function which per-
mits expression of the a phenotype (dilute seed,
red brown plant), since (3 is dominant to a in
these characteristics, and as frequent recovery
of (3 action to give sectors of the (3 phenotype
(full colored seed, purple plant). The system is
apparently autonomous as all known elements
are located at the A\ locus. Localization the effect
at (3 was confirmed by separation of the unstable
(3 ((3m) from x by crossing over in a heterozygote
of a (3m with a homologous segment carrying
only a null component of the A\ locus. The (3m
crossover has a recessive phenotype (colorless
seed, brown plant) with frequent sectors of |3
phenotype.

Included among several variations from the
original case are those in which (1) the muta-
bility has left (3 and appeared at a to produce am (3
and (2) the mutability remains at (3 but also has
appeared at a to produce am |3m. Kernels ex-
pressing the latter are phenotypically colorless
with dots of both dilute (a) and of full color ((3).
On these seeds it can be observed that changes

44

Section 4 — Gene action

occur independently at a and (3 each having its
own particular frequency and character.

From these observations it is clear that a type
of instability of obscure origin is able to appear
at a compound locus and then to move back and
forth between the components of this locus,
affecting each as though it were a separate gene.

4.26. Variegation following Hybridization between
Nicotiana tabacum and N. otophcra.
D. U. Gerstel, and Joyce A. Burns (Raleigh,
U.S.A.)

Different mechanisms seem to cause chloro-
phyll, bud anthocyanin and flower anthocyanin
variegations obtainable after hybridization of
TV. tabacum with N. otophora. The first two types
can be recognized only in later generations after
recombination has taken place. Carmine-coral
flower variegation occurs as a rule in first gener-
ation hybrids between coral N. tabacum and
N. otophora: this type has been sufficiently in-
vestigated for a preliminary report. It differs as
follows from the variegations described in the
literature: Coral spots on carmine background
are not due to chromosome loss since chromo-
some numbers in variegated and self-coral petals
of the same plant are identical; furthermore,
coral branches on a variegated plant produce
variegated test-cross progeny. Certain similarities
with v-type position effect can be noted, but
structural rearrangement is not required for
coral spotting to occur. Paramutation is obli-
gatory in certain heterozygotes of maize and the
changed allele is passed on to the progeny while
carmine-coral variegation seems to occur hapha-
zardly and the changed form is not transmitted
sexually, as stated. As yet incomplete genetic
studies indicate that a single factor, cov, from
N. otophora is involved which is dominant over
coral (co) of N. tabacum and which thus far
appears to be autonomous since no controlling
element could be separated by backcrossing and
stable carmine types could not be obtained. One
may hypothesize that there exists in N. tabacum
(chromosomes or cytoplasm) a repressor element
which at times inactivates cov in somatic cells.
During the reproductive cycle the potentiality of
cov for pigment formation is restored.

4.27. Studies on Amylase in Drosophila meianogaster
by Agar-electrophoresis. Hideo Kikkawa and
Zenichi Ogita (Osaka, Japan).

It has been found by the senior author (1960)
that the amylase activity in D. meianogaster is
controlled by a semidominant gene located at

about 80 on the second chromosome. However,
the amylase activities differ from one another
according to the strains used.

By using an agar-electrophoretic method
modified by the junior author, amylases in
various strains have been analysed. Seven amy-
lase bands were found as a whole on a zymogram.
But each strain showed a characteristic pattern in
amylase bands. For instance, Amy+ strain showed
only one clear band (No. 1), whereas Amy"
strain showed two clear bands (No. 2 and No. 6).

Fi individuals between any two strains which
showed different patterns in amylase bands, gave
a mixed type of parental patterns on a zymogram.
No additional band has been detected except the
parental ones. This result suggests that each
allelic gene produces its own amylase protein in
the cytoplasm, and also that no hybridization
occurs between components of the enzyme
proteins.

4.28. Studies on the Genetic Control of Xanthine
Dehydrogenase in Drosophila meianogaster.
R. P. Kernaghan and A. Chovnick (Storrs,
U.S.A.).

Xanthine dehydrogenase activity in Drosophila
meianogaster is controlled by at least three
functional units (ry, ma-1, and bz). Mutation in
any of these units produces a pleiotropic phe-
notype including absence of xanthine dehydro-
genase activity (Forrest, Classman, and Mitchell,
1956; Glassman and Mitchell, 1956; Glassman
and Pinkerton, 1960; Hadorn and Schwinck,
1956; Nawa, Taira, and Sakaguchi, 1958). The
inactivation of antibody specific to xanthine
dehydrogenase suggests that ma-11 contains
greater amounts of cross reacting material
(CRM) than ry'2 (Glassman, and Mitchell, 1958).
The data on the fine structural organization of
rosy (Chovnick et ah, 1962) and the description
of ma-12 (Schaiet, 1962), which complements
neither ma-11 nor bz, requires a re-examination
of the CRM question.

Extracts of wild type and mutant stocks were
partially purified with Sephadex. Enzyme ac-
tivity was determined as the increase in optical
density at 395 mu due to the reduction of added
thionicotinamide — adenine dinucleotide mediat-
ed by the conversion of 2-amino, 4 hydroxpteri-
dine to isoxanthopterin. Mutant extracts were
tested for their ability to remove specific anti-
enzyme activity from antibody preparations.
Ma-1 x and bz are positive while ma-12 is negative
with respect to CRM activity. Several rosy
mutants (ry1, ry2, ry8, ry9, and ry23), were sub-
stantially negative. An anti-ma- 11 antibody

45

Section 4 — Gene action

preparation inactivated the wild type enzyme
whereas an anti-ry2 antibody preparation was
ineffective. A discussion of these data will be
presented.

4.29. A Possible Explanation for Some of the Differ-
ences between Drosophila Mutants lacking
Xanthine Dehydrogenase. Edward Glassman
(Chapel Hill, U.S.A.).

Both the maroon-like {ma-1) and the rosy (ry)
eye color mutants of Drosophila melanogaster
lack detectable amounts of the enzyme, xanthine
dehydrogenase. However, when extracts of these
two mutants are mixed together and incubated
for 90 min at 30°C, xanthine dehydrogenase
activity is produced in the absence of protein
synthesis. W It is postulated that there is a
/Ha-7+-complementing substance in ry extracts
and a rv+-complementing substance in ma-1
extracts, and that these complementing substan-
ces interact to produce active xanthine dehydro-
genase. Preincubation of each of the mutant
extracts at various temperatures has shown that
the /no-/+-substance is very much more stable to
mild heat treatment (35° to 50°C) than is the
0>+-substance. If this difference in stability
occurs in vivo, it would explain some of the differ-
ences between the ma-1 and ry mutants. Thus,
there is no maternal effect at the ry locus be-
cause the ry+-substance is too unstable to persiss
in the egg. (2) In addition, the difference in effectt
of gene dosage at these two loci can also be ex-
plained by the relative instability of the /-.^-sub-
stance. The more stable wa-/+-substance will
pile up in the cell to such an extent, that even if
its level drops in response to lower doses of
ma-l+, it will still be in excess, and no change in
xanthine dehydrogenase activity will occur. Thus
only the ry locus has a gene dosage effect on the
enzyme activity; the ma-1 locus does not.

This research was supported in part by a
research grant (GM 08202-03) from the National
Institutes of Health.

1. Proc. Nat. Acad. Sci. 48, 1491.

2. Proc. Nat. Acad. Sci. 48, 1712.

4.30. Gene Control of Eye Pigments in Mormoniella.

G. B. Saul (Hanover, U.S.A.).

Wild type eye color results from the presence
of at least three pigments separable by chromato-
graphy. Two of these, a yellow and a red pig-
ment, are present in scarlet-eyed mutants but not

in a black-eyed mutant type; the other, a purple
pigment, is absent from scarlet-eyed wasps but
present in the black-eyed type. Spectrophoto-
metry and chemical tests indicate that the yellow
and red pigments may be pteridines; the nature
of the purple pigment is unknown, but it does not
appear to resemble the brown ommochrome of
Drosophila.

Three loci contain mutations for black eye
color. Mutants of one of these contain only
purple pigment and accumulate isoxanthopterin
and 2-amino-4-hydroxypterine; the second group
of mutants contain all eye pigments of wild type
and do not accumulate the pteridines; and wasps
containing mutations at the third locus contain
all eye pigments of wild type and also accumulate
the pteridines.

Scarlet-eyed mutants result from changes at
any of four known loci. They cannot be distin-
guished from each other by color or chromato-
graphy, but have separable phenotypes when also
containing mutant genes for black eye color.

4.31 . The Genetics of Esterases in Drosophila melano-
gaster. Theodore R. F. Wright (Baltimore
U.S.A.).

Ogita W reports the existence of a genetic
factor (ali ) on the 3rd chromosome which lowers
the esterase activity (methyl butyrate used as the
substrate) of homogenates of whole adult flies.
Homogenates from ali-/ali~ individuals have
only 15 per cent as much activity as homogenates
from ali+/ali+ individuals. Heterozygotes, ali+/
ali~, have intermediate activities; approximately
55 per cent of ali+/ali+. Wright <2) reports that
two codominant alleles which control the struc-
ture of an esterase designated as Esterase 6 are
located at 36.8 ± on the 3rd chromosome.
Starch gel electrophoresis of homogenates show
that homozygotes for the allele, Est 6F, produce
a form of Esterase 6 which migrates faster then
that produced by homozygotes for the other
allele, Est 6s. The heterozygote, Est 6F/Est 6s,
produces both fast and slow forms.

Because both of the above genetic systems are
concerned with esterase production and because
both are located on the 3rd chromosome, it be-
came desirable to investigate whether or not the
two systems are interrelated. Three of Ogita's
strains, one ali+/ali+ and two ali-/ali-, are homo-
zygous for Est 6s. Using p-nitrophenyl acetate
as a substrate, the total esterase activities of the
Est 6F and Est 6s strains (which are equal to one
another) are comparable to that of the ali+/ali+
strain. These data are not conclusive, however,
and data comparing not total esterase activities,

46

Section 4 — Gene Action

but Esterase 6 activities of the various strains and
of offspring from crosses, will be presented in an
attempt to show whether or not the two systems
are interrelated.

1. Botyu-Kagaku, Scientific Insect Control 26,
93, 1961.

2. Amer. Zool. 1, 476, 1961.

4.32. In Vitro Interconversion between Mutant Forms
of the pH 7.5 Esterase in Maize. Drew
Schwartz (Cleveland, U.S.A.).

Six esterase isozymes distinguished by elec-
trophoretic mobility are formed by the three
alleles of the E locus in maize. Three isozymes
FFS NN and SS are formed by the EF, EN and
Es alleles when in homozygous condition and in
addition three hybrid isozymes FN, NS, FS
occur in the three heterozygous combinations.
The FS hybrid isozyme migrates at the same rate
as the NN band. Recent results indicate that the
difference in the migration rates between the
isozymes are due to a positively charged group
associated with the enzyme. By treating extracts
with sodium borohydride, the single pH 7.5
esterase band found in homozygotes is converted
into a series of bands, all less positively charged.
These new bands show migration rates identical
to those produced by the various E alleles in
homozygous and heterozygous combinations. In
addition a new band migrating slower than the
SS band is also seen. Treatment of FF extracts
yields bands which are electrophoretically in-
distinguishable from the FF, FN, NN (or FS),
NS and SS isozymes. Treatment of NN extracts
reveals four bands, NN, NS, SS, and the new
slow band. Treatment of the SS extracts yields
only two bands, the SS and the new slow band.
When a mixture of EF/EN and EN/ES extracts
which contain all five isozyme types are treated,
no new bands are seen except for that at the new
slow position. These data are consistent with the
hypothesis that F, N and S are associated with
decreasing numbers of the positive charge group
and that sodium borohydride removes varying
numbers of the charged groups thereby convert-
ing the more positively charged isozymes into
the less positively charged enzyme types. The
other esterases and proteins in the same extracts
are not effected by the borohydride.

4.33. Two Forms of Tyrosinase from Each of Two
Different Mouse Melanomas. Jean B. Burnett
and George F. Wilgram (Boston, U.S.A.).

The early work of Brown and Ward which
resulted in the preparation of a soluble tyrosinase
from mouse melanotic tumor has been the basis
for current studies leading to the further puri-
fication and elucidation of the properties of this
enzyme.

Harding-Passey and B-16 tumors are grown
in Swiss white and C-57 black mice respectively.
Harding-Passey and B-16 tumors each contain
tyrosinases which are electrophoretically sepa-
rable into two components; Ta, a tyrosinase of
greater anodic mobility and T|, a tyrosinase of
lesser anodic mobility. At pH 8.6, Ta (H-P) and
T* (B-16) are tyrosinases of identical mobility
while T^ (H-P) and Tf (B-16) are tyrosinases of
also identical but of lesser anodic mobility. T£
and T| of both tumors are also separable by
column chromatography. An additional purifi-
cation o fapproximately 10-fold is obtained using
a DEAE-Sephadex ion exchange column. Cha-
racteristically, the enzymes have the same
Michaelis constant and maintain characteristic
differences in specific activity during the course
of purification. Tryptic or chymotryptic digests
of the purified enzymes (i.e. fingerprinting)
suggest differences in amino acid sequence and
composition.

These properties of the tyrosinases suggest
that differences in primary structure are most
likely in the inactive protein moiety of the en-
zyme while the primary, and probably tertiary,
structure of the active sites are the same or very
similar.

4.34. Studies on the Biosynthesis of Tyrosinase in
Neurospora. Helen Macleod, Marguerite
Fling, and N. H. Horowitz (Pasadena,
U.S.A.).

The tyrosinase of TV. crassa is a crystallizable,
copper-containing enzyme of molecular weight
30-35,000. It has been the subject of a number
of genetic and biochemical studies. (*) The en-
zyme is not produced in rapidly growing cultures,
but is formed during the sexual phase of the life
cycle, or in vegetative cultures when growth is
inhibited by starvation or by amino acid analogs
(e.g. ethionine, fluorophenylalanine, D-aromatic
amino acids). When wild-type cultures growing
on minimal medium are starved by transferring
them to phosphate buffer, tyrosinase synthesis
starts after a lag period, attains its maximal rate
at about 24 hr after transfer, and ceases at about
60 hr after transfer. At this time, tyrosinase con-
stitutes from 1 to 5 per cent of the extractable
proteins. This induction of tyrosinase synthesis is
much reduced in a mutant (ty-1) which is not

47

Section 4 — Gene Action

linked to the structural gene (T) of tyrosinase.
The induction is pH dependent. It occurs opti-
mally at pH 6 and is inhibited at pH 8. Inhibition
at pH 8 is due to the loss of amino acids and
calcium from the cells. The induction is inhibited
only slightly by actinomycin-D, a drug which
blocks DN A-dependent RN A synthesis and which
strongly inhibits the growth of Neurospora. This
result suggests that messenger-RNA synthesis
for tyrosinase occurs during the period of active
growth, but is not used for tyrosinase synthesis
while growth is occurring.

1. See Cold Spring Harbor Symp. Quantitative
Biol. 26, 233, 1961.

4.35. Comparative Genetic Studies of Tyrosinase
Synthesis in Neurospora crassa and Bacillus
subtilis. K. E. Fuscaldo, V. Del Vecchio and
W. Kaczmarczyk (New York, U.S.A.).

The genetic control of tyrosinase production in
Neurospora crassa is being studied. Two strains,
15300-1 31a and 15300- 138 A, had been isolated
by other workers and characterized for their
ability to produce tyrosinases. Three electro-
phoretically separable enzymes'were found which,
however, do not differ with respect to their
Michaelis constants or relative turnover numbers.
It appears from previous work that the three
forms of the enzyme were present in homoca-
ryons. In order to clarify this system, matings
were made between the two strains and extensive
tetrad analyses carried out to establish the mode
of transfer of the genetic information regulating
tyrosinase synthesis. It has been suggested that
the T locus in Neurospora controls the equili-
brium among several interconvertible tyrosinases
and therefore is concerned with the terminal
stages of tyrosinase synthesis (Fox et al.). Other
workers have interpreted their results to con-
clude that the T locus determines the structure of
the enzyme and two other loci, ty-1 and ty-2,
exert a regulatory control on the tyrosinase
system (Horowitz et al.).

A corollary study was undertaken to examine
the genetics of tyrosinase production in another
organism. B. subtilis was chosen for this investi-
gation since it had been reported that a variety
(niger) produced a black pigment when grown
on tyrosine supplemented media. The decision
to pursue the tyrosinase studies on this organism
was predicated on the fact that bacteria through
their ability to participate in the phenomena of
transduction and transformation offered ex-
cellent tools for the genetic analysis' of an appa-

rently complex system. Further, since these or-
ganisms possess a "chromosome" which is
relatively simple when compared to those found
in higher organisms, it may be possible to dis-
tinguish the two control mechanisms on that
basis.

4.36. A Number of Loci associated with the Reduction
of Nitrate to Nitrite in Aspergillus nidulans.
B. M. Rever and D. J. Cove (Cambridge,
Great Britain).

In a sample of 1200 mutant strains produced
by u.v. irradiation of conidia of a prototrophic
strain of the fungus Aspergillus nidulans, 40 were
unable to grow on nitrate, but were able to use
nitrite as a nitrogen source. Pairs of mutant
strains were tested for complementary growth in
heterokaryons. On this basis, the mutants fell
into nine separate classes. The study of diploid
growth complementation has now begun. The
results of preliminary experiments suggest that
there may be a difference between heterokaryon
and diploid complementation with respect to
certain pairs of mutants.

It is known that all forty mutants lack normal
nitrate reductase activity. In the few cases so far
investigated, complementary heterokaryons and
diploids show detectable nitrate reductase
activity.

Linkage studies on representatives of the nine
complementation groups indicate that at least
six separate loci are involved, none of which is
closely linked to another. The loci may be allocat-
ed to their linkage groups using haploidization
techniques.

A discussion of these results will be made in
conjuction with the discussion of the biochemical
aspects of this system in our later paper.

4.37. Biochemical Genetical Studies on Nitrate
Reduction in Aspergillus nidulans. D. J. Cove
and B. M. Rever (Cambridge, Great Britain).

The reduction of nitrate to nitrite is thought,
in many organisms, to be catalysed by a single
enzyme, nitrate reductase. This enzyme activity
has been detected in extracts of mycelium of
prototrophic strains of Aspergillus nidulans, and
can be assayed quantitatively, in two ways. The
rate of oxidation of NADPH2, with which the
enzyme is linked, may be observed spectrophoto-
metrically, or the nitrate produced may be esti-
mated colorimetrically. The enzyme activity is
inducible.

48

Section 4 — Gene Action

The isolation and genetical analysis of 40
mutant strains unable to grow on nitrate, but
able to use nitrite, has been described in a previ-
ous paper. These 40 strains involve mutations in
at least 6 loci. All have been shown to have
abnormal nitrate reductase activity, when grown
in conditions inducing appreciable activity in
prototrophs.

These findings may be explained in several
ways. The conversion of nitrate to nitrite may
proceed through a number of intermediates, each
conversion being affected by a separate enzyme.
Alternatively, the reduction may be catalysed by
a single complex enzyme consisting of several
polypeptide sub-units, the structure of each sub-
unit being determined by a separate gene. It is
also possible that some of the loci have a regu-
latory function, or control the transport of nitrate
into the cell.

These hypotheses are not mutually exclusive,
and combinations of any are possible. However,
it appears that the mechanism controlling the
conversion of nitrate to nitrite in Aspergillus
cannot be of the one regulator — one operon
type already known in bacteria.

4.38. The Inhibitory Effect of Sucrose-Sorbose-Agar
on a Possible Invertase Mutant of Neurospora
crassa. T. H. Pittenger and T. G. Brawner
(Manhattan, U.S.A.).

One of the most widely employed techniques
of the Neurospora investigator is the use of 0.1
per cent sucrose and 1 per cent sorbose to induce
colonial-like growth of the mycelium. Although
the conidial viability of many strains is quite
high on such media, a mutant strain has recently
been isolated that is inhibited by this media. The
inhibitory effect of conidial germination and
growth of this mutant is one compounded of
three components of the media: 0.1 per cent
sucrose, 1 per cent sorbose, and 2 per cent agar,
and the full effect is expressed only when these
components are autoclaved together with the
basic salts. Some of the characteristics of this
inhibition follow. If glucose is substituted for
sucrose in the media, there is no inhibition. In
the absence of sorbose there is no inhibition. The
sucrose-sorbose medium is not inhibitory in the
absence of agar. Furthermore, the inhibition may
be relieved if the sucrose, sorbose, and basic salts
are autoclaved separately from the agar and then
combined. Similarly, if these components are
autoclaved separately from the agar, and the
agar is then added and the mixture autoclaved
again, there is no inhibition. If mutant conidia
are germinated in liquid media supplemented

with either sucrose or glucose and then plated on
sucrose-sorbose-agar medium, they do not form
colonies if the incubation period is less than 20 hr.
If, however, the pretreatment is extended to
24 hr or more, then inhibition is relieved. A
hypothesis to account for these and other obser-
vations will be discussed.

4.39. Neurospora Studies of Pantothenates produced
by Enzymatic and by Chemical Syntheses.
Robert Fuerst, Lilli Li-chun-Li and Caro-
lyn McFall (Denton, U.S.A.).

The in vitro synthesis of pantothenate with
wild type mycelium of Neurospora crassa Em
5256A was demonstrated by Wagner and Gui-
rard (1948). Acetone-dried mycelium of panto-
thenicless mutants were found to possess the
same enzyme system as wild type for the synthesis
of pantothenic acid from pantoyl lactone and
beta alanine. A new homolog of pantothenic acid
was prepared by incubating acetone-dried powder
of either Neurospora crassa pantothenicless
5531 A, 74A-Y153-M66, or commercial brain
extracts, with a substrate consisting of pantoyl
lactone and gamma-aminobutyric acid. The re-
sulting active metabolite was named gamma-
pantothenate. Wild type enzyme was not active
in the production of this compound. Purifications
of the active enzyme were successful, as well as
its separation from an enzyme cofactor. Gamma-
pantothenate supported the growth of Lacto-
bacillus arabinosus as well as a pantothenicless
Neurospora strain. Its chemical and physical
characteristics were determined. Also metabolic-
ally active were chemically prepared synthetic
calcium-gamma pantothenate and in part so-
dium-gamma-pantothenate. A Neurospora mu-
tant, able to grow on calcium-gamma-pantothe-
nate, was crossed to other strains. The results of
these matings will be discussed.

Supported by the N.I.H., U.S. Public Health
Service research grant, CA 03853-06 CY.

4.40. Glutamine Metabolism and Glutamine-requiring
Mutants of Neurospora crassa. E. Reich and
S. Silagi (New York, U.S.A.).

A number of mutant strains of independent
origin, requiring glutamine for growth, have
been recovered from conidial populations under-
going "inositolless death". No complementation
has been observed between the six different

49

Section 4 — Gene Action

strains obtained to date. Concentrations of 500
mg/1. L-glutamine (D-glutamine is ineffective) are
required to sustain optimal growth of all strains
bearing the glm~ allele. Even at these glutamine
levels there is an abnormally long lag phase, and
the growth rate is less than wild-type at all stages,
although the final yield of cell mass may be
normal.

Since glutamine functions as a nitrogen pre-
cursor for purines, amino acids, vitamins and
glucosamine, the potential sparing action of these
metabolites on the glutamine requirement has
been investigated. No significant sparing action
has been observed for any glutamine-related
metabolites, singly or in combination.

Through appropriate crosses a number of
strains have been obtained containing combina-
tions of the glm- allele with other gene-con-trolled
deficiencies affecting biosynthetic steps in which
glutamine normally participates. The nutri-
tional properties of these strains will be described
and discussed. The level of glutamine synthetase,
the enzyme responsible for glutamine synthesis,
has been recorded in various strains containing
mutational alterations which might be expected
to affect intracellular glutamine concentration.
These results will also be described.

The glm gene is located on linkage group V,
where it is very closely linked to inos (approxi-
mately 2 per cent recombination) and probably
distal to it.

4.41. Some Characteristics of the Protein Determined
by the am locus in Neurospora crassa. R. R.

Burk and J. A. Pateman (Cambridge, Great
Britain).

Amination deficient {am) strains of Neurospora
crassa lack both NADP linked glutamate de-
hydrogenase and alanine dehydrogenase activi-
ties. The purified glutamate dehydrogenase pro-
tein has alanine dehydrogenase activity. This is
interpreted to mean the am locus determines a
protein which has both glutamate and alanine
dehydragenase activity.

When the protein is dissolved in 0.05 m pH 7.4
o-phosphate buffer the glutamate dehydrogenase
reaction rate is initially low and subsequently
increases to a level dependent on the assay
system. In contrast the alanine dehydrogenase
activity is initially high and decreases. When the
protein is in pH 7.4 o-phosphate buffer of 0.1 or
greater molarity the initial rate of the glutamate
reaction is high. This suggests that at pH 7.4 in
a buffer of molarity 0. 1 or greater the protein is
in a form which favours glutamate dehydro-

genase activity while in 0.05 or less the form of
the protein favours alanine dehydrogenase
activity.

It is possible to distinguish three components
in purified glutamate dehydrogenase by inverse
filtration on a 5 per cent agar column. One of
these probably corresponds to the 28,000 mole-
cular weight sub-unit of Fincham and Codding-
ton. It does not possess enzyme activity. Another
component corresponds to their 220,000 m.w.
component, and the other behaves as if its
molecular weight is greater than 800,000. The
relative amounts of the two larger components
seem to be affected by the concentration of the
buffer in which they are applied to the column,
higher concentrations favouring the larger com-
ponent.

4.42. Partial Revertants at the am locus in Neurospora
crassa. J. A. Pateman and R. R. Burk (Cam-
bridge, Great Britain) and J. R. S. Fincham
(Hertford, Great Britain).

The amination deficient, am mutants N. crassa
do not possess normal NADP linked glutamic
dehydrogenase or alanine dehydrogenase activity.
Backmutant strains were produced from am- 3
by ultraviolet irradiation and then assayed for
glutamic dehydrogenase activity. A total of six-
teen strains were found to possess less than 20
per cent of normal enzyme activity. A genetic
analysis has shown that all the backmutations are
in or close to the am locus. A biochemical in-
vestigation of the glutamic dehydrogenase pro-
duced by each of the partial revertants showed
that the partial revertants can be classified into
six distinct groups. The members of each group
produce a unique variety of glutamic dehydro-
genase with respect to the following criteria:
thermostability, thermal activation, substrate
affinities and substrate activation. A study was
made of enzyme complementation between each
of the partial revertant groups and a number of
the original am mutants. In some cases the partial
revertants show different enzyme complementa-
tion to that shown by the am-3 mutant from which
they were derived. The partial revertants have
been assayed for alanine dehydrogenase activity
and none of them possess wild type alanine de-
hydrogenase activity. There are significant differ-
ences between some of the partial revertants
with respect to their alanine dehydrogenase
activity. The significance of the genetical en-
zymatic and complementation characteristics of
the partial revertants will be discussed.

50

Section 4 — Gene Action

4.43. Structural Gene for Ornithine Transcarbamy-
lase in Neurospora. R. H. Davis and W. M.
Thwaites (Ann Arbor, U.S.A.).

Previous workers have been unable to isolate
mutants of Neurospora lacking the enzyme or-
nithine transcarbamylase (OTC), which cata-
lyzes the conversion of ornithine to citrulline in
the metabolic pathway leading to arginine. The
mutation s, recognizable by its effect upon pyri-
midine- and prolinere-quiring phenotypes but not
by a nutritional requirement, has been shown to
contain 3 percent of normal OTC activity (1).
Using conidia of the s strain, many OTC-less
mutants were isolated after u.v. irradiation. They
were non-complementing, and segregated as single
gene mutations when mated with wild type;
s was not recovered in such crosses. Extracts of
an OTC-less mutant did not interfere with OTC
activity of wild or 5 extracts. The locus repre-
sented by 5 and OTC-less mutants is denoted
arg-12 and is concluded to be the single structural
gene for OTC. The locus is genetically distinct
from mutations at the arg-2 and arg-3 loci which,
like arg-12 mutants, are stimulated by citrulline
and arginine but not ornithine. A similar search
for OTC-less mutants starting with wild type
conidia yielded only one OTC-less mutant, which
was allelic with s. The difficulty in isolating
mutants of this class from wild type may be due
to a high OTC content of conidia formed by
wild type.

Research supported by U.S. National Science
Foundation.

1. Davis, Genetics 47, 351-360, 1962.

4.44. Ornithine Transcarbamylaseless Mutants of
Neurospora. V. W. Woodward (Houston,
U.S.A.).

The reaction coupling carbamyl phosphate
with ornithine to yield citrulline is catalyzed by
the transferase, ornithine transcarbamylase
(OTC). The genetic control of this enzyme has
been obscured by the finding that mutants arg-2
and arg-3, mutants heretofore assumed to be
"blocked" between ornithine and citrulline, con-
tain wild-type amounts of OTC, and by the dis-
covery that the suppressor mutation, s, which
relieves certain pyr-3 mutants of their pyrimidine
dependence, contains no more than 3 per cent
of wild-type OTC activity. Attempts to isolate
additional mutants lacking OTC have taken two
courses: (1) since s (reduced OTC) suppresses
certain pyr-3 mutants, a search was made for

further suppressors. Many OTC-less mutants
were found, all of which require arginine, or
citrulline, but none of which suppress pyr-3,
despite the fact that the pyr-3 OTC-less double
mutants were isolated on minimal medium. The
OTC-less mutants are not allelic with arg-2 or
arg-3. (2) Using 5 as the conidial source, the
filtration and selective plating technique was used
to isolate arginine-s double mutants. From such
double mutants six of the known arginine-
requiring mutants and two OTC-less mutants
were recovered. Physiological and genetical
properties of these mutants will be discussed.

Research supported by grant number RG-
8653, National Institutes of Health.

4.45. Quantitatively and Qualitatively Altered Phenol-
oxydases in Podospora anserina, due to Mu-
tations at Non-linked Loci. Karl Esser (Co-
logne, Germany).

Wild strains of the ascomycete Podospora
anserina produce melanin pigments on suitable
media. The early steps in the formation of these
pigments are catalysed by phenoloxydases. Two
different phenoloxydases have been found in the
wild strain si-: a diphenoloxydase (laccase) and
a monophenoloxydase (tyrosinase). The latter is
formed in only very small amounts compared to
laccase. Both enzymes can be distinguished by
their substrate specifities.

During the past years we have obtained about
40 more or less pigmentless mutants, most of
which have occurred spontanously. By 5-6 sub-
sequent backcrosses with wild strain si-, these
mutants have been made highly isogenic. Some
of the mutants are allelic. They all differ from
the wild strain by a single gene and could be
localized in 5 of the 7 linkage groups. With these
deficiences in pigment formation concomitant
defects in fertility are always observed. The
normal cycle of development of the mutants is
blocked at different stages.

Since all of the mutants exhibit different de-
grees of phenoloxydase activity, we wanted to
know whether this is due to quantitative or to
qualitative alterations of the enzymes. In order
to distinguish between the two alternatives, we
have performed a preliminary serological test:
enzyme extracts of the mutants were assayed for
cross reaction with antibody against partially
purified wild type enzymes (CRM test). Some of
the mutants showed considerable deviations of
the Enzyme/CRM ratio which is 1 for the wild
strain. Therefore, the concerned enzymes seem

51

Section 4— Gene Action

to differ qualitatively from the wild enzymes.

The phenoloxydases of three of these mutants,
belonging to different linkage groups and having
different morphogenetic blocks, have been
further investigated biochemically. Methods
applied were: behavior in ammonium sulfate
fractionations, substrate specificity and heat in-
activation. The experiments have established
both quantitative and qualitative alterations of
the two enzymes.

The implications of these findings will be short-
ly discussed with respect to gene-enzyme relations
and to the genetic basis of morphogenesis.

4.46. Purification and Properties of Tryptophan Syn-
thetase from Neurospora crassa. M. Carsiotis,
E. Appella and S. R. Suskind (Baltimore,
U.S.A.).

A purification procedure has been developed
employing Sephadex G-100 and G-200 which
permits routine preparation of tryptophan syn-
thetase of N. crassa. The enzyme is about 95 per
cent pure by physical criteria. It has an S20 of
5.9 and a diffusion coefficient of 4.92 10 7 cm2
sec-1. The Svedberg molecular weight of about
122,000 is in good agreement with the value
obtained by Archibald equilibrium sedimen-
tation. These protein preparations have been
used for physical and chemical studies in an
effort to provide a structural basis for previous
genetic, enzymological and immunochemical
observations. The results from amino acid com-
position studies, fingerprint analysis of tryptic
peptides, and from ultracentrifugation in 5m
guanidine — HC1 are consistent with the existence
of a quaternary structure for tryptophan synthe-
tase, comprised of two or more subunits. Such a
structure could explain the phenomenon of inter-
allelic complementation in this system, as well
as the occurrence of CRM -negative mutants.

This research was supported by Grant C-03080
of the National Institutes of Health.

4.47. Genetic Control of Tryptophan Accumulation in
Neurospora. David R. Stadler (Washington,
U.S.A.).

Wild-type Neurospora fails to grow on medium
supplemented with 4-methyltryptophan (4MT)
due to the inhibition of one or more enzymes
required for the synthesis of tryptophan. Resis-
tant mutants have been selected after u.v. or

nitrous acid treatment, and the twenty-two
mutants examined to date are alike in the charac-
teristics which follow. Resistance results not
from any direct change in the regulation of tryp-
tophan synthesis, but from the loss of the ability
to accumulate 4MT (or tryptophan) from the
medium. Under conditions in which wild-type
accumulates tryptophan to about 15 times the
external concentration, the mutants only ap-
proximate the external concentration. The wild-
type accumulation is inhibited by sodium azide.
The mutants are all in the same genetic region of
linkage group IV, less than one map unit from
co-4. The mutants are recessive; heterocaryons
between sensitive and resistant strains are sen-
sitive. Preliminary studies of one mutant indicate
that the same gene is responsible for uptake of
several other amino acids.

4.48. A Modifier Mutation affecting Tryptophan
utilization by Tryptophan Auxotrophs of Neu-
rospora crassa. B. Maling, P. St. Lawrence,
L. Altwerger and M. Rachmeler (Berkeley,
U.S.A.).

A mutation (nwd-5) which allows growth of
tryptophan auxotrophs of Neurospora crassa on
media containing yeast extract (YE) or Bacto-
peptone (BP) without added tryptophan segre-
gates independently of the td locus and is neither
locus nor allele specific, td; mod-5 cultures
require tryptophan and show no detectable res-
toration of tryptophan synthetase activity, mod-5
activity is not specific for tryptophan auxotrophs;
it affects the growth of a lysine and of an aro-
matic mutant.

The amount of free tryptophan in YE and BP
is sufficient for greater growth of td stocks than
is observed; mod-5 appears to permit utilization
of this tryptophan. Acid hydrolysis of BP and
YE removes growth activity for modified strains.
Sephadex chromatography of BP and YE yields
fractions with activity for both td and td; mod-5
cultures. The active substance in these fractions
has been tentatively identified as tryptophan.

Differential growth on enriched media may be
due to greater sensitivity of unmodified strains to
inhibitors. Some BP sephadex fractions appear
to have a differential inhibitory effect on td and
td; mod-5 cultures. The nature of the inhibitor
and the mechanism of the increased resistance of
td; mod-5 strains to it is not known. However,
differential responses of modified and unmodified
strains to tryptic digests of (3-lactoglobulin in-
dicate that the inhibitor may be a protein con-
stituent. Equivalent growth of both strains is
obtained at low concentrations of digest but

52

Section 4 — Gene action

growth of unmodified mutants is inhibited sev-
erely at high concentrations.

4.49. Genetic and Biochemical Studies of Cytochrome
C deficient Mutants of Yeast. Fred Sherman
and Harry Taber (Rochester, U.S.A.).

In a previous investigation, a single-gene mu-
tant of yeast was found which had a decreased
concentration of cytochrome c. The cytochrome
c isolated from this mutant (cyi-i) was different
from wild type (cyi-i) cytochrome c by a number
of criteria. W

A rapid method was developed for examining
the cytochrome spectrum of yeast strains at
-190°C in order to obtain other mutants con-
trolling cytochrome c. After examining over
14,000 strains that were treated with either ultra-
violet light or nitrous acid, ten mutants were
isolated that had a decreased amount of cyto-
chrome c but normal or near normal amounts of
cytochromes a and b (defined as cy). All ten
mutants were crossed to wild type (CY) and the
original cyi-i mutant, and the sexual progeny
analyzed. Also, diploids were constructed from
all pairwise combinations of the eleven cy mu-
tants. The results clearly indicated that six loci
were involved.

In addition to the above strains, two mutants
were found having a mutation of a cy gene and a
"loss" of the cytoplasmic factor which controls
the formation of cytochromes a and b.

The cytochrome c from some of the cy mutants
has been purified and compared to wild type
cytochrome c. These mutants have approximately
20 per cent of the normal amounts of cytochrome
c, but little or no change in respiration. Electro-
phoresis and chromatography studies of the
cytochromes c are now in progress.

1. Slonimski and Sherman, in preparation.

4.50. Nature of a Suppressor Mutation affecting
Adenylosuccinase. Joseph S. Gots and Edith
G. Gollub (Philadelphia, U.S.A.).

Mutations occurring in the ade-B locus of
Salmonella typhimurium lead to the loss of
activity of adenylosuccinase, a bifunctional de-
succinylating enzyme required for two sequential
reactions in the biosynthesis of adenylic acid. A
revertant of one of these mutants was found to
be, by phage-mediated transduction, the result
of a nonallelic suppressor mutation. We previ-

ously reported that the adenylosuccinase activity
of the suppressor mutant differed from that of
the wild type by a number of physicochemical
properties and that the differences indicated that
the ade-B mutation created an altered protein
whose enzymic inactivity could be partially
activated by the suppressor mutation. More
recent findings make this explanation unlikely.
Seven new ade-B mutants have been isolated and
identified by recombination analysis as distinctly
different single-site alleles of the ade-B locus.
These fall into four complementation groups as
determined by abortive transduction. Intro-
duction of the suppressor gene into any of these
mutants leads to a suppressor phenotype. This
suggests that the suppressor mutation allows an
unrelated enzyme to take over the function of
adenylosuccinase. Support for this thesis was
obtained by showing distinct immunological
specificities between the wild type and suppressor
enzymes serving as antigens. The suppressor en-
zyme is apparently not related to other known
desuccinylating enzymes (e.g. argininosuccinase).
Attempts to identify the normal function of the
wild type allele of the suppressor locus are being
made by searching for (a) natural repressors of
the suppressor enzyme and (b) secondary mu-
tations in the suppressor locus which could create
a complete loss of function.

4.51. Linkage Groups of Genes controlling Isoleucine,
Valine, and Leucine Biosynthesis in Bacillus
subtilis. C. Anagnostopoulos and M. Barat
(Gif-sur-Yvette, France).

Genetic analysis was carried out by transfor-
mation on a certain number of auxotrophic mu-
tants of B. subtilis obtained by u.v. irradiation
and requiring either isoleucine (//"), both iso-
leucine and valine (il-val-) or leucine (leir). The
i'/~ strains were shown to bear mutations on the
structural gene for the enzyme threonine deami-
nase. The il-val markers are the result of single-
step mutations affecting the genes of the enzymes
common to both isoleucine and valine pathways.
The leir mutants are blocked in the last steps of
leucine biosynthesis.

All /'/ markers were found to be linked to each
other but unlinked to the il-val or leu markers.
The il-val and leu markers on the other hand are
linked and can all be carried on the same piece
of transforming DNA as shown by reciprocal
crosses and experiments involving double mutants

It seems therefore that in B. subtilis the bio-
synthesis of isoleucine valine and leucine follows
the same pathways as determined in other micro-
organisms. Some of the genes controlling the last

53

Section 4 — Gene action

steps of these pathways are clustered; the thre-
onine deaminase gene lies outside that cluster.
A map of this segment of the B. subtilis genome
which bears the il-val and leu markers is being
constructed.

Results of biochemical and genetic studies on
the regulation of these pathways will also be
presented.

4.52. Non-genetic Control of Amino-acid Substitutions
in the Biosynthesis of the Antibiotic Polypep-
tide Tyrocidin. B. Mach (New York, U.S.A.).

Tyrocidin is a decapeptide antibiotic known to
exist in three forms, A, B, and C, differing by
known single amino-acid substitutions involving
phenylalanine and tryptophan. The occurrence of
these forms might be under direct genetic control,
possibly as the result of mutations. However,
unlike the amino-acid replacements observed in
proteins, they might result from environmental
effects.

The relative amount of the three forms of
tyrocidin synthesized by Bacillus brevis under a
variety of conditions, either by single bacterial
clones or by large volumes of culture, was deter-
mined after incorporation of C14 amino-acids and
purification of the polypeptides. It was found
that the molecular forms of tyrocidin synthesized,
and therefore their specific amino-acid sequence,
depend on the availability of the amino-acids
involved in these substitutions. Variations in the
amounts of these amino-acids in the culture
medium result in drastic changes in the ratio of
tyrocidin A, B and C synthesized, and even in
the induced production of a new polypeptide,
tyrocidin D.

Some aspects of this environmental control
over amino-acid substitution and its possible

application to the biosynthesis of other types of
polypeptides will be discussed.

4.53. The Colour and Fragrance in Plants and Their
Inheritance. John Politis (Athens, Greece).

In 1911 we commenced a study of the forma-
tion of anthocyanin in plant cells, which up to
that time was unknown.

In that paper we had shown that the antho-
cyanin in certain flowers first appear in the cyto-
plasm inside special corpuscles, which we had
named cyanoplasts. In geminating seedlings of
Raphanus sativus and other Cruciferae, antho-
cyanin is formed principally in the cells of the
subepidermal layer of the cotyledons and hypo-
cotyl axis. Each of these cells in its early stages
contains a nucleus, chloroplasts and a spherical
cyanoplast developing a red colour due to antho-
cyanin.

The fact that the cyanoplasts often appear near
the nucleus and the fact that the colours of the
flowers as well as other substances (tannins,
chlorogenic acid, etc.) are inheritable characters,
led us to suggest that a cyanoplast is a gene,
escaped from the nucleus, and able to multiply
by budding and to produce anthocyanin.

The fragrance of flowers and of other plant
parts is also an inheritable character. Researches
made by us on the glandular hairs of different
Labiatae and Compositae proved the concomit-
tant secretion of a bitter substance and an
essential oil.

In order to explain the relation between the
above two substances as well as the transmission
of this inheritable character to the progeny, we
believe that a gene which comes out from the
nucleus is the cause of the production of a bitter
heteroside, from which through enzyme action
an essential oil is produced.

54

SECTION 5

MUTAGENESIS

5.1. Application of Radiation Denaturation of DNA
in Combination with Chemical Mutagens to
Affect the Mutational Process. R. I. Salganik
(Novosibirsk, U.S.S.R.).

It has been established that some mutagens
(hydroxylamine, hydrazine, dimethylsulfate and
others) react much more intensively with heat
denaturated DNA than with the native one; but
applying these mutagens alone, excepting heat
denaturation of DNA, under the conditions of
our experiment, does not result in significant
breaking of hydrogen bonds in DNA. The
hydrogen bonds in double-strand structure of
DNA can also be broken by means of radiation
energy (ultraviolet radiation). This effect can
be realized not only in vitro but in vivo as well
and besides that it can be dosed. The raising of
the dose of the ultraviolet radiation increases
the number of denaturated regions in DNA. It
is reasonable to suppose that those regions of
DNA in which are prevailing weaker A-T bonds
and which are less saturated with stronger
G-C bonds are broken in the first place.

It has been shown that radiation denaturation
also increases the ability of DNA to react with
investigated chemical mutagens.

The selectivity of biological effect of applied
mutagens may be raised in connection with
their ability to react in denaturated regions of
DNA with certain nitrous bases (hydroxylamine
-> cytosine, hydrazine -> thymine, dime-
thylsulfate -> guanine).

Stepped irradiation with denaturating doses
of ultraviolet rays in combination with certain
chemical mutagens has been applied for affecting
the frequency and the spectrum of mutations in
bacteria.

mutagenic alkylating agent methyl methane-
sulfonate (MMS). The first is a local "de-
naturation" which occurs upon treatment of
DNA with either MMS, ultraviolet light, X-rays,
or when DNA is denatured by heat and then
annealed. This local reaction sensitizes the DNA
to inactivation by an enzyme found in B.
subtilis and in Micrococcus lysodeikticus. DNA
inactivation is apparently due to strand breakage.

The second reaction results in alkylation of
the DNA. DNA carrying alkyl group(s) is active
for transformation and can replicate in vivo.
Alkylated DNA is exponentially inactivated by
heating well below the thermal denaturation
temperature. This inactivation is probably due
to loss of alkylated purines since enzymatic,
ultracentrifuge and thermal denaturation studies
indicate that alkylated and heated (50°C) DNA
need not contain single strand breaks. Apurinic
DNA is, therefore, inactive for transformation
and (we presume) for replication in vivo.

Mutation induced by alkylating agents must
be due to mispairing while the alkyl group is
still part of the DNA molecule rather than at the
apurinic stage since apurinic DNA does not
replicate. This hypothesis is supported by the
results of studies on the reversion specificity of
lac~{rz~) strains of Escherichia coli obtained
by one of us (N.S.); lac~ mutations induced by
the alkylating agent ethyl methanesulfonate
behave as though they were transitions rather
than the mixture of transitions and transversions
expected if mutation occurred at the stage of
apurinic DNA.

This work was supported by grants from the
National Institutes of Health (U.S.A.) and from
the National Science Foundation.

5.2. Chemical and Biological Effects of the Reaction
of Deoxyribonucleic Acid with Mutagenic
Alkylating Agents. B. S. Strauss, R. Wahl,
M. Maillis, J. Coston and N. Schwartz
(Chicago, U.S.A.).

Transforming deoxyribonucleic acid (DNA)
obtained from Bacillus subtilis undergoes at
least two reactions when treated with the

5.3. Chemical Mutagenesis on Separated DNA.

S. E. Bresler (U.S.S.R.).

1. By means of bacterial transformation we
are able to make a quantitative study of chemical
mutagenesis on separated and purified DNA.

To know the conditions of quantitative assay
for transforming activity of DNA the homogeneity

55

Section 5 — Mutagenesis

of a population of competent Bacillus subtilis
bacteria was studied. These experiments were
performed by means of genetic measurements
(double transformation with two unlinked
markers) and by means of registration of
radioactive DNA uptake by competent bacteria
(the track autoradiographs method of Levinthal
was applied to study the homogeneity of DNA
uptake). The results showed a great inhomogenei-
ty of a B. subtilis population in transformation.
The knowledge of these details permitted us
to choose the conditions for quantitative
measurements.

2. Chemical mutagenesis on purified DNA by
means of nitrous acid and u. v. -irradiation was
studied. Genetically altered DNA molecules
were registered by means of transformation of
a recipient strain. Kinetics of mutagenesis and
inactivation of DNA were measured and princi-
ples and equations of physical chemistry were
applied to both processes. Both reactions are one
hit processes, requiring one effective collision
with the mutagen.

3. The efficiency of mutagenesis in vitro
depends on the ratio of two kinetic constants —
rate of mutagenesis versus rate of inactivation.
The reaction of inactivation was studied in
detail. It was shown that many genetic loci are
inactivated simultaneously.

Possibility for reactivation of chemically
modified DNA was studied. Reactivation can
be performed by means of fusion and annealing
of DNA in solution (Doty — Marmur's method)
together with homologous but genetically
unmarked DNA, or by a slight fragmentation
of damaged DNA with ultrasound.

These facts give some new points to the
mechanism of chemical attack on DNA macro-
molecules.

5.4. The Mutagenic Effect Produced by Radiation
and Chemical Agents on Extracellular Phages.
A. S. Krivisky (Moscow, U.S.S.R.).

The phage s(I Escherichia coli SK, isolated by
the author, is endowed with a unique capacity to
show hereditary variation when irradiated in
extracellular state. Below are exposed the results
of experiments serving to induce plaque mu-
tations of phage Sd acted upon by u. v. -rays,
ionizing radiation and nitric acid. The ap-
pearance of mutations should not be attributed
to selection, multiplicity reactivation or host-
reactivation. When the mutagen is applied to
the host cell only, no phage mutations arise.
The capacity to produce mutations in response
to the in vitro action of mutagenes is charac-

teristic of various strains of phage Sa. The
mutants induced appear in equal numbers when
plated on different strains of host cells. The
initial plaque of the mutant induced contains
only phages of this mutant type. It may therefore
be concluded that the capacity to produce
hereditary variation when exposed to mutagenes
in vitro is inherent in phage particles; these
mutations arise as a result of a direct action
produced by the mutagen on the DNA of a
resting phage, and are phenotypically realized
directly after the first intracellular replication
of the phage DNA.

The mutations do not revert after the action
of the mutagen by which they were induced; such
reversions may, however, arise spontaneously.

The examination of lethal and mutagenic dose
response curves shows that the relation between
these two types of damages is typical of each
mutagen applied. The molecular mechanism of
damages seems to be identical in both cases,
since radioprotectors are equally efficient in the
case of lethal or mutagenic effect.

The highest mutation ratio per surviving
phage (~2-3 per cent) is caused by u. v. -rays.
The effect is somewhat smaller in the case of
nitric acid, and still smaller in response to various
sources of ionizing radiation. The mutagenic
effect due to u.v.-rays is characterized by the
presence of a maximum corresponding to the
viability of phage~10"2-10-3; a further increase
of the dose leads to a sharp drop of mutation
ratio per survivor. No such effect could be
observed in our experiments with a temperate
phage kappa of Serratia marcescens in which an
increase of mutation ratio is observed after u.v.
irradiation up to a viability ^10-6. The causes
responsible for these differences in the behaviour
of different phages are being analysed.

In contrast to other phages, mutations failed
to arise after u.v. -irradiation of the vegetative
intracellular phage Sa.

The possible molecular mechanism of muta-
genesis of phage Sd and the prospects of its
application as a model for radiogenetic investi-
gation are discussed.

5.5. Induced Mutations in Extracellular Actinophages.

S. I. Alikhanian and N. M. Mkrtumian
(Moscow, U.S.S.R.).

Induced mutations in bacteriophages described
in literature include mutations obtained by
exposing to various mutagens: (a) phage-
infected cells, (b) phages and cells simultaneous-
ly, (c) host cells only. Phage mutations obtained
after single reproduction of preliminarily mu-

56

Section 5 — Mutagenesis

tagen-treated phages on the bacterial cell were
recently reported. Thus, these were mutations
found in the descendants of the phages treated.
Induced phage mutations arising directly after
the treatment of bacteriophages were also
reported. There are no reports, however, on
such cases with actinophages, nor are there
reports on obtaining induced mutations in
actinophages in general.

The authors of the present work tried to obtain
mutations in extracellular actinophages by
exposing them to mutagens and to reveal
mutations immediately after the treatment of
phages with mutagens. For this purpose the
authors used an actinophage belonging to the
phage group that affects streptomycin producing
cultures. H-mutations were studied. As the host
cultures sensitive strain A. streptomycini B-6
and resistant strain A. streptomycini No. 675
were used. Spontaneous frequency of H-type
mutations in our phage was 1 per 103 of all
inoculated corpuscles. As mutagens u. v. -light,
nitrous acid and sarcolysine hydrochloride were
used.

Repeated tests have shown that sarcolysine
hydrochloride pronouncedly increases the fre-
quency of H-type mutations which exceeded in
some experiments the frequency of spontaneously
occurring mutations byor der 2 or 3, whereas
u.v.-light and nitrous acid did not produce any
notable increase of the mutation frequency.

Thus, it was the first case of obtaining induced
mutations in actinophages and, what is more
important — in extracellular actinophages.

Further studies of sarcolysine as a mutagen
were carried out with several derivatives of this
preparation which differed from sarcolysine
hydrochloride in an additional amino acid
chain. A number of polyfunctional chlore-
thylamines were also studied.

The fact that all the mutagens used cause
mutations in extracellular actinophages may
serve as a ground for a new interpretation of the
mode of phage mutation.

5.6. The Action of Some Mutagenic Agents on
Bacteriophage 0X-174. J. H. van de Pol
and G. A. van Arkel (Utrecht, The Nether-
lands)

The inactivation of bacteriophage OX-174 with
nitrous acid (Tessman, 1959) and ultraviolet
rays (Setlow and Boyce, 1960) follows a single-
hit kinetics. It has also been found for the inac-
tivation of T4 with hydroxylamine by Freese and
coworkers (1961). We likewise found for 0X-174,
when treated with nitrous acid and ultraviolet

rays, an inactivation curve that was single-hit.
Upon treatment of the phage by hydroxylamine,
however, an inactivation curve was obtained
that extrapolated at zero time to approximately
2. In contrast to expectations the inactivation of
OX-174 by hydroxylamine thus appears to be
a double-hit event. Results will be presented of
experiments performed in order to test possible
explanations of the curves.

Another notable finding was that the in-
activation constant depended on the way the
phage was grown, presumably on the medium
used for propagation. The nature of this differ-
ence in sensitivity will be discussed.

The mutagenic action of hydroxylamine was
studied by determining the number of host
range mutants appearing on C/0S, a phage-
resistant mutant of E. coli C (Van de Pol,
Vendrig and Van Arkel, 1963; cf. Van Arkel and
Van de Pol, these abstracts). A method has been
developed for the detection of the induced
mutants. The number of mutants was measured
as a function of the dose, and was compared to
the values calculated assuming either single- or
double-hit kinetics of mutagenesis.

The effect of mutagenic agents on isolated
0X-DNA and on double-stranded phage DNA is
under investigation.

5.7. Synergistic Mutational Effects Between Radi-
ations and Chemicals. Delbert M. Shankel
(Lawrence, U.S.A.).

When cells of Escherichia coli are impinged on
membrane filters, subjected to nonlethal dosages
of ultraviolet light and subsequently incubated
in media containing certain chemicals, e.g.
caffeine, theophylline, theobromine, a great
increase in mutant numbers occurs. This increase
is much greater than the total obtained from the
two treatments employed separately. These
"synergistic" effects of the chemicals can be
prevented partially by the inclusion of very
small amounts of other chemicals such as
cysteine or 2-animo,4-sulfonamido-phenyl-disul-
fide in the post-irradiation phenotypic expression
medium. The synergistic activity of the caffeine-
type compounds appears to be related to certain
features of their chemical structure. The amount
of ultraviolet dosage plays a crucial role in the
synergistic effect; nonlethal dosages providing
for the maximal synergistic effect, while in-
creasing dosages result in the gradual disap-
pearance of the synergistic activity. Radioactive
tracer experiments indicate that the synergistic
chemicals are not incorporated into the nucleic
acids of the cells. No synergistic relationship

57

Section 5 — Mutagenesis

between X-irradiations and chemicals has, so
far, been observed. Possible mechanisms for
this unusual type of biological activity are
discussed in view of the available evidence.

5.8. Mutation during and after Synthesis of DNA.
Herbert E. Kubitschek (Argonne, U.S.A.).

In tryptophan-limited continuous cultures of
Escherichia coli B/l, try, the rate of caffeine-
induced mutation to resistance to bacteriophage
T5 is constant, independent of growth rate.
However, mutation rate is proportional to
growth rate for cultures limited with glucose,
lactate, succinate or phosphorus. These differ-
ent responses do not appear to be due to differ-
ent cellular concentrations of caffeine nor to
death or reversion of mutants before expression
of the mutant phenotype. The simplest interpre-
tation of the two kinds of response is that the
mutagen affects DNA either during or after
synthesis. If the mutagen acts only during the
formation of DNA then, as predicted by the
error hypothesis, mutation rate should be
proportional to the rate of replication of DNA,
and hence to growth rate. Alternatively, if the
mutagen acts after formation of DNA the rate of
mutation should be constant since DNA is
present in relatively constant amount. The prima-
ry evidence supporting this interpretation is that
responses to two other mutagens are in agreement
with expectation. There is evidence that the
first, ultraviolet light, acts upon DNA rather than
its precursors. If so, mutation rate should be
independent of growth rate for cultures limited
either with glucose or with tryptophan. This
result is found. For the second, 2-aminopurine,
mutation is expected to result from the incorpo-
ration of this base analog into DNA. Hence
mutation rate is expected to be proportional to
growth rate for either limiting factor. This
proportionality is also observed.

Work supported by the U.S. Atomic Energy
Commission.

5.9. Elective (Specific Directed) Mutation in Bacteria.

Francis J. Ryan and Stephen D. Cetrulo
(New York, U.S.A.).

The demonstration by Nagata that the
chromosome in synchronously dividing E. coli
Hfr replicates synchronously and with a polarity
from the hind (F containing) end allows the

following experiment. Similarly synchronized
bacteria are exposed to short pulses of a base
analogue which is mutagenic only upon in-
corporation. Only those genes which are repli-
cating during the pulse are expected to undergo
induced mutation. Preliminary experiments
bear out this expectation. Applications of this
technique to genetic problems, such as mapping,
will be discussed.

5.10. New Derivatives of (3-chloro- ethyl- amine as
Active Mutagenic Factors. S. I. Alikhanian
and M. Oganesian (Moscow, U.S.S.R.).

It is known, that a number of (3-chloro-ethyl-
amine derivatives, such as embichin, chloram-
bucil and sarcolysine are highly mutagenic.

We have studied for genetic purposes a large
series of previously not investigated amino acid
derivatives of (3-chloro-ethyl-amine, synthesized
in the laboratory of I. L. Knuniantz. Thirty-four
new compounds included two derivatives of
p-di (2-chlorpropyl) amine, one derivative of
/j-chloro-ethyl-sulfide and thirty-one derivative
of n-di (2-chloro-ethyl)amine. These agents
belong to monofunctional, bifunctional, tri-
functional and tetrafunctional compounds. This
means that all derivatives had an "attacking"
(3-chloro-ethyl group with the exception of some
derivatives having as an "attacking" 2-chlor-
propyl group instead of 2-chloro-ethyl group.

From 37 compounds tested we selected seven
most active compounds which were studied
more thoroughly. Among them methyl-bis-
(P-chloro-ethyl)-amine (embichin), /»-propyl-
(bis-(3-chloro-ethyl)amine (chlorambucil), and
bis-((3-chloro-ethyl)-amino-phenyl-alanine (sar-
colysine) were studied previously with respect to
their mutagenic action on Neurospora, Penicilli-
um chrysogenum, D. melanogaster, phage T4 of
E. coli and other organisms.

Compounds Nos. 6 (Ethyl ether of S-(3-
chloro-ethyl-dl-cysteine hydrochloride), 31 (Bis-
/chlorpropyl/phenylacetic acid), 32(N-formul-
sarcolysine), and 33 (N-acetylsarcolysine) were
the most active among the new compounds
studied by us.

For comparative purposes highly active
mutagenic factors, such as ultraviolet light,
diethylsulfate, and ethylenimine were used.
Strain H-6 of Actinomyces olivaceus served
an as object in our experiments. The muta-
tions were esti mated according to three cha-
racteristics, that is methionine deficiency (M-),
streptomycin sensitivity (Ss), and vitamin B12
synthesis (B12). According to the first two
characteristics the frequency of back mutations

58

Section 5 — Mutagenesis

to M+ and that of direct mutations to Sr per
10" colonies was estimated. According to the
third characteristic variations with respect to
the level of vitamin formation were determined.

The results of the study showed that compound
No. 6 by its effect with respect to the frequency
of mutations in loci M and S is equal to embichin,
chlorambucyl, sarcolysin and ultraviolet light;
10-20 times superior to diethylsulfate with
respect to the mutation frequency and much
more superior to these mutagens by the action
on induced variation with respect to vitamin
formation. Compounds Nos. 31, 32 and 33 by
their effect on methionine deficiency and vitamin
formation are 5-6 times superior to all these
mutagens, including ultraviolet light.

It should be noted that these compounds
have a different effect on the two loci. Compound
No. 32 increases the mutation frequency by
37.000 times with respect to methionine de-
ficiency as compared to the frequency of spon-
taneous mutations, thus being 70 times superior
to ultraviolet light by its mutagenic effect on
the same locus, whereas with respect to strepto-
mycin sensitivity this compound increases the
frequency of mutations only by 150 times as
compared to the frequency of spontaneous
mutations, thus being 7 times inferior to the
ultraviolet light with respect to the mutation
frequency induced by this mutagen. No such
markedly differentiated effect on the two loci was
observed in compounds Nos. 31 and 33.

On the basis of these results it is possible to:
(1) discuss some problems of the mechanism of
chemical mutagenesis; (2) consider the possi-
bility of differentiated mutability of various
genes under the action of the same mutagenic
factor and (3) assume that new compounds with
a strong mutagenic and specific effect are found.

5.11. Comparative Studies on the Mutagenic Action
of Carcinogenic Substances, Nitrous Acid and
Diethylsulfate in Saccharomyces cerevisiae.
F. K. Zimmermann, H. Marquardt and
R. Schwaier (Freiburg, Germany).

Certain carcinogenic substances have been
described that are supposed to act by producing
diazoalkanes which alkylate cellular structures.
Since mutations can be induced by alkylation
also, we tried to compare the possible mutagenic
action of carcinogens to mutagens of known
mutational pathways: Nitrous acid as a transi-
tional and diethylsulfate as a transversional
agent. The carcinogenic substances were:
N-nitroso-N-ethylurethan (ethylating), N-nitro-
so-N-Methylurethan (methylating).

The mutational activities were checked in a
backmutation test with four haploid, adenine
requiring strains of Saccharomyces cerevisiae
all mutated spontaneously at the ade-locus.
The mutagenic activities are compared on a
modified mutational/lethal hit base.

All carcinogenic substances are potent mu-
tagens, the methylating compound being more
efficient than the ethylating one. There is a
striking difference between diethylsulfate and
N-nitroso-N-ethylurethane. In two strains only
the latter is active, in one strain diethysulfate
alone and in one both are active together. The
ethyl and the methyl nitroso compound, how-
ever, parallel each other qualitatively in all
strains. Excepting for one strain there is a fair
coincidence in activity with nitrous acid.

Mutational differences between diethylsulfate
and N-nitroso-N-ethylure then may be due to dif-
ferences in chemical reactivity. But the similarity
of the nitroso compounds among each other and
to some extent to nitrous acid might indicate that
the mutagenic action resides in a mechanism
different from alkylation.

5.12. Mutagenesis in Corynebacterium. J. Necasek
(Prague, Czechoslovakia).

The possibility of demonstration of the relative
specificity of mutagens was studied by inducing
biochemical mutants in the strain Corynebac-
terium VUA 9366. This strain is biotin dependent,
produces large amounts of glutamic acid and
has a very low spontaneous and induced mu-
tation rate. Nitrogen mustard, ethyl methane-
sulphonate and ultraviolet light were used as
mutagens. Biochemical mutants were isolated
by the penicillin method. Mutants with identical
dependences isolated in a single experiment were
classifie das caused by a single mutation. On the
whole 25,000 isolates werein vestigated after ap-
plication of the three mutagens mentioned above.
Nearly 1 per cent nutritionally deficient mutants
was obtained, in which approximately one half
required specific amino acids for growth.

The results indicate that there exists a quali-
tative difference between the mutagenic action
of alkylating agents and ultraviolet light. With
respect to arginine biosynthesis the alkylating
agents induce mutants requiring arginine (step 3)
and mutants requiring either arginine or citrulline
(step 2). On the other hand the ultraviolet light
induces mutants requiring also arginine and
mutants requiring either arginine or citrulline
or ornithine (step 1). The difference in frequencies
of mutations blocking step 1 and step 2 was
statistically significant. It is concluded that the

59

Section 5 — Mutagenesis

relative specificity of mutagens may exist not
only on the allele level, but also on the intergenic
one, and that different mutagens may act with
different intensity on individual whole loci.

5.13. Comparison of Ultraviolet and Ethyl Methane
Sulphonate induced Mutations of Adenine
Loci in Saccharomyces cerevisiae. W. P. Cos-
tello, E. A. Bevan and M. W. Miller
(Oxford, Great Britain).

Mutations at the adenine-2 locus in S. cere-
visiae cause the accumulation of a red pigment.
If an adenine-2 strain is treated with mutagenic
agents a small percentage of white colonies is
observed among the survivors. These are due to
either reversion to wild-type at the adenine-2
locus, or to a mutation at a locus prior to ade-
nine-2 in the pathway of adenine synthesis, which
prevents the accumulation of the red pigment. By
reference to their complementation reactions
with standard strains, these double mutants
can be classified into five adenine loci.

Two of these loci, adenine-5 and adenine-7, as
designated by Roman, are very closely linked.
Our data suggest that the adenine-5/7 complex
may be interpreted as a single locus showing
complementing sub-units.

A total of 27 u.v. and 69 EMS-induced mutants
of the general adenine-5-7 type were isolated and
classified further on their complementation inter-
reactions. Of the u.v.-induced mutants 4 were
ad-5, 21 ad-7, and 2 ad-5/7, the double mutant : of
the EMS-induced mutants 27 were ad-5, 39 ad-7,
and 3 ad-5/7, indicating a degree of specificity
for the ad-5 type mutation by EMS.

Survival-mutation studies for buffered and
unbuffered EMS treatment indicate that while
the overall mutation rates of the two are compa-
rable with respect to survivors, EMS buffered
at pH 5.8 kills only 40 per cent over a period of
2 hr whereas unbuffered EMS in saline, in which
the pH falls from 7.0 to 2.8 over 2 hr, kills 80 per
cent.

5.14. The Pattern of Some Biochemical Mutants of
E. coli after X-rays and Cancerogenic Sub-
stances. A. Rosener and H. A. Kunkel
(Hamburg, Germany).

By means of methods published by Lederberg
and by Holliday it is possible to identify various
biochemical mutants of bacteria produced by
mutagenic agents and to analyse the distribution
of the different types. In our investigations

on the radioresistant strain E. coli B/r most of
the spontaneous mutations to auxotrophy are
serine/glycine-dependent. Other biochemical
mutants occur just sporadically. Nearly the
same distribution is observed after X-irradiation.
Further experiments were cairied out with several
nitrogen-compounds. These substances which
belong to the group of nitrosamines or nitro-
samides were proved to be highly cancerogenic
by Druckrey et al. Furthermore they are organ-
otropic, too, and produce malignant tumors in
different organs according to their chemical
structure. After application of these agents
a specific pattern of biochemical mutants is
observed which differs from the "spectrum"
obtained by X-irradiation or spontaneously in a
typical way. These results are discussed and
compared with the findings of similar experi-
ments which were carried out on Drosophila
in our laboratory, simultaneously.

5.15. Reversion Patterns among Genetic Sites in
Salmonella typhimurium on Treatment with
Chemical Mutagens. A. Eisenstark and
Ruth VanSickle (Manhattan, U.S.A.).

Initially, auxotrophic mutants of Salmonella
typhimurium LT2 were obtained by induction of
prototrophic cells with ultraviolet, X-rays,
neutrons, nitrous acid (NA), 2-aminopurine
(AP) and diethylsulfate (DES). The collection
also included a number of spontaneous mutants.
Approximately 400 of the single-site auxotrophic
mutants were examined for reversion to proto-
trophy following treatment with a series of
chemical mutagens. Among the observations
were: (1) the method of induction of forward
mutation influenced the reversion pattern in a
distinct manner; (2) base analogues failed to
revert over half of the NA mutants, indicating
that NA was not restricted to transition capa-
bility; (3) N-Methyl-Ni-nitro-N-nitrosoguani-
dine reverted the same sites as DES, but the two
showed distinct quantitative differences for
certain specific sites; (4) 4,5,6 triaminopyrimi-
dine-sulfate-hydrate was highly mutagenic, but
reverted a smaller number of sites than did AP;
(5) deletions were not observed when either AP
or DES were used to induce mutation in the
prototrophic strain; (6) reversion patterns were
independent of either lysogeny or the presence
of a second mutation within the strain tested;
(7) a large fraction of apparent reversions did
not involve restoration of true wild-type, indi-
cating possible mutation at a suppressor locus.
These could be easily scored as stable small-
colony-formers when plated on minimal medium.

60

Section 5 — Mutagenesis

A number of these suspected suppressor mutants
were tested, using transduction experiments, to
determine whether each was site-specific, locus-
specific, or non-specific.

5.16. Genetic Analysis of Adenine — 1 Revertants
in the Fission Yeast Schizosaccharomyces
pombe. C. H. Clarke (Edinburgh, Great
Britain)

Ultraviolet induced adenine — 1 auxotrophs
isolated by Prof. U. Leupold have been used in
reverse mutation studies with S. pombe. Adenine-
independent revertants of spontaneous, HNO2
and u.v.-induced origin have been obtained and
tested for the presence of suppressor mutations.
Revertants giving no adenine-requiring recom-
binants among 300-500 progeny ascospores,
after backcrossing to wild-type, are classified
as "reversions".

One adenine-1 mutant, number 40, gives
spontaneous revertants which are mainly of a
suppressor type, while most HNO2 and u.v.-
induced revertants of this same mutant are
"reversions". Another temperature-sensitive
mutant, adn.-l, 199, gives revertants spontane-
ously and with HNO2 treatment which are
mostly "reversions", u.v.-induced revertants of
mutant 199 are however predominantly of the
suppressor type. Furthermore the appearance of
HNG"2-induced "reversions" of mutant 199 is
only weakly, that of u.v.- induced suppressor
revertants strongly, inhibited by the addition of
L-methionine to the minimal medium upon
which adenine-independent colonies are scored.
These two differences between HNO2 and u.v.
induced revertants of the same mutant may be
related. The results indicate an unusual example
of mutagen specificity for individual alleles.

Full details and results will be submitted for
publication to Genetical Research in due course.

5.17. The Use of the Suppressors of Methionine 1 for
the Study of Mutation in Aspergillus nidulans.

Lorna J. Lilly (London, Great Britain).

During a survey of spontaneous reversion
rates from nutritional requirement to protrophy
in Aspergillus, it was found that the change from
methionine requirement to prototrophy oc-
curred with exceptionally high frequency (approx.
IO-5).

These reversions from methionine requirement
can be shown to be due to suppressor genes, all
those so far tested being unlinked to the methio-

ninei locus. The suppressors have various
visible (morphological) phenotypes ("A", "B",
"C", etc.). If each phenotype is determined by a
single cistron, these suppressors could be used for
the simultaneous visible scoring of forward
mutation in several cistrons.

The genetics of some of the phenotypes which
occur with highest frequency are being investi-
gated. The results of crossing one strain of type
"A" with one of type "B" indicate that each
strain carries a single suppressor and that these
suppressors are unlinked. Crosses between
strains of type "A" show that at least two
unlinked loci can independently determine
this phenotype. Crosses between the other
phenotypes are in progress and the suppressors
are being located.

If two unlinked loci control the "A" phenotype
this phenotype is not suitable for the visible
scoring of mutation in a single cistron. This
result may affect the interpretation of results
obtained by workers who are using this system
to study chemically induced mutation.

5.18. Kinetic Studies of Chemically Induced Reverse
Mutations in Neurospora crassa. H. G. Kol-
mark and B. J. Kilbey (Edinburgh, Great
Britain) and S. Kondo (Misima, Japan).

The experiments were designed to follow the
effects of chemical concentration and time of
treatment on mutation induction. Conidia
from the strain K 3/17 ad-3A, 387ol, were
treated with diepoxybutane (DEB), or ethyleno-
xide(EO) in aqueous solution. (1)The temperature
was kept at 21° or 22°C during treatment.
Treatments with DEB were carried out for
varied periods with concentrations varying
from 1.0 x 10-2 to 3.16 10"1 m. At the
higher concentrations of DEB it was found
that the frequency of adenine reverse mutations
among survivors was determined by the product
of time and concentration (the "dose"). After
a short initial period the frequency is approxi-
mately a linear function of (cone. time)2. This
result suggests that the induction of adenine
reversions by DEB has a two-event mechanism.
It seemed possible that this mechanism was due
to the existence of the two reactive epoxygroups
in the molecule of DEB. However, similar
experiments with EO, which has only one
epoxygroup, gave the same kinetics. It therefore
seems more likely that the basis for a two-event
mechanism resides in the induced genetic change.
The monofunctional compound, EO, is by far
the more efficient when similar doses of the two
compounds are compared. Mutation frequency

61

Section 5 — Mutagenesis

is independent of time ("dose rate") at the
higher concentrations. At the lowest concen-
trations used, there is a decreased response to
a given dose.

1. Consult: Kolmark and Kilbey, Z.f. Verer-
bungslehre 93, 356-365, 1962, regarding
techniques.

5.19. Mutation and Crossing-over Induced by Mono-
and Bifunctional Alkylating Agents. G. Mor-
purgo (Rome, Italy).

The ability of methyl bis ((3-chloroethylamine,
2-chlorotriethylamine hydrochloride, diepoxybu-
tane and ethylene oxide) in inducing mutation
and mitotic crossing-over in a diploid strain of
Aspergillus nidulans has been tested.

Mono- and bifunctional compounds are
equally active in inducing mutation. Only
bifunctional compounds are able to induce high
frequency of somatic crossing-over. These
effects on crossing-over can be attributed to the
cross-linkage of non sister chromatids.

5.20. Genetic Analyses of Photo-reactivation, Dark-
reactivation and Resistance to Ultraviolet Light
in Escherichia coli. P. van der Putte and
A. Rorsch (Rijswijk The Netherlands).

The genetic constitution of mutations in
Escherichia coli which affect the radiation
sensitivity of the strain has been studied. One
mutation leads to loss of the tendency to form
long filaments after u.v. irradiation, a second
to loss of the ability to repair u.v. lesions in the
DNA of the cell in the dark (dark-reactivation)
and a third mutation to loss of the ability
to repair u.v. lesions by subsequent irradiation
of the cell with visible light (photo-reactivation).

By conjugation experiments with auxotrophic
mutants it is proved that all three properties are
chromosomally determined and may be located
as distinct markers on the bacterial genome.
Methods to screen large numbers of recombi-
nants for the markers which affect the radiation
sensitvity of a strain will be described. The
results will be published in full in the Biochim.
Biophys. Acta.

5.21. Reactivation of U.V. Irradiated Escherichia coli
K-12 (A) by Treatment with EDTA. Renana

Ben-Gurion (Ness-Ziona, Israel).

When lysogenic bacteria are irradiated, the

prophage genome passes from a state of repres-
sion to an active state, phages are being pro-
duced, and the lysogenic bacteria are killed in
this process. It appears, therefore, that induction
(a process as yet not fully understood) renders the
lysogenic bacteria more sensitive to irradiation
than their nonlysogenic derivates. Because of the
higher sensitivity of lysogenic cells to irradiation,
these organisms could perhaps be suitable tools
for the study of irradiation effects.

When the lysogenic bacterium Escherichia coli
K-12 (A) (strain 169) was irradiated with ultra-
violet light and then transferred to a medium
containing EDTA, this resulted in a partial
reactivation of the lysogenic bacteria. The num-
ber of survivals was increased and the production
of phage decreased, as compared with the irra-
diated controls. The composition of the bacterial
growth medium, as well as that of the medium
to which the bacteria were transferred after
irradiation, had a marked effect both on the
number of surviving cells and on the reactivation
resulting from the treatment with EDTA.

5.22. X-ray and Ultraviolet Light Mutagenesis in a
Threonine Requiring Escherichia coli K12 stud-
ied by Recombination Techniques. T. Kada
and H. Marcovich (Paris, France).

During the mating process in E. coli K12, the
chromosome of the male cell (Hfr) is transferred
into the female (F ) without any detectable
amount of cytoplasmic material. This system, the-
refore, is a workable one for determining wheth-
er the initial step in X-ray or u.v. mutagenesis is
a cytoplasmic or nuclear one. This work deals
with crosses between E. coli Hfr thr~33leu+ sen-
sitive to T6 phage and E.coli F-thr_33leu- resistant
to T6 phage. Having irradiated the male before
mating with the female, we scored the reversion
to threonine independence among the recombi-
nants possessing the resistance to T6 and having
received in addition the leu+ character. The thr
and leu determinants are closely linked, and leu
serves here as a scoring factor which assures that
the thr region of the male chromosome has been
injected into the female. When the male is irra-
diated with X-rays, the mutant character can be
immediately transferred to the zygote. With
u.v. irradiation, no such immediate transfer is
seen. This transfer takes place only afterwards,
increasing with time up to 100 per cent in 24 hr
culture in broth. In reciprocal irradiation in-
volving the female, it appears that the male
genetic material is mutated if injected in a u.v.
irradiated female, but not if X-ray is used. These

62

Section 5 — Mutagenesis

results suggest that X-ray mutagenesis, in the
example studied here, is exerted directly on the
chromosome, and that u.v. mutagenesis involves
a rather long-lasting cytoplasmic step before the
mutant character is integrated in the chromos-
some.

5.23. The Effect of U.V.-Light and X-rays on the Varia-
bility of Bacillus licheniformis. F. Paleckova
(Prague, Czechoslovakia).

The relationship between irradiation dose,
killing effect and production variability of a
bacitracin-producing strain of B. licheniformis
was studied. The organism was exposed to u.v.-
light for 4, 8, 16, 32 sec; the survival curve was
sigmoidal. In experiments with X-ray treatment
doses of 200,000 r, 400,000 r, 600,000 r were used.
The survival curve was linear. The effect of these
mutagenic factors on the variability of the anti-
biotic-producing ability of the strain was studied
and submitted to statistical analysis.

5.24. A Region of the Chromosome of Escherichia coli
K12, abnormally Resistant to Ultraviolet.

F. Joset and H. Marcovich (Paris, France).

Studies dealing with the effects of radiations
on genetic recombination in E. coli K 12 have
shown that a region of the male chromosome is
abnormally resistant to ultraviolet and a radia-
tions. This region, located between the determi-
nants for Tryptophane and Histidine, represents
about one-fourth of the whole chromosome.

No genetic markers have yet been found in
this region, as compared to about 100 for the
rest of the chromosome; assuming a random
distribution of the markers this fact might have
a biological signifiance.

Two suggestions can be made which could
explain the resistance to u.v. and the absence of
genetic markers:

1. This region might be almost completely
empty of DNA. This hypothesis seems to be
ruled out by 32P labeling experiments, which
show a normal sensitivity to radio-phosphorus
per unit length. These experiments have shown
the existence of a second series of lesions in-
duced by 32P. These lesions can be transferred to
the female during a cross, and prevent the in-
tegration of the labelled material in the zygote
chromosome.

2. If not absent, the DNA of this region might
have an abnormal base composition. The mole-

cular basis of u.v. damage is thought to be mostly
a dimerization of thymine. As this region is
relatively insensitive to u.v., one may think that
thymine dimers are less likely to be formed,
either because of a low thymine content, or be-
cause of the absence of neighboring thymine-
molecules.

Experiments using base analogues such as 5-
bromouracil, are being done to test this possi-
bility.

5.25. The Effect of Temperature on the Response to
Ultraviolet of Neurospora crassa. B. J. Kilbey
(Currie, Great Britain).

The temperature of the conidial suspension
during ultraviolet irradiation has been found to
influence the mutagenic response of the strain
K 3/17 ad-3A 38701, inos 37401.W A dose of
ultraviolet administered at 3-4°C may produce
two to three times the number of adenine rever-
sions compared with a similar dose administered
at temperatures between 25°C and 30'C. Survival
and inositol reversions do not appear to be
greatly influenced by this difference in tempera-
ture.

The evidence at present available suggests that
a temperature sensitive recovery process is acting
to repair adenine reverse mutations. Interest is
centred on the specific nature of this process and
it is hoped that this may be clarified by the study
of other ad-3A mutants and inos mutants which
respond to ultraviolet light, and by an examina-
tion of the effect of varying the experimental
conditions.

1 . A full description of the characteristics of this
strain may be found in Zeit. fiir Vererbungs-
lehre 93, 356-365, 1962.

5.26. Genetic Studies of a Mutation to Radioresistance
induced by 32P decay in Saccharomyces cere-
visiae. E. Moustacchi, H. Hottinguer-de
Margerie, and H. Marcovich (Paris, France).

Different agents (32P decay, ultraviolet light,
mustard-gas, ionizing radiations) induce in hap-
loid Saccharomyces cerevisiae a mutation to-
wards resistance to the lethal action of ionizing
radiations. DNA content per cell of these resistant
strains show that they are still haploid. This is
confirmed by the fact after that sporulation of
diploid strains from a cross between a radio-
sensitive and a radioresistant strain the viability

63

Section 5— Mutagenesis

of the spores and the segregation of the mating
type are normal.

The tetrad analysis of these strains shows that
the resistant character segregates from the radio-
sensitive one with a ratio of 2: 2. In other words,
one genetic determinant is responsible for this
character.

Radiosensitivity (S+), which is the wild type, is
dominant over radioresistance (S), The diploid
cells (S+ S") are less resistant than the resistant
haploid parent (S~). Their survival curve is super-
imposed on that of the diploid wild type (S+ S+).

Different sites (three at least) are implicated in
this character; the survival curves of some S"S"
diploids are identical with that of the S+S+ and
the others are identical with that of the S ~; the
latter diploid cells are homoallelic and the
former are heteroallelic.

The size of the target is about l/20th of the
whole genome. This estimation is made through
a dose-effect relationship for the induction of
this mutation by 32P decay or by X-rays.

When the S~ character is introduced into a
cross, deviation from the 2: 2 segregation ratio
for different biochemical markers is observed:
there is a large excess of prototrophs. These
deviations may be accounted for by a 32P induced
mutation of a suppressor gene.

5.27. Determination of Radiation-induced Mutation
Rates of Recessive Lethal Alleles in Saccha-
romyces. Wolfgang Laskowski and Klaus
Haefner (Berlin, Germany).

After irradiation with small doses of u.v. and
X-rays diploid Saccharo?nyces-strams were iso-
lated, which proved by tetrad analysis to be
heterozygous for a single recessive lethal allele
( + /1). The radiation sensitivity of some of these
strains was compared with the sensitivity of the
diploid parent strains homozygous for the cor-
responding wild type alleles (+/+). From these
data the u.v. and X-ray induced rates for the
mutation of wild type alleles to the recessive
lethal condition ( + ->1) were calculated and
found to be several orders of magnitude larger
than mutation rates from prototrophic to auxo-
trophic condition.

Detailed data will be published in Z.fiir Ncitui-
forschung.

5.28. A Hereditary Propensity to Untimely Death in
Yeast and its Temporary Reversal by Radiation.

Allen P. James (Chalk River, Canada).

A respiratory-deficient strain of yeast is un-
stable when cultured on a medium usually con-
sidered optimal for growth. The instability of
these "vegetative petites" is characterized by the
production of moribund cells. Analyses involving
the study of individual cell lines have demon-
strated that clones derived from a single cell
frequently differ in their degree of instability. In
general, the rate at which moribund cells are
produced increases in successive generations;
extinction of the strain is prevented only by
occasional and temporary reversals of this trend.
Complete stability can be obtained either by
mutation or by culturing the strain on a minimal
medium. The tendency to extinction can also be
reversed temporarily by exposure of the cells to
ultraviolet light but not to X-rays. The result of
experiments designed to uncover the underlying
mechanism and influence of radiation are dis-
cussed.

5.29. The Mutagenic Activity of Ethyleneimino Deri-
vatives with Different Numbers of Reactive
Groups. H. LiJERS and G. Rohrborn (Berlin-
Dahlem, Germany).

We compared the mutagenic activity of tri-,
bi-, and monofunctional ethyleneimines for
studying the relation between the number of
reactive groups and the degree of mutagenic
action in this class. We also tested three of their
basal compounds without any groups of alky-
lating ability.

The tested compounds were as follows: tri-
functional: (A) tris-ethyleneimino-benzoquinone-
1,4, (B) tris-ethylene-thio-phosphor-amide; bi-
functional: (C) 2,5-di-n-propoxi-3,6-bis-ethyl-
eneimino-benzoquinone-1,4, (D) 2,5-bis-ethyl-
eneimino-benzoq uinone- 1 ,4 ; monofunctional :

(E) dipiperino-phosphoric-acid-ethyleneimine,

(F) 4-ethyleneimino-naphtoquinone-l,2, (G) 2-
ethyleneimino-5, 6, 7, 8-tetrahydro-naphto-
quinone-1,4; without ethyleneimino groups: (H)
p-quinone, (I) 1, 3, 5-triazine,(K)2,4,6-triamino-
1, 3, 5-triazine.

Using the Berlin wild stock of Drosophila me-
lanogaster the rates of sex-linked recessive lethals
have been determined by the Base method in
broods of three days after having fed or injected
the Berlin wild males with aqueous solutions of
the test substances. With respect to the mutagenic
effects on the postmeiotic stages of the sperma-
togenesis there are striking differences between
the different compounds. Trifunctional ethyl-
eneimines proved to be more mutagenic than
bifunctional ones, and these had a higher effi-
ciency than the monofunctional ethyleneimines.

64

Section 5 — Mutagenesis

The differences between these three groups are
significant. The three basal compounds did not
show any distinct mutagenic effect. Moreover, we
got differences between the mutation rates of the
postmeiotic stages within single groups. So A
proved to be more mutagenic than B, and E more
than F and G. All ethyleneimines also induced
mutations in the meiotic stages of the spermato-
genesis. In premeiotic stages a mutagenic efficacy
could be established for A, C, and D, but not for
E, H, and K.

5.30. Somatic and Gonadal Mosaicism in D. melano-
gaster. E. A. Carlson and J. L. Southin (Los

Angeles, U.S.A.).

Simultaneous experiments in chemical muta-
genesis using the specific visible dumpy (2, 13.0)
and using sex-linked lethals have demonstrated
a considerable degree of mosaicism for both
approaches. The two methods are not directly
comparable because only gonadal tissue is as-
sayed in the sex-linked lethal test, while both
somatic and gonadal tissue may be assayed for
the specific visible test. The use of brood analysis
(postmeiotic and premeiotic sperm) for both
assay systems indicates a shift from postmeiotic
mosaically occurring mutant individudals to
premeiotic "complete" (non-fractional) mutant
individuals. This is expressed by modification of
the premeiotic F2:F3 sex-linked lethal frequency
and by the presence of both somatic and gonadal
complete mutation in most of the premeiotic
dumpy mutants. These studies have enabled us
to calculate the fraction of a gonad carrying the
mutant tissue, the number of individuals carrying
mosaic sex-linked lethals, and the total mutation
frequency for agents inducing mosaicism. A
theory which predicts the ratio of F2:F3 sex-
linked lethals from the analysis of the mutant
classes in a specific visible test has also been
inferred from the data presently available.

Sex-linked lethal experiments have demon-
strated an F2 (gonadal complete) mutation fre-
quency of 11.3 per cent and an F3 (from F2
gonadal mosaic) mutation frequency of 3.8 per
cent. The number of F2 gonadal mosaics in the
total F2 females sampled is 9.21 per cent. The
portion of gonad containing sex-linked lethals is
44 per cent. It can be shown from our data that
the cells forming the polar cap are non-randomly
selected and probably do not consist of more
than four cells in most instances. The F2:F3 mu-
tation frequency for 1-4 day sperm is 1.8; for
5-6 days 1.1 : for 7-9 days 4.1 ; for 10-11 days 7.9;
for 12-14 days 24.8 and for 15-18 days 13.8. The
mean F2:F3 sex-linked lethal frequency for all

broods is 3.0. These results are corrected for
spontaneous sex-linked lethal frequencies. The
mutagen used for this study is monofunctional
quinacrine mustard (I.C.R.-100).

5.31. The Mutagenic Activity of Formaldehyde-
treated Ribonucleic Acid in Drosophila mela-
nogaster. Thomas Alderson (Cambridge,
Great Britain).

In experiments using a chemically-defined
axenic treatment medium, it has been shown that
formaldehyde exhibits no mutagenic activity
towards Drosophila melanogaster larvae unless
ribonucleid acid is present in the treatment
medium. Further analysis of this phenomenon
has shown that adenylic acid is the active
component of ribonucleic acid uniquely con-
cerned. The presence of adenylate in the treat-
ment medium is apparently necessary for the
mediation of the mutagenic activity of formal-
dehyde, whether adenylate is present as the free
mononucleotide or bound in ribonucleic acid
polynucleotide. An in vitro reaction product of
formaldehyde with adenylate appears to be the
effective mutagenic alkylation.

Since formaldehyde-induced mutagenesis has
not previously been attained in the absence of
free formaldehyde, experiments have been
carried out to determine whether the necessary
formation of alkylated-adenylate is itself a
sufficient condition for the mediation of the
mutagenic activity of formaldehyde. Ribonucleic
acid treated with formaldehyde under conditions
used in mutation tests and dialysed free of
unbound formaldehyde, was found to be decided-
ly mutagenic towards Drosophila larvae. Ribo-
nucleic acid similarly handled, but not treated
with formaldehyde, showed no mutagenic
activity. No difference is observed between the
two series in the developmental time of the
larvae or in the emergence of imagines. It would
thus appear that the in vitro alkylation of
adenylic acid by formaldehyde is both a neces-
sary and sufficient condition for the mediation
of the mutagenic activity of formaldehyde.

5.32. The Elimination of a Specific Chromosome
Segment by Nucleic Acids in Drosophila
melanogaster. O. G. Fahmy and Myrtle
J. Fahmy (London, Great Britain).

Macromolecules (nucleic acids, proteins and
ionic polymers) were shown to be mutagenic as
regards the induction of small chromosome
deletions (Minutes). Three grades of phenotypic

65

Section 5 — Mutagenesis

expression were distinguished: Minute - slight,
M(s), with only the macrochaetes reduced in
length and thickness; Minute- intermediate, M(i)
with more extreme bristle effect, accompanied
by some abnormality of wings, eyes or body;
Minute - extrem, M(e), highly pleiotropic with
several external features grossly affected. Radi-
ation induced Minutes showed all grades of
expression with a preponderance of the M(i)
class, whereas those of macromolecules were
either M(s) or M(e), with the M(i) class virtually
absent.

A correlation was established between the
phenotypic expression of the Minute and the
genetic position of the chromosome deletion
causing it. In a sample of 165 M(e) mutations
induced by various macromolecules all but 3
(which werenearer the M(i) class) were on the
IVth chromosome. The deletion involved the
proximal 4/5th of the salivary gland IVth
chromosome, and covered the majority of its
marker genes, including the most proximal:
sparkling-poliert (spaP°l.) and eyeless (ey). In
contrast, of the 34 M(s) mutations studied, 33
were on the large autosomes (Ilnd or Illrd).
The proportion of the M (e) class among the
Minutes induced by various macromolecules
was different, reaching a maximum of 95 per
cent with DNP and a minimum of 29 per cent
with polymethacrylic acid. This indicates a
differential distribution of the chromosome
deletions with the various macromolecules.

The frequency of induction of IVth chromo-
some deletions with DNA was compared with
that occuring with radiation. Among 47 fertile
Minutes induced by DNA, 38 were on the IVth
chromosome (81 per cent) and all covered
spaP°K ; whereas with 46 X-ray Minutes recovered
in a comparable experiment only 13 occurred on
the IVth (28 per cent), 12 of which covered
spa?01. These two distributions are very sig-
nificantly different (x2 [i] =26.0, P<10"6)
indicating a high degree of selective gene elimi-
nation with DNA.

5.33. The Genetic Effects of Labeled DNA Precursors.

W. D. Kaplan, H. D. Gugler and K. K. Kidd
(Duarte, U.S.A.).

Sex-linked recessive lethals have been induced
in Drosophila males by feeding tritiated thymi-
dine to larvae. In three experiments feeding was
restricted to 8 hr to limit the incorporation of
the isotope. The most heavily labeled germ
cells appeared in the brood sperm utilized for
the first of three 3-days broods tested for induced
lethals. In one experiment, during which larvae

were permitted to feed on labeled food through-
out larval life a higher frequency of labeled
sperm bundles was obtained and also heavier
labeling of individual sperm.

Induced mutation rates reflected the degree
of incorporation, and mutations were restricted
to those broods with labeled sperm.

The physical characteristics of the beta e-
missions of tritium - - low energy and short
mean path — led us to test the distribution of
the induced mutations. The pattern is non-
random and differs significantly from those
obtained by the use of X-irradiation. The region
of the X-chromosome from 1 to 20 is relatively
free of mutations, whereas the regions between
30-35, 50-55, and 60-65 have a higher number
of induced lethals than would be expected at
random.

Mutations have also been induced by tritiated
deoxycytidine. These mutants will be localized
and the distribution compared to the one
observed with H3 thymidine. If the pattern of
induced mutations reflects the varying fre-
quencies of the pyrimidine bases along the
length of the chromosome, the pattern induced
by H3 deoxycytidine should differ from that
observed with the use of H3 thymidine.

5.34. Incidence of Mutations in Drosophila melano-
gaster raised from Flies fed on Irradiated
Medium. S. Nirula, M. S. Swaminathan,
A. T. Natarajan and R. P. Sharma (New
Delhi, India).

Earlier studies at the Indian Agricultural
Research Institute on the effects of culture
media, potato mash and fruit juices irradiated
with ionizing radiations on mitosis in root
meristems of barley, Vicia faba and onion
grown on them have revealed that such irradiated
products may have radio-mimetic effects. In
view of the obvious bearing of these data on
assessing the wholesomeness of food material
sterilised through ionizing radiations, a study
was initiated in Drosophila melanogaster to
ascertain whether the mutational load is en-
hanced in flies fed with irradiated food.

The basic medium consisting of glucose agar
and yeast is irradiated with 150 k rad of gamma-
rays (sterilizing dosage) at a 160 curie Co60
source. Young Oregon-K flies (soon after
emergence) are fed on this irradiated medium.
The flies are allowed to breed and parent flies
are killed about 7 days later. The male flies
which emerge out of the larvae fed exclusively
with irradiated food are then used for conducting
M-5 tests on normal culture medium for further

66

Section 5 — Mutagenesis

F1 and F2 breeding. The experiments have been
repeated thrice.

Sex-linked recessive lethals have been found
only in the families derived from the irradiated
medium-series (6.44 per cent). A wide range of
phenotypic changes was a striking feature of
the F2 families from the irradiated medium
cultures (their frequency ranging from 0.13-
0.37 per cent against 0.018-0.095 per cent in
control on population basis). Some of the
mutant flies, viz. curly wings, yellow body, and
another with dominant wing mutation were
found to breed true. A few changes like half
thorax, rotated abdomen and absence of neck
belong to the non-inherited group of abnormali-
ties of Morgan (Bridges and Sturtevant 1925) they
were observed only in the irradiated medium
series. The implications of these studies will be
discussed.

5.35. Experiments with Chemical Compounds which
Reduce or Increase Mutagenic Effects of
Ionizing Radiations. H. A. Kunkel and
A. Rosener (Hamburg, Germany).

This paper deals with investigations on three
chemical compounds well known as radiopro-
tecting agents with regard to somatic radiation
damage: cysteine, aminoethylisothiuronium
(AET) and 5-hydroxytryptamine (serotonin).
Experiments were carried out on Drosop/iila and
on E. coli. In Drosophila the rate of ray-induced
sex-linked recessive lethals was tested after X-
irradiation by means of the Muller-5-Method.
In the experiments on E. coli the rate of back-
mutations was observed of a leucine-dependent
and of a streptomycine-dependent strain to
prototrophy. Cysteine considerably reduced the
X-ray induced back-mutations in E. coli. No
influence, however, was observed in Drosophila.
Serotonin reduced the rate of mutations in
Drosophila as well as in E. coli. Protection,
however, was only small. After AET the ray-
induced rate of mutations significantly increased
although this compound per se has no mutagenic
effect. Lastly experiments are reported with
bromodeoxyuridine which is incorporated into
DNA instead of thymidine and provides in-
creased radiosensitivity in some cases.

mutation process and in the investigation of the
essence of the natural biochemical protection
of the cell.

The class of antimutagenic substances consists
so far of very few compounds. The need for
new antimutagens is great. For the solution of
this problem we have founded our investigations
on the following principles:

1. The search for new antimutagens in the
group of substances of universal protection,
i.e. protecting the cell from possibly many various
effects. Kihlman has shown that streptomycin
protects against ultraviolet and y-rays and
a-particles. Our experiments with streptomycin
have shown that it is an antimutagen.

2. Searches in the group of substances which,
being protective against radiation, are at the
same time a natural and an active cellular
metabolite. The aminoacid arginine, responding
to these requirements, was also found to be an
antimutagenic compound.

The antimutagenic effect of streptomycin,
cysteamine and arginine has been proven on
onion roots and broad beans, which had been
protected by these substances from the occurrence
of spontaneous chromosome rearrangements.
The cytological analysis of the antimutagenic
effect of the compounds studied has shown that
it is connected with a decrease in the frequency
of primary breaks and not with the processes
of the re-union of fragments. In experiments
with recessive sex-linked lethal mutations we
have shown that streptomycin produces an
antimutagenic effect upon the occurrence of
spontaneous gene mutations.

Depending on the concentration of the anti-
mutagenic compound it may, in optimum
concentrations, produce an antimutagenic effect,
in high doses it may prove to be mutagenic, in
average and minimum concentrations it may be
neutral.

As a rule, in certain concentrations the antimu-
tagens acquire properties of substances which
are able to protect the chromosomes against the
effect of ionizing radiations. It is shown that the
character of this protection is different for
different phases of the cellular cycle.

Of great importance in the problem of muta-
gensis is the analysis of their differential effect
on various genes.

5.36. The Problem of Antimutagens in Connection
with Mutagenesis and Chemical Protection.

N. P. Dubinin (Moscow, U.S.S.R.).

The problem of antimutagens plays a
paramount role in the control of the spontaneous

5.37. Methods for Estimating Differential Radio-
sensitivity. 1. 1. Oster and E. Pooley (Philadel-
phia, U.S.A.).

Although marked differences in the sensitivity
of the various stages of germ cell development

67

Section 5 — Mutagenesis

to ionizing radiation have been recognized,
conflicting estimates of the relative amounts of
damage have been reported by some investi-
gators. Fortunately, it is becoming in-
creasingly apparent that such discrepancies
are in large measure, if not entirely, due to the
siphoning off of cell samples representing
different stages of development (and therefore
different intrinsic radiosensitivities) at the time
of treatment. Not only would such mixtures
result in false estimates of the relative radio-
sensitivity of the various stages but it is known
that many external and/or internal factors 1 are
capable of altering the degree of heterogeneity
of any particular sample of cells which is being
analyzed. The extent to which this occurs is
often exceedingly variable even within the same
experiment.

By utilizing a combination of techniques we
have been able to avoid these difficulties and
have made cytogenetical analyses of cells repre-
senting homogeneous stages of development at
the time of treatment. These methods include
the use of our so-called multi-purpose stocks
(i.e. strains which allow for the detection of
several different types of genetical damage in the
offspring of the same treated individual), his-
tological examination of the germ tract of the
treated individuals, and a sampling technique
that involves the treatment of immature stages
which contain a preponderance of germ cells in
a similar stage of development. The clear-cut
pattern of germ cell radiosensitivity as regards
various types of genetical damage which has
emerged for Drosophila is in general agreement
with that obtained more recently for the mouse
by Dr. L. B. Russell.

This parallelism suggests that the mecha-
nism(s) underlying differential radiosensitivity
may be quite widespread.

In addition to a comprehensive report of the
results obtained with radiation, comparisons
will be made with the data obtained by treating
the reproductive cells with chemical mutagens.

This work has been supported by grant
AT (30-l)-2618 from the U.S. Atomic Energy
Commission.

1. E.g. C. Auerbach, Sensitivity of the Droso-
phila testis to the mutagenic action of X-rays.
Z. indakt. Abstamm.- u. Vererb.-Lehre 86,
113 (1954).

5.38. The Rates of Visible Mutations in Sequential
Broods of Irradiated Drosophila melanogaster
Males. Aloha Hannah-Alava (Turku, Fin-
land).

The brood pattern of recovered mutations was
determined for three different types of visibles-
dominants and Minutes in all chromosomes
and recessives at specific third-chromosome
loci --in the progeny of D. melanogaster males
of two genotypes: wild-type, Oregon-RS, males
(Exp. I) and males heterozygous for 12 third-
chromosome recessive markers (Exp. II), mated
singly and sequentially to females of an appropri-
ate genotype for detecting mutations at specific
loci and/or crossovers. The males in Exp. I
were 2-24 hr of age and in Exp. II 24-48 hr of
age at the time of treatment (3000r hard X-rays)
and initial mating. The sequential broods
derived from these males, mated up to 24 days,
totaled 94,081 and 42,951 Fi offspring for the
irradiated and control series respectively.

Except for the first brood (1-3 days) in which
there was a lower incidence of mutations in the
progeny of the older males than the younger
males, the curves of induced mutational response
were not significantly different in the two
experiments. In the broods following the period
of excessive sterility (6-8 days) 113 mutations
were recovered from 46,922 offspring of the
irradiated males. The slopes of the curves in the
premeiotic broods (11-24 days) suggest that one
of the consequences of radiation of the early
gonial stages is elimination of the germ cells
with potential visible mutations to a greater
extent than elimination of germ cells with either
induced crossovers or recessive lethals with no
visible phenotype.

Research supported by IAEA contract No. 31
and USAEC contract No. AT (30-1) 2690.

5.39. The Induction of XO Males, by Irradiation
of Drosophila. melanogaster Males. B. Leigh

(Leyden, the Netherlands).

It has been found that, after the irradiation of
adult males, there is a difference in the sperm
samples which show the highest frequencies of
lethals and translocations, and those which
show the highest frequency of XO males. A set
of experiments were carried out to investigate
this discrepancy. Twenty-four-hour old males, of
the genetic constitution Xc2yB; sc8 Y, were
treated with doses from O-3000 r. A brood
pattern of five 2-day broods was used, with six
females per male per brood. The Fi was scored
for XO males, non-disjunction females, and

68

Section 5 — Mutagenesis

sex ratio. The highest frequency of XO males
was found in sperm sampled on the 7-8th days
after irradiation, presumably corresponding to
young spermatocytes, and this was not accompa-
nied by any comparable increase in the number of
non-disjunction females. From the structure of
the ring-X it is expected that most breaks will
result in loss of the chromosome. This is sup-
ported by the fact that the same dose of irradia-
tion produces more XO males from the ring-X
than from a rod-X. The dose effect curve for the
production of XO males is a straight line, one-
hit event. A comparison of this dose effect
curve with that of the change in sex ratio gives
further evidence that XO males are mainly
produced by loss of the X chromosome. A
hypothesis to explain the different peaks for
the two types of breakage-involving effects, XO
males and translocations, will be discussed.

Sponsored by the Institute for Radiopathology
and Radiation Protection.

5.40. The Brood Pattern of X-ray Induced Crossing-
overs in Drosophila melanogaster Males.

Jaakko Puro (Turku, Finland).

sperm production to the control level by the
12th day. Furthermore it appears that the high
incidence of crossovers, on the 10th day, is the
result of induction of crossovers in definitive
(secondary) gonia.

Research supported by IAEA contract No. 31
and USAEC contract No. AT(30-1) 2690.

5.41. An Analysis of X-ray induced Non-disjunction in
Drosophila. A. J. Bateman and Ann C. Chand-
ley (Manchester, Great Britain).

It is common to distinguish between two forms
of meiotic non-disjunction: at first and second
division respectively. This is a gross simplifi-
cation. There are many possible mechanisms re-
sulting in non-disjunction. Some of them are
recognisable by their genetic effects. An analysis
of matroclinous females in Drosophila, produced
by X-rays, has been made in an attempt to deter-
mine which modes of non-disjunction are, in fact,
operating in their production. Though the
results are ambiguous, some definite conclusions
can be drawn.

The pattern of induced crossing-overs in
Drosophila melanogaster males was studied using
a hybrid stock of 12 recessive markers in the
third-chromosome. Adult males, after treatment
with 3000 r X-rays, were mated singly and
sequentially (at 2- or 3- day intervals for the
first three broods and daily from the 7th to the
24th day) to females of an appropriate genotype
for detecting crossovers. Fertility and fecundity
of the treated males, after decreasing to the
lowest level on day 8, had a marked recovery
on day 10 followed by a small depression on
days 11 and 12; no differences between the
experimentals and controls were found for any
of the rest of the broods. The number of tested
Fi offspring totaled 51,964 from 46 treated and
25,778 from 1 1 control males.

With rare exceptions, proven crossovers were
first detected on the 9th day but the highest
incidence was on the 10th day after treatment.
Clusters of crossovers, many of which continued
in several broods, began on the 11th or 12th day.

The evidence from the continuing crossover-
clusters substantiates the hypothesis proposed
by the author (1962) from clusters of recessive
third-chromosome lethals, that the treated pre-
definitive gonia — on an average five in number
per male, basing the estimation upon the size of
clusters — are responsible for the recovery of

5.42. Dose-dependence of X-ray Induced Non-dis-
junction in Drosophila melanogaster Females.

Ann C. Chandley and A. J. Bateman (Man-
chester, Great Britain).

The frequency of induced non-disjunction in
female germ cells has been estimated on eleven
successive days following irradiation with X-
rays. Doses of 1 , 4, 6 and 8 k rad have been used
and the results indicate a non-linear dose-de-
pendence both for female (XXY) and male (XO)
exceptions.

Over the first six days, the frequency appears
to increase with the square of the dose. In spite
of the frequency of male exceptions being 4-5
times as great as that of the female exceptions,
the shapes of the dose-dependence curves are
closely similar.

After day 6, when the female and male ex-
ceptions have equal incidences, the dose-depend-
ence departs even further from linearity.

5.43. Induction of Mutations and Cell Killing in
Irradiated Drosophila Spermatogonia. Per Of-

tedal (Montebello, Norway).

Treatments of Drosophila spermatogonia with

69

Section 5 — Mutagenesis

acute doses of 55r, HOr, 160rand 3 lOr gave a clear-
ly non-linear dose-effect curve for sex-linked re-
cessive lethals. In order to explain the results, a
mathematical model for the irradiated cell pop-
ulation has been formulated, considering cell
killing, variation in sensitivity to killing and to
mutation induction with cell cycle stage, and
length of cell cycle. Thus, the frequency of mu-
tations observed will be equal to the product of:
dose absorbed by sensitive cells number of
sensitive cells : sensitivity of sensitive cells
survival of sensitive cells, divided by: total
number of cells in population less killed sensi-
tive cells. In this first approximation, effects on
resistant cells have been disregarded. According
to this model, protraction of doses in this dose
range should lead to higher yields of mutants.
Protraction of 144 r, 267 r, and 542 r over 8 hr
(four spermatogonia! cell cycles in the irradiated
embryo), have given results in support of the
hypothetical model, showing a linear increase in
mutation rate with dose, with a slope higher than
2 10-5 sex-linked lethals per r, indicating a sen-
sitivity at least as high as for mature sperm, for
doses lower than a few hundred r.

The work has been supported by The Nor-
wegian Cancer Society.

5.44. Modification of the X-ray induced Rate of Sex-
linked Lethals by Nitrogen Post-treatment and
Fractionation of the Dose in Drosophila melano-
gaster. A. D. TATES(Leyden, The Netherlands).

Male flies carrying a ring X-chromosome, were
exposed to a flow of purified nitrogen for 25 min,
immediately after having received a dose of
1450 r (55 r/sec). Results of five independent ex-
periments show a significant increase of mutation
frequency due to post-treatment in spermatids
and late spermatocytes, 9.5 per cent lethals (3744
tested chromosomes) were found after irradiation
without post-treatment and 10.8 per cent lethals
(5190 chromosomes) with N2 post-treatment;
normal deviate = 2.21 and/> = 0.0139. A possible
interpretation of the results is that N2 post-
treatment blocks repair of pre-mutational dam-
age.

In two experiments a third group of flies re-
ceived N2 post-treatment after a delay of 25 min.
A comparison of the results of delayed versus
direct post-treatment shows a significant de-
crease of the mutation rate in the group with
delayed post-treatment. The percentage for the
direct treatment is 9.54 per cent (2222 chrom.)
and for the delayed treatment 7.15 per cent (2545

chrom.); normal deviate 2.538 and P = 0.0055.
This result suggests that in young spermatids and
late spermatocytes repair, under these experi-
mental conditions, is completed within 25 min.
Five experiments were carried out to investi-
gate whether dose fractionation favours repair.
The mutagenic effects of a dose of 1350 r given
in five equal fractions, seperated by two hour-
intervals, were compared with that of unfractit
onated irradiation. The results show a significan-
decrease of mutation frequency due to fraction-
ation, in the same germ cell stages where N2_
post-treatment had been effective. Percentages
of lethals for the fractionated and unfractionated
groups are 8.63 per cent (4994 chrom.) and 9.77
per cent (4676 chrom.) respectively (normal
deviate 3.01 and P = 0.0013).

5.45. Oxygen Effects and Dose Fractionation in the
Developing Germ Cells of Drosophila virilis.

Mary L. Alexander (Austin. U.S.A.).

One-day-old males of D. virilis were treated
with lOOOr of X-rays while under 74.7 lb pressure
of argon gas and in a gaseous atmosphere of
45 lb argon + 19.7 lb oxygen. X-rays were given
at a rate of lOOOr/min as a single dose and as two
doses fractionated by a period of 40 min. Ad-
ditional tests were made with 2000r under 64.7 lb
of argon. Postmeiotic, meiotic and premeiotic
germ cells were tested for induced biological
damage.

Dominant lethals in postmeiotic sperm and
spermatid cells were increased by the presence of
oxygen. In sperm and spermatids, there were no
differences in the percentages of dominant lethals
or translocations with dose rate changes in argon
or when oxygen was present. These results
differed from those observed for D. melanogaster
where dose fractionation in argon, but not oxy-
gen, increased translocations, sexlinked lethals
and dominant lethals in spermatids. The pre-
meiotic cells of virilis, as melanogaster, showed
an enhancement in dominant lethals with dose
fractionation in both anoxic and oxygenated
gases. Enhancement with dose fractionation in
virilis appears to be limited to later sperma-
togonia! stages. The stem cells do not respond to
dose fractionation.

Enhancement of lethals in premeiotic cells has
been observed in anoxic and oxygenated atmos-
pheres of gases in both species. Differences in
response of spermatid cells in virilis and melano-
gaster may be due to oxygen metabolism or
other metabolic differences in the two species.
Speimatogenic cysts are not prevalent in young
males of either species and the differences cannot

70

Section 5 — Mutagenesis

be explained by mixed populations of resistant
and sensitive spermatid cells.

Work supported, in part, by Contract No. AT-
(40-l)-3014 with the United States Atomic
Energy Commission.

5 .46. Effect of Penicillin Feeding on the Reduction of
Radiation Induced Mutation Rate in Drosophila
melanogaster. Toshio Shiomi (Chiba, Japan).

It has been reported by Burdette (1961) that
D. melanogaster, grown on medium containing
penicillin, exhibits a diminished X-ray induced
mutation rate. The present work reports the
following results: a wild type D. melanogaster,
Canton-S, was grown in a culture medium con-
taining penicillin G (20,000 units per ml); 24 hr
old virgin males hatching from these cultures
were irradiated with X-rays. The induced sex-
linked recessive lethal mutations in mature
sperms were tested by the Muller-5 method.
X-ray doses used were 500, 1000, 2000, and 3000
r.

(a) The radiation induced mutation rates in
penicillin-fed Drosophila are found to be about
one-half of those for flies grown in normal
culture conditions.

(b) The radiation dose-effect relationship re-
mains linear.

(c) Penicillin containing medium lengthens the
growth period of flies by nearly one day as com-
pared to the normal culture, and the rate of
emergence is higher than in the control.

(d) The feeding of penicillin during the first
half of the larval stage is more effective in re-
ducing the induced mutation rate than when fed
during the second half.

The interpretation of this action of penicillin
on the radiation induced mutations, especially
its effect in the early stage of feeding, is difficult.
One might assume that the metabolic patterns of
the larvae are affected by penicillin or its deriv-
atives, and the genie material itself or its sur-
roundings are changed to more radioresistant or
easily recoverable conditions. Whether the effect
is caused by penicillin itself or by its degraded
forms remains for future work.

5.47. X-ray Induced "Dominant Lethals" in Insemi-
nated Eggs of Drosophila. (a) Experiments in the
stage between completion of meiosis and be-
ginning of cleavage. H. Ulrich (Zurich, Swit-
zerland).

In insects, pre-adult mortality of the progeny
of irradiated parents is thought to result from
radiation induced "dominant lethals" in the
genome of the maternal or paternal gametes.
These "dominant lethals" can be either gene or
chromosome mutations. Similar radiation effects
(e.g. embryonic mortality) are found if instead of
the gametes the progeny (e.g. young embryos) is
irradiated. This raises the question whether in
both types of experiments pre-adult mortality is
the result of "dominant lethals" (i.e. alterations
in the genome) and/or "physiological effects"
(i.e. effects in cell components others than
chromosomes).

During the last 10 years we have accumulated
quite a number of experimental results on in-
seminated Drosophila eggs. X-rayed after depo-
sition in the stage between completion of meiosis
and beginning of first cleavage. Most data (e.g.
differential radiosensitivity of cell parts with or
without a nucleus, the one hit dose action curves
for mortality and recessive lethals, influence of
oxygen during irradiation, etc.) were consistent
with the assumption that the most important
radiation effects are alterations in the genome
which behave like dominant lethals (probably of
a one hit type predominantly). However, new
results (experiments with cold post treatment and
the analysis of radiosensitivity of eggs and em-
bryos with different chromosomal constitutions)
indicate that, at least some of the radiation effects
have to be considered as "physiological effects".

5.48. X-ray Induced "Dominant Lethals" in Insemi-
nated Eggs of Drosophila. (b) Experiments with
different stages between insemination and end of
second cleavage division. F. E. Wurgler
(Zurich, Switzerland).

The nature of X-ray induced mortality in in-
seminated Drosophila eggs has been studied by
analysing the radiosensitivity in different stages
of meiosis and early cleavage. The highest sen-
sitivity was found during late anaphase/early
telophase of meiosis II and each cleavage division
analysed so far. Sensitivity was lowest when the
nuclei were in interphase.

Dose action curves for the different stages vary
greatly in shape and slope. The one hit curve
found for egg samples containing all stages
between completion of meiosis and beginning of
cleavage (see Ulrich, preceding report) could be
shown to result from superposition of different
non linear dose effect curves. This result invali-
dates the strongest argument supporting the
hypothesis that radiation induced mortality is
of a one hit type.

71

Section 5 — Mutagenesis

It has been shown by various authors that in-
sect embryos developing from irradiated gametes
can die at different stages of development. In
Habrobracon and Drosophila different lethal
syndromes seem to result from different kinds of
nuclear damage as shown by Von Borstel (1961).

Different lethal syndromes can also be distin-
guished after irradiation of \r\sem\naXtdDrosophila
eggs. The dependence of the relative frequencies
of different syndromes after irradiation in specific
stages of nuclear divisions and the dose depend-
ence of the different syndromes have been re-
corded. The results indicate that radiation induced
genetic effects alone can hardly explain all the
facts.

5.49. The Restitution of Radiation Induced Breaks
in Structural Heterozygotes of Diosophila.

E. Goldschmidt, E. Barak, N. Bernstein and
R. Falk (Jerusalem, Israel).

Males of Drosophila melanogaster carrying the
Cy (II L) inversion and their structurally homo-
zygous brothers were given an X-ray dose of
5000 r. The salivary glands were scanned in
larval offspring of both groups of males, and all
breaks induced in euchromatic sections of the
entire chromosome set were located. The rate of
induced aberrations was higher in the Fi pro-
duced by Cy fathers.

The hatchability of eggs fertilized by irradiated
males of both groups was studied in order to
compare the brood patterns and the overall rates
of "dominant lethals" induced during the pre-
meiotic stages up to the first meiotic anaphase.

It has been suggested that the loop configu-
ration in the heterozygous bivalent facilitates
non-homologous contact and thus promotes
abnormal restitution. A more comprehensive
hypothesis proposes that each of two perfectly
synapsed homologues provides a "splint" for the
normal restitution of its partner. Hence asynapsis
per se will interfere with the normal repair of
breaks. On this assumption the structural heter-
ozygosity of one bivalent, promoting asynapsis
in non-homologous elements, may be responsible
for the increased production of abnormal resti-
tutions in the entire chromosome set.

5.50. Recessive Lethals in the IV Chromosome of
Drosophila melanogaster. H. J. Belitz (Berlin,
Germany).

After X-ray treatment of ey2/ey2 males of
Drosophila melanogaster with doses of 3, 4.5 and

6 kr respectively 30 chromosomes with recessive
lethals and 1 1 with semilethals were found. In
allelic tests these 41 mutated chromosomes were
outcrossed with each other. Two chromosomes
contained two lethals each. Among the 43 mu-
tations no more than 25 occupy one locus; the
others are small deficiencies: 12 at least comprise
two loci, 4 each three loci, and 2 four and sixteen
respectively. By aid of these overlapping defi-
ciencies a part of this chromosome could be
mapped over a range of about 20 loci including
the locus of ci. Among the induced lethals two
clusters were found : one consisted of 5 mutations
induced in a spermatogonium and occupying
probably one locus; the other was composed of
two lethals, which gave a positive result in the
allelic test, but proved to be overlapping small
deficiences including the locus of ci.

5.51. Crossing-over in Males of Dorsophila Induced
by Radio Frequency Treatment. George
H. Mickey (Ridgefield U.S.A.).

Although crossing over normally does not
occur in the male germ cells of Drosophila, reports
in the literature indicate that this phenomenon
has been induced by treatment of male flies with
high temperature, X-rays, gamma-rays, ultra-
violet rays, formaldehyde and nitrogen mustard.
Our experiments yielded significant numbers of
recombinants in the progeny of heterozygous
(3ple) male flies treated with radio frequency of
20 megacycles. Recombinants frequently oc-
curred in clusters and usually without comple-
mentary classes. The distribution of exchange
regions was nonrandom. The induced crossovers
appear to have occurred in spermatogonial cells
rather than in spermatocytes.

5.52. Different Types of Mortality Including Prolon-
gation of Female Lifetime after X-raying
Drosophila melanogaster Imagines. H. Nothel
(Berlin, Germany).

The influence of X-rays on the lifespan of
Drosophila melanogaster imagoes has been
studied by application of 17 different doses
(100 KV, 0-125 kr) to 1-2 days old males and
females of the Berlin wild stock.

A prolongation of female lifespan is induced by
doses >4 kr, it culminates at 11 kr in 150 per
cent of the mean survival time of unirradiated
females. The prolongation is not accompanied
by alterations in locomotion or copulation of
the females concerned and is independent from

72

Section 5 — Mutagenesis

radiation effects in the males mated with them.
Measurements of fecundity have proved the
prolongation to be correlated with the radiation-
induced sterilization of the females. This has
been confirmed furthermore by investigations
with unirradiated virgin females having a low
fecundity, with females of a special strain
showing a high radiation resistance with regard
to fecundity, and with chemically (by TEM)
sterilized females.

In the males mean survival-time, Tso, Tioo, and
the time of the highest mortality decrease
linearly when plotted logarithmically against a
raster of linearly increasing doses. The females
show in the same raster corresponding curves,
except a sharp increase caused by the prolon-
gation of lifespan. This makes the LD50 of
females (96 kr) twice that of males. The linearity
of these curves is interpreted with an always
identical type of damage. In both sexes the
normal distribution of the mortality rate over
the days after irradiation is altered at doses [>90
kr. A second peak appears at the third day,
growing with increasing doses. Corresponding
to this, at 90-100 kr the gradient of the declining
survival time curve is strengthened. So a new
type of mortality seems to be manifested at
high doses. In agreement with the radiation
syndrome at these doses it is interpreted as a
central-nervous-death.

5.53. Cytological Interpretations of Five Types of
Induced Modification in the Oviposition-pattern
of the Wasp Habrobracon. Daniel S.
Grosch (Raleigh, U.S.A.).

In contrast to Diptera where ovaries may not
mature for several days, at eclosion, differen-
tiated and transitional cells are present in the
sequential series provided by the polytrophic
ovariole of the parasitoid wasp Habrobracon.
Furthermore, ovariole sinvariably number only
four, and quantitative modifications of the
pattern of egg deposit may be traced back to the
cellular state of their contents.

When eggs are plotted against days, control
females quickly reach a sloping plateau main-
tained for about 15 days until the sharp senile
decline occurs. Low and moderate radiation
exposures result in (1) a two-humped curve.
The interposed valley, which can dip to tempo-
rary infecundity, attests to the vulnerability of
transitional cells which undergo five successive
mitotic divisions. High doses of radiation destroy
the oogonia as well as transitional cells. This
results in (2) a rapid decline to permanent in-

fecundity within the first week. Single-meal
ingestion of metal cations and classic organic
enzyme inhibitors produce a constant deficit
throughout life, reflected as (3) a curve lower and
parallel to that of controls. A general debility of
somatic tissues ensues rather than a direct effect
upon the gonads. Ingested animetabolites can
also give type 3 curves, but more typical is (4>
an initial lag phase of up to five days resulting
from oocyte retardation and degeneration
attendant interference with nurse cell function.
Nuclear disturbance is involved and may be
obtained with agents influencing either protein
or nucleic acid synthesis. Aureomycin, metho-
trexate, DON, or FUDR give similar type 4
curves when used in proper concentration.
Antimitotic agents provide still another type of
curve (5) characterized by a compensatory
deposit of temporarily arrested cells which make
up for earlier deficits.

5.54. Virus-host Relationship and the Effects of X-ray
Induced Mutants in Heterozygous Condition. R.

C. Baumiller (Woodstock, U.S.A.)

The only reported phenotypic effect of the
"virus'' sigma has been to make its Drosophila
host sensitive to CO2. L'Heritier (J> has reported
strains of virus carrying flies which differ
according to the stability of the virus-host
relationship. The experiments, presently com-
municated, examined the effects during develop-
mental stages of X-ray induced mutants in
heterozygous condition on several of these
strains W.

Sensitive (S) and sibling cured (R) virgin
females were collected from the strain to be
tested and mated to sibling cured males who
had (X) or had not (U) received 3000r of X-rays.
In a cured line the presence of the virus can no
longer be demonstrated. After a two-day mating
period the females were placed in net-enclosed
cylinders and eggs were collected over one-half
hour periods. Each hour, beginning with the
21st hour after oviposition, the number of larvae
hatching were scored.

The effect of mutants in heterozygous condi-
tion on time of egg hatching varied according
to the stability of the virus in the strain tested.
The reasons for this difference in effect will be
discussed.

A portion of this work was supported by a
grant to Dr. I. H. Herskowitz from the Atomic
Energy Commission (Contract AT (ll-l)-633)

73

Section 5 — Mutagenesis

while the author was a Postdoctoral Fellow of
The National Foundation.

1. Adv. Virus Res., 1958.

2. For the effects of such mutants in the wild
type Oregon-R strain of Drosophila see
Baumiller, Genetics, 1963.

5.55. Effects of Irradiated Chromosomes on Viability
in Heterozygotes and Hemizygotes. Raphael
Falk, Nehama Ben-Zeev, and Shula Baker
(Jerusalem, Israel).

Drosophila males were irradiated with an
X-ray dose of 2000 r and mated according to a
design that permitted the determination of
viability effects in both the heterozygotes and
hemizygotes carrying the irradiated X-chromo-
some. Apart from the induced lethals and semi-
lethals, the milder quasi-normal viability mu-
tations could also be detected in the hemizygotes
for irradiated chromosomes.

In crowded cultures, when deleterious mu-
tations caused a decline in the number of males
hemizygous for the irradiated chromosome, the
remaining genotypes competed for the available
space. If the efficient competitors were heter-
ozygous for an irradiated chromosome as well,
the induced viability effect was offset in the final
outcome by their advantage in competition. The
heterozygotes for irradiated chromosomes thus
exhibited a spurious increase in viability as
compared with their controls which carried
unirradiated chromosomes. When intra-culture
competition was kept at a minimum it could be
demonstrated that the induced mutations had an
adverse effect on the viability of heterozygotes.

The heterozygotes carrying semi-lethal mu-
tations were the most seriously affected. The
heterozygotes for quasi-normal chromosomes
were considerably less viable than the controls,
their viability being reduced at least as much as
that of heterozygotes for lethals.

This work was supported by grant GM 8258
from the U.S. Public Health Service.

5.56. Further Data on Overdomi nance.

Wallace (Ithaca, U.S.A.).

Bruce

in heterozygous condition in flies (1) otherwise
homozygous for their second chromosome,
(2) heterozygous for two different chromosomes
obtained from the same locality, and (3) hetero-
zygous for chromosomes obtained from widely
separated localities. Two chromosomes from
each of three localities were used for these
studies; radiation levels were 0 r, 250 r, 750 r,
and 2250 r.

At the time this is written statistical analyses
are incomplete. Nevertheless, the data suggest
that the mean viability of homozygotes is
improved by the new heterozygosity (confirming
the report made at the Xth Int. Cong, of Ge-
netics) while that of heterozygous individuals i&
lowered. The implications of these observations
will be discussed.

Studies have been completed on the average
viability effects of radiation-induced mutations
(second chromosome; Drosophila melanogaster)

5.57. Transitory Increase in Genetic Load in Ir-
radiated Laboratory Populations of Drosophila
melanogaster. H. L. Carson (St. Louis,
U.S.A.).

Four replicate populations (two control and
two experimental) of se ss es k ro stock were
maintained for three years in vial populations
in which food, space and change schedule were
rigidly controlled. Populations produced by
this design are small (not more than 200 adults)
and are equilibrated under strong natural
selection. Population size and production were
measured weekly. The experimental populations
received radiation treatment; the dose was
given over a period of two years and totalled
65,000 r units of X-ray. Rate of administration
was 1000 r/week with three periods of 10, 10,
and 20 successive weeks during which radiation
was suspended. No radiation was given in the
final year. Genetic loads carried by each popu-
lation were measured three times during the
final year. Random lots of eggs from each popu-
lation were collected, counted and then adults
were reared from these eggs under ideal con-
ditions. Yields of adults from the irradiated
populations were significantly below those of
the controls at the first measurement. This
indicates that these populations were carrying
genetic loads due to the history of radiation.
These differences, however, were absent after
one year, indicating that the loads due to
radiation had disappeared. This effect is ascribed
to the efficiency with which natural selection
removes new deleterious mutants from small
populations. At the termination of the experi-
ment, population sizes of the experimentals did
not differ significantly from the controls.

74

Section 5 — Mutagenesis

5.58. Fitness of Irradiated Populations of Drosophila
melanogaster. K. Sankaranarayanan (New
York, U.S.A.).

Males in experimental populations of D. mela-
nogaster were treated with acute doses of X-rays
(2000 r, 4000 r, 6000 r and 7000 r) and the effects
of irradiation on population fitness were studied
under conditions of relaxed selection, the relax-
ation being obtained mainly, by minimizing
larval competition. In one population, males
received an initial dose of 7000r units and no
further irradiation in subsequent generations.
At other dose levels, there were populations
where the males received X-irradiation (1) at
generation O only and (2) at generation O and
in every subsequent generation. From the latter,
sub-populations were derived where irradiation
was stopped after 5, 10, and 15 generations. The
components of fitness investigated are (1)
hatchability, (2) viability from the larval to
the adult stage, and (3) viability from the egg to
the adult stage.

An important finding that confirms the results
of earlier investigators is the observation that the
radiation induced mortality (dominant lethal
effect) is mainly (but not exclusively) at the egg
stage. A second finding is the surprisingly rapid
recovery to the control level in hatchability and
viability rate following the cessation of ir-
radiation.

A detailed analysis of the second chromo-
somes is in progress to get an estimate of the
radiation-induced genetic load and to gain an
insight as to how much of the genetic load is
adaptively incorporated into the gene pool of the
population.

Work done during the tenure of a Columbia
University Pre-Doctoral Fellowship (1961-62)
and Boese Fellowship (1962-'63).

5.59. Alterations of Viability Characters in an
Irradiated Population of Drosophila. V. Boch-
nig (Berlin, Germany).

Alterations of viability of Drosophila melano-
gaster have been studied after X-raying a
population in its consecutive generations. The
population used had been derived from the
Berlin wild stock. In each generation 500 males
and 500 females (1-5 days old) have been irradi-
ated with an acute dose of 2100 r. Up to more
than thirty generations of irradiatian viability
characters have been studied in intervals of
about five generations by testing lifespan,

fecundity, and fertility after application of
different X-ray doses ranging from 0-100 kr.
The results obtained show that in each of the
three characters studied the irradiated population
reached a significantly higher degree of viability
than the Berlin wild stock under all experimental
conditions. The higher fitness of the irradiated
population is explained by an effect of heterosis.
A great number of mutations must have been
induced by the irradiations and accumulated in
the population. As a consequence a higher degree
of heterozygosity must have been reached.

5.60. Mutational Recurrence or Genetic Compen-
sation of Lethals in Drosophila Natural and
Irradiated Populations? M. L. Reguly and
A. R. Cordeiro (Porto Alegre, Brazil).

The total frequency of 30.3 per cent lethal II
chromosomes of D. willistoni from an irradiated
"A" population do not differ from the control
"E" population, 30.5 per cent (x2 = 2.17)(714
strains). This analysis was made 77.5 generations
after radiation. The "A" population exhibited
6 out of ten retested lethals and the "E" 3 of
these.

Nevertheless, three lethal loci are exclusively
found in "A" irradiated population. The allelism
tests we performed and the calculated average
mutation rate per locus in the II chrom. of
willistoni, 0.000008 obtained from the data of
Dobzhansky et al (1) allows the estimation, with
Wright's formulae, of the equilibrium levels for
each lethal assuming complete recessivity.

According these calculations the six more
frequent induced lethals, still present in popu-
lation "A" and the three spontaneous alleles of
"E" population are at 10 to 24.4 times above the
expected values.

The lethal survival in nature, at such high
frequencies, can only be explained by mutation
pressures many times above the normal average
observed or by overdominant alleles providing
genetic compensation or both, by mutational and
balanced effects. We suggest that selection of
modifiers and or overdominant allelic forms,
tends to buffer (balance) the recurrent lethal
producing loci.

1. Genetics 37, 650 1952.

5.61 . Changes of Reproductive Performance of Droso-
phila willistoni at Two Inbreeding Levels. A. J.

Centeno, M. L. Reguly and A. R. Cordeiro
( Porto Alegre, Brazil).

75

Section 5 — Mutagenesis

From 608 wild inseminated females 123 out-
bred strains with inbreeding of 0.26 and 124
sibmating strains with f =0.65 (at 6th generation)
were obtained.

At the 5th generation each strain have been
divided in two strains each with 15 pairs of flies.

For each strain one culture was submitted to
600 r of a Co60 gamma source.

The relatively outbred strains gave a total of
40,126 flies in the control (K) and 41,363 from
the irradiated parallel cultures (R).

An hierarchal analysis of variance showed no
significant difference between treatments (F =
0.002, F.05 (1244) = 3.89). The same non-
significant result was obtained at the more
inbred strains in a total of 28,158 flies in the
control and 27,100 Fi of irradiated strains.

A paired analysis of irradiated-control failed
to show any significant overall increase of the
irradated or the control. Considering all the
paired strains in which a betterment of repro-
ductive performance was observed after radia-
tion, a parcel of 51.22 per cent among 123 strains
and 45.16 per cent among 124 strains, contrib-
uted to a significant / value.

We have indications that several strains do
increase performance after radiation. The aver-
age effect on the total population is certainly
counterbalanced by the others that shows a
significant decrease.

produced an overall enhancement of egg-adult
viability; K (d) == 32.53 per cent, R(d) = 44.44
per cent for 10,650 eggs fertilized by control and
irradiated spermatozoids. Nevertheless, the sper-
matozoids produced from irradiated spermatides
and spermatocytes showed a slight decrease in a
total of 1 1,230 eggs, K (d) == 32.5 per cent R (d)
= 30.8 per cent.

The increase of egg-adult viability among the
(d) level "low" class is K (d) = 7.53 per cent,
R (d) == 40.85 per cent, placing its average via-
bility over that of "high" class after radiations:
K (d) == 62.95 per cent, R (d) == 33.74 per cent.
The variance increased significantly among the
irradiated spermatozoid block, considering that
the extremes (high and low) of viability regressed
toward the average more among the radiated
than between the control the median class
showed the greater increase in variance.

Our results partially support the Wallace n
and Wallace and Dobzhansky 2l results. Con-
sidering that the more commonly induced mu-
tations are the ones that recur most in natural
conditions also, and consequently, are more
likely to be co-adapted in balanced genie systems
of a race gene pool, these are probably the same
that improved the "low" class strains.

1. Evolution 12, 532.

2. Genetics 47, 1027.

5.62. Restoration, Without Selection, of Balanced
Genetic Load by Radiation of Drosophila Inbred
Strains. A. R. Cordeiro, M. L. Reguly and
A. J. Centeno (Porto Alegre, Brazil).

5.63. Persistence of Lethals in Irradiated Natural
Populations of Drosophila willistoni. E. K.

Marques, H. Winge, M. Napp and C. M. P.
Maciel (Porto Alegre, Brazil).

Starting with wild inseminated D. willistoni
from natural populations, four levels of inbreed-
ing were obtained: (a) outcross, about "zero
level" (150 strains); (b) outcross with sib mating
last (5th) generation, level: 0.39 (101 strains from
608); (c) sib mating 5 generations, level: 0.69
(116 strains from 608), and sib mating 41 gener-
ations, near level 1.0 (1 strain from 50). Signifi-
cantly different egg-adult viabilities were observed
among these series: (a): 68.5 per cent (b): 46.9
per cent (c): 29.7 per cent = (d): 32.5 per cent.

Levels (b), (c) and (d) strains were sub-cultured
in two simultaneous and parallel series of replic-
ations, one (R) received 600 r from a Co60 source,
the other (K) was submitted to the same conditi-
ons, except radiation.

A total of 127,010 eggs have been counted in
these experiments. The most pertinent results are:
at the (b) and (c) levels the (K) did not differ
from (R). Nevertheless at the (d) level radiation

An isolated wood in the grassland region of
R. G. Sul (Brasil) received during one year six
releases of about 71,000 individuals Fi of irra-
diated samples from a laboratory population
originated from the same wood. A total of
45,000 r from a Co60 gamma source was delivered
to these six samples. The Fi of the last irradiated
sample, 6Ri, as well as the samples collected in
the isolated wood, 5 generations (N6R0 and 15
generations (3N6Ri) after the release of 6Ri
have been genetically analysed for the II and III
chromosomes.

The 6Ri exhibited a significant overload of
lethals (II chromosome: 69.75 per cent; III chro-
mosome: 30.87 per cent in regard to its control
values from another unirradiated isolated wood
(II: 26.34 per cent; III: 19.02 per cent). The alle-
lism increased for each sample: II: 6Ri = 6.39
per cent and Control = 1 .02 per cent ; III : 6Ri =
4.00 per cent and Control = 0.29 per cent. This

76

Section 5 — Mutagenesis

increase persisted five generations in nature:
N6R1: II = 3.82 per cent; III = 3.22 per cent.
This high allelic frequency was maintained in spite
of the return to control values of the III chro-
mosome lethals. The II chromosome lethal
frequency was still significantly higher than the
control (44.44 per cent). The allelism tests inter-
samples (6R1 N6R1 : II = 5. 1 8 per cent ; III =
3.03 per cent and 6R1 :< Control: II == 0.22 per
cent; III = 0.07 per cent allowed us to estimate
the radiation induced lethal persistence at the
N6R1 to be 75.82 per cent for the II and 89.51 per
cent for the III chromosome.

The same type of calculations made for the
3N6Ri gave 1 1 .48 per cent for the II and 5.30 per
cent for the III chromosome, as values of lethal
persistence from the 6R1, 15 generations after
under natural selection. There are indications that
several components of the adaptive value were
depressed (l) and that chromosomal inversions
might "protect" some lethals (2). The persistence
of some lethals suggests their complete recessivity
as even some balanced effects.

1. Marques and Maciel, Experientia 17, 404.
1961.

2. Cordeiro, Experientia 17, 405, 1961.

6.64. Genetic Effects of y-Irradiation on the Percent
Adult Emergence of Drosophila melanogaster.
J. C. DeFries and R. W. Touchberry (Ur-
bana, U.S.A.).

Newly emerged males and females from two
populations (randombred and inbred) derived
from Luce's wild type strain were subjected to
various dosages of y-irradiation (0, 500, 1000 or
1500r) and mated in all possible combinations
within populations, resulting in a four X four
factorial arrangement of treatments. Three mat-
ing pairs were included in each subclass of two
replicate experiments, requiring a total of 96
mating pairs per generation in each experiment.
Eggs were collected over a 10-day period follow-
ing treatment and the percent of the offspring to
emerge as adults was determined. In addition to
these data of generation one (offspring of treated
flies), similar data were obtained for generations
two and three, resulting from full-sib matings of
generations one and two, respectively. Somatic
and genetic effects would be expressed in gener-
ation one, whereas only genetic effects would be
expressed in subsequent generations.

Highly significant linear depressing effects
resulting from treatment of both males and
females were observed in the data of generation

one, with the magnitude of the effect being greater
for males than females. Highly significant effects
resulting from treatment of the original males,
although smaller in magnitude than in the first
generation, were again found in the data of
generations two and three; however, no effect
was evident from treatment of the original fe-
males in these later generations.

This investigation was supported in part by
Research grant GM-07951-03 from the Public
Health Service.

5.65. The Sex Ratio in the Offspring of Irradiated
Avian Males. M. Vojtiskova, V. Matousek
and A. Lengerova (Prague, Czechoslovakia).

Birds proved to be advantageous material for
studying the radiation effect on the sex ratioin
the offspring of irradiated homogametic and non-
irradiated heterogametic parents, since

(a) they can be bred in large numbers under
standard conditions;

(b) in contrast to mammals, the homogametic
sex in avian species is male; this circumstance
makes it possible to extend the followup of the
dose-effect relationship to a higher dose range,
since the sterilizing dose for males is much higher
than for females. In addition, each irradiated
individual has a large number of offspring, which
is very useful from the aspect of the requirements
of statistical analysis.

In the present experiment a total number of
1 1,874 birds was used to estimate the shift in the
sex ratio after the X-ray irradiation of males.
Within the dose range 0-600 r the shift was found
to lie between the limits of 1 10"5 to 8 10~5
per 1 r of irradiation with a confidence coefficient
of 0.95.

5.66. Influence of Fast Neutrons on Frequency of
Lethal Mutations in the Reproductive Cells of
Male Mice. M. D. Pomerantzeva (Moscow,
U.S.S.R.).

The males of white mice were totally irradiated
by fast neutrons (with average energy ~ lMeV)
in the horizontal canal of a nuclear reactor. The
average dose rate of fast neutrons was 350 rad/hr,
the concomitant dose of y-rays was 85 rad/hr.
The following doses of fast neutrons were used:
17. 34, 57, 114, 171 and 228 rad. Immediately
after irradiation and on the 18th day after irra-
diation each male was kept for 3 days with 2 to

77

Section 5 — Mutagenesis

4 non-irradiated females. The females (328) were
dissected on the 13th to 16th day of pregnancy
for counts of corpora Iutea, implantations, and
post-implantation losses. The results obtained
show that the treatment both of the spermatozoon
and of spermatids led to the exponential depend-
ence of the dominant lethals frequency upon the
dose. The average probability of one dominant
lethal appearance per dose unit (mutation rate)
was 6.0 x 10-3 for the spermatozoon irradiation
and ll.OxlO-3 for the spermatid irradiation.
The dose which leads to the death of 50 per cent
of embryos, calculated by probit-method, was
106 ± 10 rad for the spermatozoon irradiation
and 64 ± 9 rad for the spermatid irradiation.
Thus the spermatids appeared to be ~ 1.7 times
more genetically radiosensitive than the sperma-
tozoon.

The probability of dominant lethal appearance
which leads to the death of embryos before im-
plantation was ~ 0.3 both for the spermatozoon
and spermatid irradiation.

The comparison of the results obtained with
the data in the literature leads us to the conclusion
that in the case of the mature spermatozoon irradi-
ation the genetical effect of fast neutrons is about 4
times higher than that of X-rays. In the case of
the spermatid irradiation the RBE of fast neu-
trons is somewhat lower.

5.67. A Dose-rate Effect in Mice with Chromosomal
Mutations. A. G. Searle and R. J. S. Phillips
(Harwell, Great Britain).

Lyon, Phillips and Searle have shown that
1200 r acute X-irradiation of mouse spermatogo-
nia delivered in two equal fractions 8 weeks apart,
leads to a decrease of 1 5 per cent in litter-size of
offspring at birth. This is due to the induction
of dominant lethals acting around embryonic
implantation. Translocations, leading to semi-
sterility (or complete sterility in the male if sex-
linked), were induced in about 4 per cent of
survivors. The dominant effects of 1 200 r chromic
gamma-irradiation of spermatogonia, spread over
12 weeks at 100r/week, have now been investi-
gated, using the same mouse stocks. Results so
far show that the dominant lethal effect is much
reduced, with a decrease in litter-size of only
about 2 per cent in the irradiated series. In ad-
dition, the rate of induction of translocations is
decreased to about 1 per cent.

Two different phenomena are probably re-
sponsible for the lower yield of chromosomal
mutations: (i) a reduction in the magnitude of
the non-linear component of the dose response
when the dose-rate is low, and (ii) a greater capa-

city for "repair" with low than with high dose-
rate irradiation, as found with specific locus
mutations.

5.68. The Influence of Radiation on the Genetic Con-
trol of Neonatal Mortality in Swine. D. F. Cox

(Ames, U.S.A.).

Duroc and Hampshire swine are used in a
continuing experiment designed to measure the
genetic effects of radiation. The first generation
offspring produced by normal males and females
are compared with offspring from males given a
single exposure of 300 r X-irradiation at least six
months prior to matings with untreated females.
Present data include 951 litters representing
9640 pigs.

Survival from birth to 42 days was 72 per cent
in the control group compared with 68 per cent
in the group sired by irradiated males. No signi-
ficant changes in the number born, the sex ratio
or the number of late fetal deaths have been
found. The rate of mortality was determined in
three intervals of postnatal life; day one including
those born dead, the period between day one and
day six, and the period from day six through
day 42. The rations of the mortality rates in the
irradiated group to those in the control group for
the three intervals studied were 1.090, 1.529
and 1.020, respectively. The mortality rates in the
offspring from irradiated males exceeded the con-
trols in each interval but the period between the
first and sixth day after birth appears particularly
sensitive to the genetic effects of radiation. Inter-
pretation of this pattern of mortality resulting
from paternal irradiation will be presented on
the basis of information concerning the breed,
sex, and individual genetic factors contributing
to variation in the probability of survival.

This work is supported by Contract AT(1 1-1)
707 from the U.S. Atomic Energy Commission.

5.69. Heterosis in the Response of Mouse Embryos to
Irradiation. Donald J. Nash and John W.
Gowen (New Brunswick and Ames, U.S.A.).

Mice of three inbred strains, BALB/Gw, K,
and S, and the hybrids derived from them were
exposed in utero on day 6 J, 10|, 14| or 17^ of
gestation to single whole-body doses of X-rays
of from 20 to 320 r. Data were obtained on the
incidence of malformations at birth, postnatal
growth from birth to maturity, life span, and

78

Section 5 — Mutagenesis

lifetime reproductive function when treated mice
were mated to untreated mice. Comparison of
the two general types of progeny, inbreds and
hybrids, revealed significant differences among
these genotypes. Following treatment with 160r
at 10£ days gestation 64 per cent of the inbred
embryos were born with external malformations
compared to 29 per cent of the hybrid progeny.
In addition, the incidence of stillborn births
among inbred progeny after 160r at 10| days was
greater than that of the hybrids (100 per cent
vs. 64 per cent). The general response of the
inbreds and hybrids also was observed in those
treatments which produced the most noticeable
effects on growth and reproductive performance.
For example, after 320 r at 17^ days gestation ir-
radiated mice later produced a mean number of 24
progeny in a one year period compared to 63
progeny in the controls. Of particular significance
is the fact that all of the inbred embryos that had
received this treatment later were sterile. Half of
the hybrid progeny produced litters however.
Results indicate that heterotic effects may exist
during prenatal stages of development.

This work has received assistance from Con-
tract No. AT (11-1) 107 from the United States
Atomic Energy Commission.

5.70. An Attempt at Genetic Extinction of a Small
Hybrid Mouse Population by Gonadal Irra-
diation. Earl L. Green (Bar Harbor, U.S.A.).

Genetic extinction of a population may be
claimed only if a non-irradiated generation, one
or more generations after the last irradiated
generation, fails to reproduce. This experiment
was an attempt to extinguish a small population
of hybrid mice (8 mated pairs in each generation)
by exposure of the gonads of the male parents,
when six weeks old, to 900 r of X-rays in each
generation. The gonadal dose of 900 r was select-
ed because it neither killed nor permanently
sterilized these hybrid mice. Seven weeks after
exposure, each male was mated to a randomly
selected non-sister female of the same generation.
All mice of this population (DX-GE) were des-
cended from a four-way cross of strains C3HeB/
FeJ, C57BL/6J, DBA/2J, and BALB/cJ.

After six generations of irradiation, genetic
extinction had not been achieved. Furthermore,
reproductive performance (fertility, number
of litters, number of offspring born and wea-
ned, average litter size) had not been greatly
affected.

5.71. Influence of Cobalt-60 continuing Irradiation
on Fertilities and Life Spans of Different
Strains of Mice W. Janice Stadler and John
W. Gowen (Ames, U.S.A.).

Continuous 22 hour a day irradiations have
now been received by up to 18 successive
generations of different strains of our mice.
Full lifetime fertilities, life spans and other
characteristics of the successive generations of
progenies are being taken. Five generations now
have completed life spans. Dosages varying
from 0.03 r to 0.10 r per hour allow reproduction
to continue. Both parental sexes as well as in
uterine development are under irradiation.
Irradiation to the progenitors prior to con-
ception, ancestral irradiation, may be separated
from that received by the mouse itself, direct
irradiation. The results showed that within
these dosage ranges mice maintain numbers in
first litters directly comparable to those observed
for unirradiated mice of like strains at corre-
sponding periods in the colony. Sex ratios were
comparable within the different strains. Strain
differences in reproductive performance under
irradiation were evident. The factors most
important to reproductive worth of both
irradiated and unirradiated mice were similar;
time when fertility was initiated, well balanced
reproductive sequence and litters adjusted to
physiological capacities of the parents. Search
for visible gene mutations showed none in the
untreated mice and one recessive, brachypod,
in the irradiated group. This mutation appearing
in the 9th B X S generation is allelic and phenoty-
pically comparable to a similar mutation from
mice receiving high dosage acute irradiation
some years earlier. Cumulative irradiation
dosages over 10 generations exceeded 1500ror
27 times the 50 per cent sterilizing acute dose for
the females, 4 times that for the males and more
than 2 times the acute lethal dose for either sex.

1. Journal Paper of the Iowa Agri-
cultural and Home Economics Experiment
Station, Ames. Project Nos. 1180 and 1187.
Assistance has been received from Contract
AT(ll-l) 107 Atomic Energy Commission,
U.S.A.

5.72. X-irradiation Effects on Lifetime Repro-
ductivities of Different Strains of Mice.

John W. Gowen and Janice St ADLER^CAmes,
U.S.A.).

Effects of single dose X-ray irradiations to

79

Section 5 — Mutagenesis

young adult mice on total lifetime fertilities
and progenies, as distributed by litter sequences
and strains, have shown profound differences
between the sexes. Males of 8 inbred strains
fertilized up to 25 litters in 2390, whereas females
had up to 15 successive litters in 707 conceived.
Acute irradiations from 0 to 320r scarcely
affected male fertility or the size of litters
obtained at each successive gestation. After
320r, male fertilities, the numbers of their litters
and of their progenies within litters, decreased
orderly on an irradiation dose-dependent basis.
Irradiated female fertilities, the numbers of their
litters and the numbers of their progenies, all
decreased regularly even with the lowest X-ray
dosages of 20r. Sex differences may seem sur-
prising for each sex has nearly equal genie
materials for ion pair absorption. However,
sperm potential of the males exceeds egg
potential of females by a large factor. A large
number of sperm may be inactivated, yet sperm
capable of fertilization are still available for full
fertility. After this point is reached dose depend-
ence of fertility for both sexes becomes com-
parable. Sex ratios for the progenies of either
irradiated males or females are similar. Dose de-
pendence of the traits is affected by strain. These
data are striking contrast to those of low daily
dosages, 0.1 to 0.03r per hour, of 22 hour per
day Cobalt-60 irradiation to be presented at
this Congress.

1. Journal Paper of the Iowa Agri-
cultural and Home Economics Experiment
Station, Ames. Project Nos. 1180 and 1187.
Assistance has been received from Contract
AT (11-1) 107 Atomic Energy Commission,
U.S.A.

5.73. Cytogenetic Radiosensitivity of Various Phases
of Nuclear Cycle of Human Cells in Tissue
Cultures. N. P. Dubinin (Moscow U.S.S.R.).

The problem of the effect of ionizing radiations
on the heredity of man is one of the principal
ones in contemporary biology. In this respect
the data concerning radiogenetics of mammals
are of great importance. However, the main
direction in the experimental analysis of the
problem is determined by investigations of the
effect of radiation on the chromosomes in the
nuclei of human cells.

The study of the effect of radiation on the
cellular nucleus necessarily runs the following
trends: (a) analyses of the dependence of various
types of nuclear changes on the chromosome

structure in various phases of the cell cycle, as
well as on the relative capability or inability of
fragments to reunite; (b) various degrees of
sensitivity of chromosomes to the primary breaks
in various phases of the cell cycle; (c) the time of
appearance of different types of rearrangements
due to prolongation of the cell cycle as the
radiation effect.

The effect of 50 r of X-rays on every phase of
the cell cycle of man was studied in tissue
cultures of l|-2-months-old embryos. It was
found that the highest sensitivity relates to the
early prophase and to the second half of the
postsynthetic phase. The first half of the post-
synthetic phase was 5 times less radiosensitive
as compared with the mitotic prophase. Through-
out the synthetic phase, radiosensitivity is 2-3
times higher as compared to the first half of the
postsynthetic phase. During the presynthetic
phase, radiosensitivity falls again to the level
of the first half of the postsynthetic phase.

It was established that fusion and non-fusion
of the distal and proximal breaks, when they
occur at isoloci, proceed independently. The
fusing capacity of the distal fragments is at its
maximum at the early prophase and gradually
falls to a minimum towards the onset of the
presynthetic phase. The fusing capacity of the
proximal fragments is at its maximum at the
stage of synthesis of DNA and it falls both at the
onset of early prophase and the presynthetic
phase.

Throughout the cell cycle the chromatid
rearrangements (100 per cent at the prophase
and during the whole postsynthetic period)
are being displaced by chromosomal rearrange-
ments (100 per cent at theonset of the presynthetic
period). The presence of a mixture of chromatid
and chromosome rearrangements at the syn-
thetic phase and for the greater part of the
presynthetic phase points either to the occurrence
of an asynchronous synthesis in various cells,
or to the presence of a mechanism of chromatid
rearrangements of chromosomal origin, and
perhaps to the simultaneous action of both
these mechanisms.

The investigation of a great number of
chromatid rearrangements in the course of the
spontaneous mutation process, revealed the
occurrence of some real number of chromosome
rearrangements.

The data obtained have shown that the
knowledge of quantitative and qualitative
chromosome changes, induced by radiation at
various phases of the cellular cycle, should
determine a new approach to the very method-
ology of the investigation of radiocytogenetics
of human cells. These data also point to the
necessity of studying the effects of the dose, type

80

Section 5 — Mutagenesis

and rate of radiation. The dynamics of chromo-
some changes, induced by radiation, and the
mechanism of their occurrence at different
phases of the cellular cycle, seems to be a key to
new investigations of the protection of the
nucleus from the hazard of radiation. In this
respect, our first analyses indicate, on the one
hand, to the existence of new sites in the chemical
protection and, on the other hand, in the
mechanism of the occurrence of changes in the
nucleus of cells, induced by radiation.

5.74. On Intracellular ( Chromosomal) and Organismal
Mechanisms of Control in Mammalian Radio-
sensitivity. J. J. Kerkis (Novosibirsk,
U.S.S.R.).

According to frequency of cells with chromo-
somal aberrations appearing for 1 r while irradiat-
ing in vitro embryo fibroblasts of human, guinea
pigs, mice, rabbits and hamsters with the doses
from 5 to 50 r all these mammals arrange
according to the decreasing radiosensitivity as
follows: guinea pig= man < mouse < rabbit <
hamster. These organisms have the same arrange-
ment according to increasing LD 50/30 inherent
in them and according to chromosome radio-
sensitivity of bone marrow cells in vivo. It is
doubtful whether these coincidences are acci-
dental and appears to indicate the leading role
of nuclear structure in the control of general
radiosensitivity of organism. This point of view
is also confirmed by the data obtained in our
laboratory on conformity of cellular and general
protection degree by using chemical radio-
protectors.

It is known that adrenalectomy removes
strain differences on radiosensitivity in mice.
Direct quantitative dependency between the
activity of adrenal metabolism and radiore-
sistency takes place among mice. We have
investigated the influence of adrenalectomy upon
chromosomal radiosensitivity. It has been es-
tablished that removal of the adrenal metabolism
increases the chromosome radiosensitivity ap-
proximately for two times. The average level of
adrenal activity is genotypically conditioned. On
the other hand, the products of chemical metab-
olism have an influence upon the radiosensi-
tivity of chromosomes themselves. The adrenal-
ectomy before and after irradiation has shown
that its influence appears to express by creating
different conditions for realization initial radi-
ation damages. The genetical conditionality
of adrenal activity, its effect upon general
radiosensitivity of an organism and upon
sensitivity of chromosomes of separate cells and,

at last, higher degree of dependency of adrenal
activity itself upon the state of the central
nervous system — all these demonstrate compli-
cation of interaction of cellular and organismal
systems of the control on radiosensitivity and
will complicate the estimation of genetical
danger degree at different doses of irradiation.

5.75. The Effects of Protection Against Genetic
Damage caused by Ionizing Radiation in
Mammalian Sex Cells. N. J. Nuzhdin and
G. V. Niznik (Moscow, U.S.S.R.).

In spite of numerous investigations on the
possibility of protection against genetic damage
in mammalian germ cells, the evidence available
is rather contradictory. This is apparently
explained by the fact that owing to physiological
barriers the protective drugs used for injection
into the organism do not reach sex cells, or
while reaching, are incapable of displaying
protective action. In this connection we studied
the effects of some protective drugs used in
irradiation of spermatozoa in vitro on the rate
of dominant lethal mutations occurred in them.

The experiments were carried out on chinchilla
rabbits. Spermatozoa were given y-rays Co-60
with a dose of 800 r in the saline solution, in the
solution of (3-mercaptoethylamine (MEA), S,
(3-aminoethylisothiouronium Br. HBr (AET) in
corresponding concentrations and also in an
atmosphere of CO and N2.

Females were artificially inseminated after
mating with a vasectomized male and sacrificed
at 18-20 days of pregnancy. The ratio of the
number of live embryos to that of corpora lutea
was taken as an index of the rate of dominant
lethals.

The investigations have shown that the
insemination of females with unirradiated
spermatozoa in both the saline solution and the
solutions containing protective drugs ensured a
high percentage of embryo survival (83 per cent)
in relation to the corpora lutea of pregnancy.

The insemination of females with spermatozoa
irradiated in the absence of protective drugs led
to their high mortality rate (84 per cent). The
results obtained showed that such protective
drugs as MEA and AET, well-known for their
good action, did not protect spermatozoa from
the appearance of dominant lethals, if introduced
into the ejaculate prior to irradiation (15 to 30
min). The percentage of viable embryoes in
females inseminated with spermatozoa which
were irradiated in the presence of the above
mentioned protective drugs did not differ from

81

Section 5 — Mutagenesis

that in females inseminated with unprotected
spermatozoa.

The irradiation of spermatozoa in CO and N2
after a preliminary passage of the gases through
the ejaculate diluted with saline solution,
revealed a higher percentage of viable embryoes
as compared with irradiated controls.

The highest percentage of normally developing
embryoes was observed in females inseminated
with spermatozoa irradiated in nitrogen.

This level was almost four times as higher as
in spermatozoa irradiated in the air and only
1.5 times lower than in unirradiated controls.
The death of embryoes largely occurred in the
preimplantation period of development.

5.76. The Effect of Cysteine Pretreatment on Radi-
ation Induced Dominant Lethals in Mice.

U. H. Ehling (Munchen, Germany).

Hundred (101 C3H) Fi males 10-12 weeks
old were divided into 4 groups. Groups I and II
received an intraperitoneal injection of 15 mg
cysteine, Groups III and IV an equal volume of
saline. Groups I and III were irradiated with
600 rads of X-rays. The interval between injection
and irradiation was 25-30 min. After treatment
each male was caged separately with a hybrid
female for a period of 7 days. Every 7th day the
females were replaced. The uterine contents of
fertilized females were examined 13^ to \6\ days
postconception. The proportion of implanted
embryos in three successive matings, expressed
as percent of controls (Group 11 + IV), was in
Group 1 : 88 per cent, 90 per cent, 45 per cent and
in Group III: 87 per cent, 84 per cent, 41 per
cent. Classifying embryos as alive or dead
(including resorption sites) gave the following
mean numbers of two respective types per litter
in the successive mating series, Group I: 3.9 and
4.0; 4.6 and 3.4; 1.0 and 3.1, and Group III:
3.8 and 4.0; 4.3 and 3.3; 1.5 and 2.2. The pooled
results of both control groups are, Group I:
8.5 and 0.5, and Group IV: 8.4 and 0.5. The
results clearly demonstrate differences in the
effect of cysteine in different stages of sper-
matogenesis. More data are being collected to
evaluate the response of different gametogenic
stages. AET will be used in additional experi-
ments.

5.77. Effect of AET against the Dominant Lethality
Induced by X-rays in Male Mice. A. Leonard
and J. R. Maisin (Mol, Belgium).

C+ male mice injected or not with AET (2-(3-

aminoethylisothiureabromide-hydrobromide or
chloride-hydrochloride) were given 400 r on the
whole-body or on testes only and mated with
virgin females immediately after treatment.

At the 1 7th day of their pregnancy, the females
were dissected and the preimplantation loss and
intrauterine death rate scored. AET does not give
any protection against dominant lethality induced
by 400 r on the spermatozoa: after 400 r, the
number of post-implantation deaths increased in
the same way in protected and non- protected
groups while the preimplantation loss was the
same than in the control group.

These results and the data obtained with 400 r
and 1200 r on the dominant lethality induced in
spermatogonia and in spermatozoa will be
discussed.

5.78. Population Dynamics of Irradiated Type A
Spermatogonia of the Mouse. E. F. Oakberg
(Oak Ridge, U.S.A.).

Type A spermatogonia are the stem cells of
the seminiferous epithelium, and by the process
of stem cell renewal, maintain a constant popu-
lation while forming an unlimited number of
cells which differentiate into spermatozoa.
Since the duration of all other spermatogenic
stages is short in relation to the total reproductive
span, type A spermatogonia are the cells of
primary importance in both genetic damage and
fertility of irradiated male mammals. Degener-
ation of cells in interphase or early prophase
reduces type A cells in irradiated testes to a
small fraction of control numbers. With acute
exposures of 300 r or higher, the surviving cells
multiply before differentiation into later stages
is resumed. The population dynamics of these
few surviving cells, however, is not well under-
stood. Our present experiments were stimulated
by theobservationt1) that two 500 r doses 24 hr
apart gave a mutation rate 5 times that obtained
for 1000 r given as a single exposure. Synchro-
nization of the cell population by the first dose
fraction was suggested as a possible explanation.
Our quantitative histological analysis of mouse
testes 24 hr after 500 r support Russell's hy-
pothesis of a difference in the cell stages present.
Type A spermatogonia were almost exclusively
in interphase. A few early prophases were
counted, but no later mitotic stages were ob-
served. Experiments are now under way to
determine the relative frequencies of Gi, S and
G2 phases in spermatogonia arrested in inter-
phase by irradiation.

1 Russell, Proc.Natl. Acad. Sci. 48, 1 724-1 727, 1 962

82

Section 5 — Mutagenesis

5.79. A Comparative Genetic Analysis of the Radio-
sensitivity of Germ and Somatic Cells of
Monkeys (Macaca mulatta) and Mice.

M. A. Arsenieva, N. N. Orlova and E. D. Ba-
kulina (Moscow, U.S.S.R.).

The investigation is dedicated to a comparative
cytogenetic study of radiosensitivity of germ and
somatic (bone marrow) cells of monkeys and
mice. The animals were exposed to 10, 25, 50,
100, 200 and 400 r of X-rays.

The comparative analysis carried out at some
stages of prophase of meiosis showed that
after irradiation by the doses of 10-100 r the
genetic radiosensitivity of germinal epithelium
of monkeys was 2-25 times higher than that of
mice.

The highest radiogenetic sensitivity of sper-
matocytes I of monkeys was found at the stage
of diakinesis- metaphase I. In average, Ir. causes
at the prophase of meiosis in monkeys, the
incidence of 0.15 per cent of chromosome
rearrangements as compared with 0.06 per cent
in mice.

The cytohistological analysis of spermatogonia
of monkeys, exposed to the doses of 50-100r of
X-rays showed that the type B spermatogonia are
especially radiosensitive. The death of type A and
B spermatogonia occurs after the exposure to the
doses mentioned above, at the interphase as well
as in the moment, when their division begins. The
irradiation of spermatogonia A leads also to the
depression of their mitotic activity. There was
no renewal of mitotic activity of spermatogonia
A after 30 hr following the irradiation. Type A2
spermatogonia were found to be more radio-
sensitive than the spermatogonia Ai.

On the whole the radiosensitivity of sperma-
togonia of monkeys was found considerably
higher than that of mice.

Cytological investigations of the rate of
chromosome aberrations in bone marrow cells
of monkeys and mice after exposure to 100, 200
and 400 r of X-rays also discovered the higher
genetic sensitivity of monkeys.

The higher genetic radiosensitivity of germ and
bone marrow cells of monkeys ascertained by the
analysis, points to the necessity of more detailed
investigation of the problem of comparative
genetic radiosensitivity of mammals in vitro and
in vivo including the studies based on the cells of
man in tissue culture.

5.80. Radiosensitivity of Different Meiotic Stages in
Rabbioocytes Discovered in Blastocysts.

W. Kuhlmann (Miinster, Germany).

Most experiments concerning radiosensitivity

of different meiotic stages have been made until
now in Habrobracon, Drosophila and mice. The
aims of such investigations are

(1) to radiate the different stages as precisely
as possible and

(2) to detect the different categories of damage
which are induced in the different meiotic
stages by adequate genetic and/or cytological
methods.

For several reasons the rabbit seems to be
especially suitable for such experiments : ( 1 )
Since the ovulation is induced only by mating
(or pituitary hormons) the stages of meiosis from
dictyoten op to telophase II are precisely
predictable. (2) Since the implantation is
relatively late (7th day) the blastocysts with a
great number of calls can be recovered by oper-
ating or by sacrificing the animal on the 5th or
6th day after mating, and after adequate treating
every blastocyst gives some analysable meta-
phase-plates for chromosome study.

In order to detect anomalies it was necessary
to analyse the normal chromosome-complement
and to set up a karyogram, which revealed a
great similarity to the human chromosome-
complement, not only in number (44) but also in
the morphology — in contrast to the mouse
which has got only telocentric chromosomes.
The radiation experiments yielded the greatest
sensitivity for inducing dominant lethals as
measured by degenerating blactocysts shortly
before metaphase, that means in diakinesis,
while radiation at the time of metaphase (about
6 hr after mating) with the same dosis of 100 r
remained without detectable effect. The possible
reasons for this difference to the results in
Habrobracon — not in mice — will be discussed.
Experiments for detecting the presumably
second sensitive phase shortly after telephase II
(see Habrobracon, Drosophila, mice) are not yet
completed, likewise the chromosome-studies in
blastocysts for cytological evidence of induced
mutations and will be reported about at the con-
ference.

5.81. Studies of the Mechanism of the Effect of
FUdR on Chromosomes. Sandra Bell and
Sheldon Wolff (Oak Ridge, U.S.A.).

Fluorodeoxyuridine (FUdR) is a specific DNA
synthesis inhibitor in virus-infected Escherichia
coli W. Taylor et al. <2) have shown that FUdR-
treated lateral roots of Vicia faba have chromo-
some gaps and are shattered within 3 to 5 hr
after treatment. These aberrations were inter-

83

Section 5 — Mutagenesis

preted to be an effect by FUdR on DNA synthe-
sis.

A series of experiments was undertaken in
which roots were treated 15 min with H3-
thymidine to label cells in S and then treated
with a mixture of 10~5 m FUdR and 10~4 m
uridine. Chromosome gaps and shattering were
found in those cells treated 2 to 4 hr. FUdR
markedly inhibited the mitotic index within 4 hr.
These results were similar to Taylor's. Autoradio-
grams of these cells, however, showed that the
broken chromosomes were not labeled and
thus must have been broken during the stage
following synthesis. Thus, controsy to what was
postulated previously, FUdR appears to break
chromosomes of Vicia independently of DNA
synthesis.

When lateral roots were treated with FUdR
and uridine preceding treatment in H3-thymidine,
autoradiograms showed labeled nuclei. The
number of grains over the nuclei was not
significantly different from that in untreated
cells. In other experiments in which roots were
grown in FUdR, uridine, and 10 5 m thymidine,
there was almost as much chromosome breakage
as in the roots grown in FUdR and uridine only.
At concentrations of thymidine (10 3 m) 100
times greater than the concentration of FUdR,
the level of chromosome damage was only
slightly above the level of the control cells grown
in the absence of FUdR.

1. S. S. Cohen et al., Proc. Natl. Acad. Sci.,
U.S., 44, 1004-12, 1958.

2. Proc. Natl. Acad. Sci., U.S., 48, 190-198,
1962.

5.82. Cytological and Biochemical Analyses of
Chemically-induced Chromosome Breakage.

Norman S. Cohn (Athens, U.S.A.).

The roots of Vicia faba. Allium cepa, and
Pisum sativum have been treated with the
following agents: 5-fluorodeoxyuridine, 5-bro-
modeoxyurdidine, hydroxylamine, and certain
amino acids. Feulgen smears were prepared for
cytological analyses of chromosome breakage as
determined in metaphase and anaphase of
mitosis. In addition, chromatographic analyses
of certain of the treatments were performed using
specific methods of extraction for histone and
non-histone protein from treated and untreated
roots. It is our contention that breakage of
chromosomes must involve protein as well as
nucleic acid, and preliminary data tend to
support this. While biochemical analyses of all

treatments are yet incomplete, observations on
Vicia and Pisum indicate quantitative as well as
qualitative differences in certain amino acids,
when comparing treated with untreated material.
Treatment with amino acids alone cast doubt
on their use as radiomimetic agents, contrary to
the work of Sharma and Sharma. Using relatively
short treatment times and various periods of
recovery, 5-FUdR and hydroxylamine produce
significant levels of chromosome breakage, but
5-BUdR has little effect. These results confirm
the studies of other workers (Kihlman; Taylor,
Haut, Tung; Somers and Hsu). In addition, these
studies have indicated that FUdR induces
breaks after DNA synthesis as well as during the
synthetic phase. While reunion of broken ends is
relatively low, significant reunion does occur.
This is supported by the presence of anaphase
bridges as well as exchanges and sister union of
Isochromatid deletions observed at metaphase.

Supported in part by the U.S. Atomic Energy
Commission, contract AT (11-1) 826.

5.83. The Effects of 5-fluorodeoxyuridine (FUdR),
5-chlorodeoxyuridine (CUdR), 5-bromodeoxy-
uridine (BUdR), and 5-iododeoxyuridine (IUdR)
on the Frequency of X-ray-induced Chromatid
Aberrations in the Root-tips of Vicia faba.
B. A. Kihlman (Uppsala, Sweden).

Pretreatments with CUdR, BUdR, and IUdR at
concentration of 100u M increased the effect of
a given dose of X-rays by factors of 1.3, 1.6, and
1.9, respectively.

IUdR increased the frequency of X-ray-
induced aberrations to the same extent whether
the roots were irradiated under anaerobic or
aerobic conditions.

In contrast to the thymidine analogs CUdR,
BUdR, and IUdR, the deoxyuridine analog
FUdR produced chromosome damage in the
absence of radiation.

When X-irradiation was combined with a
FUdR-treatment which by itself did not produce
any chromosome damage, the number of anapha-
se fragments was markedly increased and the num-
ber of bridges decreased, in comparison with the
X-ray control. The FUdR treatment was equally
effective when given after as when given before
and during irradiation. These results were
interpreted as an enhacnement of the chromo-
some-breaking effect of FUdR by X-rays. The
chromosome damage produced by FUdR was
reversed by thymidine and thymidine analogs;
this is consistent with the idea that the effect of

84

Section 5 — Mutagenesis

FUdR on chromosome structure is a result of
its inhibitory effect on the synthesis of thymidylic
acid. The experiments have shown that whereas
FUdR-induced thymidine deficiency produces
fragmentation of chromosomes, the incorpo-
ration of CUdR, BUdR and IUdR into chromo-
somal DNA does not itself cause chromosomal
aberrations, but makes the chromosomes more
sensitive to the chromosome-breaking effect of
X-rays.

5.84. Ultraviolet Action Spectra for Chromosome
Aberrations of Thymidine Analogue-substituted
Mammalian Cells in Vitro. Ernest H. Y. Chu
(Oak Ridge, U.S.A.).

A clonal quasidiploid line of Chinese hamster
cells grown directly in quartz dishes were
irradiated with monochromatic ultraviolet (u.v.)
light. Previous studies (Chu, 1962) had establish-
ed the average cell generation time and dose-
effect relationships. The u.v. -induced chromo-
some aberrations were qualitatively indis-
tinguishable from those induced by X-rays in
both plant and animal materials. The u.v.
action spectrum for chromosome aberrations
reaches a peak frequency at 2652 A and approxi-
mately parallels the u.v. absorption spectrum of
nucleic acids.

For varying periods prior to irradiation,
different concentrations of 5-bromodeoxyuridine
(BUdR) or 5-iododeoxyuridine (IUdR) were
included in the growth medium. A few hours'
treatment with BUdR did not induce chromo-
some abnormalities. Prolonged exposures (24 and
48 hr) of cells to 10 5 m BUdR did not inhibit
mitosis and induced only infrequent chromosome
aberrations, although higher concentrations
caused increasingly severe damages to the cell.

Cells pretreated with BUdR for 24 hr showed
an increased sensitivity to u.v. in terms of
induced chromosome aberrations. The most
significant modification of the u.v. action spec-
trum was observed in cells which had been
exposed to BUdR for 48 hr. Particularly note-
worthy is some thirty-fold and six-fold increase
of aberration rates at 2250 A and 3130 A,
respectively. IUdR-substituted cells also had a
modified action spectrum, but IUdR- or BUdR-
substituted cells responded differently to u.v.
at various wavelenghts.

The facts that the u.v. action spectrum is
similar to nucleic acid absorption spectrum and
that direct chemical alterations of the DNA
molecules modify the u.v. radiosensitivity
strongly suggest that the initial DNA damages

probably account for the majority of u.v.-
induced chromosome breakages.

5.85. Actions of 5-bromouracil deoxyriboside on
Plant Chromosomes. F. K. S. Koo (Mayagiiez,
Puerto Rico).

The thymidine analogue 5-bromouracil de-
oxyriboside (BUdR) upon its incorporation into
cellular DNA is known to increase the radio-
sensitivity of the cells. In the present experiments
with chromosomes in root tip cells of Zebrina
pendula, Rhoeo discolor, and Allium capa, the
following additional aspects of BUdR effect
have been observed in some or all of these
species: (1) BUdR often prevents the contraction
of the secondary constriction, giving an ap-
pearance of a stretched region between the
chromosome arm and the satellite. (2) BUdR
induces breakage more readily at the centromere
than at other regions of the chromosome. Since
the constituent of the centromere is presumably
not the same as that of the genetic material along
the chromosome arms, so the mechanism for
breakage induction could be also different.
(3) BUdR is capable of inducing breakages
either in one of the sister chromatids or in both.
Based on the semi-conservative hypothesis for
DNA replication and chromosome duplication,
each chromosome is supposedly composed of one
old and one new chromatids and BUdR is
expected to be incorporated into the newly
formed chromatid. If the breakages represent
the manifestation of weakness at the sites of
BUdR incorporation in the chromatid, they
should occur only in one of the sister chromatids
in a given chromosome. The detection of
breakages in both chromatids indicates that
BUdR is also capable of disrupting chromatids
by other means. (4) In the combined treatment
with gamma-rays, BUdR interacts with radi-
ation to produce a synergistic effect in the
induction of chromosomal aberrations.

5.86. Experiments Utilizing Labeled Nucleic Acid
Precursors and Analogues in Plant Cell
Research. Harold H. Smith (Upton, N.Y.,
U.S.A.).

Investigations on the uptake and incorporation
of DNA precursors and analogues are being
made to study certain cytological, genetic and
morphological phenomena. Incorporation of
5-iododeoxyuridine (IUdR) and 2-aminopurine
into chromosomes of Vicia faba was demon-

85

Section 5 — Mutagenesis

strated by use of the tritiated analogues with
autoradiography. Replacement in DNA was
shown utilizing I131 labeled IUdR and by CeCl
density gradient centrifugation. Phasing of DNA
synthesis in Vicia faba cells was accomplished by
treatment with 5-aminouracil (5AU). Studies on
the rate of entering and leaving DNA synthesis
during 5AU treatment were made with double
labeling techniques involving H3 and C14 labeled
thymidine. Uptake of H3 deoxycytidine ap-
parently forms a pool which is used slowly in
DNA synthesis, thus providing a means for
autoradiographic studies of various other
subsequent treatments affecting rate of DNA
synthesis. Incorporation of H3 labeled arginine
into chromosomes of Vicia faba has been success-
ful and is of significance in investigations on
nuclear proteins.

In Arabidopsis thaliana the normal distribution
of DNA synthesizing cells in the shoot meristem,
as well as the morphogenetic effects of incor-
porated IUdR, have been traced with tritiated
thymidine. The inco poration of IUdR into
gametophytic tissue, .: prerequisite to mutagenic
studies, is being investigated with an I125 labeled
form of this nucleoside analogue.

Kinetin (6-furfurylaminopurine) in combi-
nation with indolacetic acid initiates tumor
formation when applied to the apical meristem of
genetically tumor conditioned seedlings of
Nicotiana suaveolens-langsdorffii. Tritiated kine-
tin is being used to investigate the fate of this
aminopurine in cells of the meristematic region.

Research carried out at Brookhaven National
Laboratory under the auspices of the U.S.
Atomic Energy Commission.

5.87. Effect of Metabolic Inhibitors on X-induced
Chromosome Breakage in the Ehrlich Ascites
Tumour. Carmen Bertucci de Lozzio and
Juan I. Valencia (Buenos Aires, Argentina).

Increased sensitivity to X-irradiation has been
found with pretreatments with chloramphenicol,
5-fluordeoxiuridine, sodium azide or oxygen, in
the Ehrlich ascites tumor ELD.

Irradiation with 2000 r at 500r/min, 240 kV,
15 mA, 1 mm Al, do not inhibit tumor growth.
When the same 2000 r were delivered after one
hour treatment with either chloramohenicol
500 Hg/ml to 2 mg/ml, 5-fluordeoxiuridine
(5-fluoruracil deoxiriboside, FUDR) 4xlO-5M,
sodium azide 0.005 m or oxygen at 30-60 mm
Hg (over the normal atmospheric tension), the
result was a permanent inhibition of the tumor

growth. The same pre-treatments before irra-
diation with 500r did not inhibit tumor growth.

The cytological analysis showed a high
frequency of chromosome aberrations in the
first post-irradiation division. All pretreatments
increased the number of breaks per cell compared
to that observed when the same dose of radiation
was given alone. The qualitative and quan-
titative yield varied in each case. For example.
FUDR produced a segmental shattering of the
chromosomes while oxygen or chloramphenicol
induced a high frequency of chromosome
fragments and few chromosome and chromatid
exchanges; sodium azide increased the frequency
of all types of aberrations, including chromatid
exchanges, but no chromosome shattering.

It is apparent that pretreatment with FUDR
induces a very typical kind of aberration by
interfering with DNA synthesis and perhaps
some other processes, which seems to be related
to the normal integration of the chromosome.
On the other hand oxygen, sodium azide and
chloramphenicol seems to act on the rejoining
mechanism by preventing synthetic processes of
restitution.

This research was supported by a grant of the
Argentine National Research Council.

5.88. The Influence of Nucleic Acid Base Analogues
on the Scales of Ephestia kiihniella. Ernest W.
Caspari and F. W. Muth (Rochester, U.S.A.).

Larvae of Ephestia kiihniella strain NCR were
injected with varying amounts of 5-bromodeoxy-
uridine(5 BDU), lug60ug per animal. The animals
developed normally, and no reduced viability
compared to the controls was observed. The
hind wings were studied for the occurrence of
aberrant scales. Abnormal scales occurred with
high frequency. In the lower range of concen-
trations, 1-10 ug, the number of aberrant scales
rises with increasing dosage, but between 10-30 ug
the number of aberrant scales remains stable.
At still higher dosages a further increase appears
probable. The production of aberrant scales is
strongly reduced or completely inhibited by
equimolar amounts of thymidine. 5-bromouracil
and 2-aminopurine produce few if any aberrant
scales. The types of aberrant scales appear differ-
ent from those induced by radiation. A charac-
teristic type called 1 ("leaf") is predominant at
low concentrations of BDU, but does not
increase at medium levels while it decreases at
higher concentration. It has not been found on
irradiated wings. Other characteristic types of

86

Section 5 — Mutagenesis

aberrations nave similar characteristics dose
curves. Frequently, two aberrant scales are
found very close together, so that they may be
regarded as the derivatives of one affected cell.
The two aberrant scales may be either of the
same type, or may be different from each other.
The former case occurs most frequently in
irradiated wings, the latter type of doublets is
more frequent in BDU-induced aberrations.
Since BDU is an analogue of thymidine, and
thymidine suppresses the described effects of
BDU, it is suggested that the effects are due to
incorporation of BDU into the DNA of the
developing cells and represent mutational
changes in the genetic material.

This investigation was supported by grant AT
(30-l)-2902 from the U.S. Atomic Energy
Commission.

5.89. Interactions between Cations and Chromosome
Damage induced with Mesyloxy Compounds.
J. Moutschen and M. Moutschen-Dahmen
(Liege, Belgium).

Chromosome aberrations induced with several
mono- and difunctional mesyloxy compounds
were modified by some cations in broad bean
and barley. Cu++ and Zn++ considerably
enhanced the rate of chromosome aberrations
induced with EMS (ethyl methane sulfonate)
and to a lesser degree MES (Methyl ethane
sulfonate) and MMS (Methyl methane sulfo-
nate). The optimal conditions for this interaction
were investigated. The effects of these ions were
observed to be lesser for difunctional compounds
Myleran and dimethyl myleran. Some cations
like Ca++ and Mg++ were inefficient with
monofunctional compounds but reduced th^
rate of aberrations with difunctional. The
modifications of the types of aberrations were
considered. One cation: A1+++ not only modified
the efficiency of Myleran but also the distribution
of aberrations. Some interpretations are given.

5.90.EfFects of Substances Blocking Heavy Metals on
Interphase Nuclei and Mitosis. Dolores An-
gulo and F. Silio (Madrid, Spain).

In our previous publication we called attention
to the action provoked on interphase nuclei by
EDTA. The abnormalities induced by this agent
encouraged us to realize another work on biolo-
gic activity of substances blocking heavy metals,

using also CNK to verify its effect on plant
species with the test of Allium cepa.

In these experiments we have used CNK, that
does not present any chelating action on Ca and
Mg, in order to compare its effects with those
produced by EDTA in relation to the belief that
this agent owes its action to the chelation of
divalent cations Ca++ and Mg++.

The effects obtained with either agent show
similar effects in many aspects such as a remark-
able swelling of the interphase nuclei that dis-
covers fine reticulate or filamentous structures
and furthermore heteropicnotic areas which may
correspond to chromocentres, observing at the
same time in mitosis the helicoidal structure
owing to the disturbance of the chromosomic
proteins. Likewise the great neatness observed in
the material under treatment is remarkable, sup-
plying favourable conditions for the study of
chromosomic structures.

On the other hand, the EDTA produces a
beginning of c-mitotic action, which effect we
have observed, though very seldom, in material
treated with CNK. However with this agent there
appear the corresponding radiomimetic effects,
which are not observed in material treated with
EDTA.

Therefore the biologic action of EDTA, che-
lating heavy metals, may be identified in some
aspects with that of CNK, which is not a
chelating substance of Ca and Mg.

5.91. The Influence of Respiratory Inhibitors on Chro-
mosome Breakage by Lasiocarpine and Mono-
crotaline. S. Avanzi (Rome, Italy).

In a previous publication (x) data on the radio-
mimetic effect of monocrotaline and lasiocarpine
in Vicia faba were reported. To test whether the
radiomimetic effect of the two alkaloids is re-
lated to respiration, lateral root tips of Vicia faba
seedlings were subjected to the following treat-
ments :

(1) simultaneous treatment with 5xl0~3 m
lasiocarpine and 0.5 xlO"3 m NaN3 for
8 hr at 15°C, followed by recovery in water.

(2) simultaneous treatment with 5<10-3 m
monocrotaline and 0.5 x 10-3 m NaN3 for
8 hr at 15°C, followed by recovery in
water;

(3) simultaneous treatment with 5 x 10-3 m
monocrotaline and 98 per cent CO for 8 hr,
followed by recovery in water.

As control material served lateral root tips
recovering in water after an 8 hr treatment with :
(i) 5xl0-3 m lasiocarpine at 15°C; (ii) with
5xl0-3 m monocrotaline at 15° or 21°C; (hi)

87

Section 5 — Mutagenesis

0.5 x lO-3 m NaN3 and (iv) 98 per cent CO. Cyto-
logical analysis took place after 24, 36, 48 hr of
recovery. It was found that NaN3, by itself in-
active, brought about a 4-fold increase in both
the percentage of aberrant anaphases and the
number of breaks per cell induced by lasiocar-
pine alone; with monocrotaline, a still stronger
effect of NaN3 was observed (6-fold increase over
the control). Also CO influenced the radiomimetic
effect of monocrotaline by doubling the per-
centage of induced aberrant anaphases.

Parallel experiments with 2,4-DNP 30' pre-
treatment with 10~4 2,4-DNP followed by a 8 hr
treatment with 5 x 10~3 m lasiocarpine and subse-
quent recovery in water indicated that this in-
hibitor only slightly enhanced the chromosome
breaking effect of lasiocarpine.

These results seem to indicate that the radio-
mimetic effect of monocrotaline and lasiocarpine
is not positively correlated with either respiration
or phosphorilation.

1. Avanzi, Caryologia, 15, 351-356, 1962.

5.92. Interaction of Chemically Induced Chromatid
Lesions in Vicia faba. Rigomar Rieger, (Ber-
lin, Germany).

After treatment of primary roots with ethyl-
methane-sulfonate, myleran and ethylalcohol in
all possible combinations of the agents two by
two and the second treatment following the first
one immediately, the number of isolocus breaks
was found to be approximately additive, whereas
the number of chromatid translocations was in
accordance with the values theoretically ex-
pected for full interaction of the breaks induced
by each of the agents. Thus it might be concluded
that all agents tested are breaking the same kinds
of chemical bonds in the chromosomes.

Two treatments with triethylenemelamine or
with triethylenmelamine and nitrogen mustard
one following the other immediately or seperated
by intervals in water up to 8 hr showed that even
after the longest interval used in both cases the
number of exchanges fitted the expectation for
full interaction of the breaks induced by the first
with those induced by the second treatment. On
the basis of the breakage-reunion-hypothesis and
on the supposition that the fractination effect is
really an expression of the time of rejoining, this
result might be interpreted as meaning that
breaks induced by the first treatment are staying
open at least for 8 hr, suggesting the bonds
broken to be of the covalent type. Full interaction

was found to be confined to recovery times giving
the aberration peak.

Because of the complexity of events leading to
aberration production, a lot of which are un-
known, interpretations of this type should be
accepted with due reserve and cannot be more
than working hypotheses for further experimental
tests.

5.93. Chromosome Breakage by Triethylenmelamine
in Vicia faba in Relation to the Mitotic Cycle.

M. Buiatti and Vittoria Nuti Ronchi (Pisa,
Italy).

Experiments were carried out on the induction
of chromosomal aberrations by triethylenmela-
mine (TEM) in relation to the mitotic cycle.
Seedlings of Vicia faba with lateral roots 5-10mm
in length, were treated for one hour with 0.5 x
10~4 TEM at 19 C, after which they were trans-
ferred either into water or into 0.5 per cent
colchicine.

Anaphase and metaphase scores at 2 hr inter-
vals after TEM treatment showed an increase in
aberration frequency from the 6th to the 45th
hour of recovery with a slight drop at 16-18 hr.

As revealed by the relative proportions of
diploid and tetraploid cells in TEM-treated and
control roots subjected to continuous colchicine
action, a mitotic delay by TEM of 8 to 10 hr was
calculated. This might indicate that cells carrying
aberrations at the first recovery hours were under
treatment at the end of G2, possibly suggesting
some mechanism of aberration induction, in
absence of DNA synthesis.

A strong decrease in mitotic index from the 4th
to the 26th hour of recovery (mitotis index: 5,61
and 3,47 respectively) was observed; at 30 hr,
no significant difference was found between
treated and control root tips (mitotic index: 10,
63 and 9,98 respectively).

Roots treated with colchicine for 40 hr sfter
TEM treatment were scored for diploid(Xi)and
tetraploid (X2) cells showing aberrations: apart
from the twofold increase in aberrations fre-
quency per cell in tetraploids, no differences in
aberration types between Xi and X2 were noticed.

Achromatic lesions, so called '"gaps", showed
a continuous increase, both at anaphase and
metaphase, up to 45 hr recovery, a time when
most cells were in the second division. This
could support the idea of "gaps" being partial
breaks. Scoring of gaps at anaphase gave, in all
cases, higher values than metaphase scoring;
this might result from the stronger contraction of
metaphase chromosomes, leading to under-
estimation of "gaps".

88

Section 5 — Mutagenesis

5.94. Radiosensitivity of the Chromosomes. N. Ta-

naka (Tokyo, Japan).

Freshly rooted plantlets of Tradescantia palu-
dosa were divided into four groups: the first
group was grown in a culture solution, aerated
and supplemented with tritiated thymidine
(2uC/ml) for 8 to 12 hr (internal beta-ray irra-
diation); the second group was treated identically
as the first group but exposed simultaneously to
gamma-irradiation at a distance 40 cm from
Co60 source (1.19 rad/hr) (both internal and ex-
ternal irradiation); the third group was exposed
only to the gamma-irradiation at the same in-
tensity as the second group (Co60 external ir-
radiation); and the fourth group served as a
control.

The root tips were fixed with acetic alcohol at
relevant time intervals. Radiosensitivity of the
chromosomes was measured in four ways by
observing frequency of the total aberrations,
fraction of sister chromatid reunion distal (SUD),
fraction of sister chromatid reunion proximal
(SUP), and fraction of erosion proximal (EP) in
the mitotic anaphases.

The treatment was carried out at room tem-
perature 26 C and the mitotic cycle was estimated
to stand around 16 hr, being Gi=2.8 hr, S = 8.4
hr, G2 = 2.8 hr, and M = 2.7 hr, where Gi, S, G2,
and M designated first growth period, DNA
synthetic period, second growth period, and
mitosis, respectively.

Radiosensitive period as measured by fre-
quency of total reunion (SUD + SUP + EP) was
found at S and G2 in the first group and at S in
the second group. Frequency distributions of
each SUD, SUP, and EP in the second group
contrasted with each other, suggesting the com-
bined irradiation enhanced SUD in S and G2
while SUP in Gi and S, and for EP in G2.

5.95. Chromosome Aberrations and the Mitotic Cycle
in Trillium. C. J. Grant (Oxford, Great
Britain).

The mitotic parameters of root tips of Trillium
grandiflorum were measured by the flash label-
ing technique, and confirmed by the binucleate
cell method. At 25 C the period before DNA
synthesis Gi, the synthetic period S, and the
post-synthetic period G2, were of two, one and
two days duration respectively.

It was found that the Xi, X2 and X3 divisions
were labelled in such a way as to confirm the
view that chromosomes duplicate before normal
cell division by a semi-conservative method.

After X-irradiation of root tips, cells con-

taining the so-called sub-chromatid, chromatid
and chromosome type aberrations are found.
Overlap of these types may occur within a single
cell, and in species with a short mitotic cycle this
makes it difficult to correlate the different aber-
ration types with the state of the cell at the time
of their induction. However the relatively long
cycle in Trillium results in little overlap and
chromosome type aberrations can be definitely
correlated with induction during Gi, Chromatid
types with G2, and sub-chromatid types with
prophase. Chemical agents induce chromatid
types during S.

5.96. Cytological Effects of Tritiated Water on Root-
tip Meristems of Allium cepa L. G. Wittmer
(Rome, Italy).

Series of onion bulbs were grown, after ger-
mination, in tritiated water having specific
activity of 2.5 mc/ml. Cytological examinations
performed at different times from exposure to
THO have shown:

progressive mitoinhibition; after 42-48 hr, the
treatment is lethal;

no physiological effect on the chromosomal
matrix: phenomena of stickiness were only
seldom observed;

strong direct effect on mitotic chromosomes,
i.e. very high fragmentation in anaphase (as
far as 90 per cent of anaphases with frag-
ments); the fragmentation is highest after
24-32 hr, and at this time also the meta-
phases show, if even in a low grade, frag-
ments; the fragments are nearly always
acentric and free, rarely attached;

these mitosis with aberrant anaphases (or
metaphases) may end their cycle and yeld
cells with 1-2 micronuclei near the nucleus;

"restitution" of these breakages induced by
(3-rays occurs quite frequently, since root-
tips transferred in tap-water after the THO
treatment show progressive normalization
of the mitotoc activity, with only rare chro-
mosomal aberrations.

The treatments were performed in the Labora-
tories of the Istituto di Chimica Farmaceutica
delTUniversita di Roma.

5.97. Effect of X-rays on Chiasma Frequency in Coix
aquatica Roxb. K. C. Subbaiah (Bombay,
India).

The present study relates to the effect of radia-
tion on chiasma frequency in Coix aquatica

89

Section 5 — Mutagenesis

(bead coix). The chromosomes of Coi.x aquatica
(n = 5) are long ( 1 10-75|Ji) with terminal knobs
and are differentiated into eu- and hetero-
chromatic regions, the latter flanking the centro-
mere on both sides. The chiasmata have been
found confined only to dichromatic segments.
All the five bivalents, two of which are nucleolar,
are mostly of the ring type with a terminalization
coefficient of 0.753.

The inflorescences were irradiated with two
series of 250 kVp X-ray doses (i) 2.5 r, 5 r, 10 r,
20 r, 40 r and 80 r and (ii) 250 r, 500 r, 750 r and
1000 r. The chiasma frequency at diakinesis fol-
lowing irradiation was determined 24 and 48 hr
after irradiation. Translocations, both ring and
chain types, involving (i) two nucleolar bivalents
(ii) nucleolar and non-nucleolar bivalents and
(iii) two non-nucleolar bivalents were scored.

While the doses from 2.5 r to 20 r failed to
produce any observable translocations, those
from 40 r and 80 r were only 2 and 4 per cent re-
spectively. The frequency of interchanges obser-
ved in the high dose series showed that they were
proportionately the highest within nucleolar;
lowest among non-nucleolar and intermediate
between nucleolar and non-nucleolar chromo-
somes. A linear relationship was found to exist
between the interchanges and the radiation dose.

Bivalents without any visible aberrations, such
as translocations, had nearly the same frequency
of chiasmata as in the control, for the entire
dosage range. However, there was a slight de-
crease in chiasma frequency in bivalents involved
in translocations. Terminalization coefficient was
found to be higher with the higher doses.

ments revealed (1) that the minimum mitotic
cycle duration in irradiated meristems was
essentially the same as the unirradiated control,
(2) that at a given daily exposure rate the per-
centage of anaphase cells with visibly damaged
chromosomes increased with increased cycle
duration, and (3) that the irradiation reduced the
number of cells per meristem below that of
controls. The decrease in the number of cells per
meristem can probably be attributed to repro-
ductive cell death due to genetic loss or unbalance
since reduction in cell number varied directly
with the percentage of cells showing chromosome
damage.

The results with Pisum suggest that (1) for
chronic exposures (up to 1000 r/day) the prob-
ability of a chromosome being damaged in-
creased with increasing duration of the mitotic
cycle, (3) the accumulated exposure per cycle re-
quired to produce a measurable decrease in the
number of cells per meristem approaches a con-
stant value, and (3) the accumulated exposure
per cycle required to produce an equivalent
number of cells with damaged chromosomes in
meristems having different minimum mitotic
cycle durations also approaches a constant value.
Thus in quantitating the factors responsible for
cellular radiosensitivity or cell survival the dura-
tion of the mitotic cycle must be considered, at
least for chronic exposures.

Research carried out at Brookhaven National
Laboratory under the auspices of the U.S. Atomic
Energy Commission.
1 . Sparrow and Evans, Brookhaven Symposia in

Biology, No. 14, 76-100, 1962.

5.98. The Influence of Mitotic Cycle Duration on
Chromosome Damage and Cell Survival in Chro-
nically Irradiated Pisum Root Meristems.

J. van 't Hof and A. H. Sparrow (Upton,
New York, U.S.A.).

An hypothesis has been advanced that for
chronic exposures the duration of the mitotic
cycle is an important factor contributing to cel-
lular radiosensitivity 0). To test his hypothesis,
experiments were performed with root meristem
cells of seedlings of Pisum sativum in which
duration of the mitotic cycle was controlled by
temperature. Seedlings were exposed to Co60
gamma-rays at exposure rates of 185, 250, 500
and 1000 r per day for 3 days. Following expo-
sure, measurements were made to determine the
mitotic cycle duration, the number of cells having
bridges or fragments at anaphase, and the
number of cells per meristem. These measure-

5.99. Mutagenic Action of Contrast Media for
Roentgen Diagnosis. A. Barthelmess and M.
Bauchinger (Munchen, Germany).

In the course of investigations on the cyto-
genetic action of substances which come in con-
tact with gonadal tissues or gametes in the human
body we tested 3,5-dijodid-4-pyridone-N-acetic
acid-diethanolamin (I) and N,N'-adipine-di-3-
amino-2,4, 6-trijodoben-zoic acid-methylgluka-
min (II). Both substances are (or have been) used
as contrast media in urography, arteriography
and venography (I and II) and hystero-salpingo-
graphy (II).

Root tips of Allium cepa have been immersed
4 and 24 hr in the solution and fixed at intervals
of 0, 24 and 48 hr after the end of treatment.
Both substances caused numerous breaks and
structural rearrangementsof chromosomes, mi-

90

Section 5 — Mutagenesis

cronuclei, release of droplets of DNA in the cyto-
plasm, and disturbance or inhibition of the reg-
ular mitotic distribution (stathmokinesis, mero-
kinesis, non-congression). The maximal effect
was obtained with both substances at a concen-
tration between 1 and 2 per cent, which allows
normal growth of the roots. However the number
of cells showing cytogenetic effects often varied
considerably between the different roots of the
same bulb.

A dose of 60-80 r of roentgen rays gives about
the same number of structural effects as 1.5 per
cent of I, but nearly no abnormalities of mitotic
distribution. No clearcut results as to a synergis-
tic or antagonistic action of simultaneous chemi-
cal and radiation treatment were obtained be-
cause of the high numerical variability of all the
cytogenetic effects mentioned above.

As the substance I is administered also in sal-
pingography (in concentrations from 30-70 per
cent) one has to take in account dysgenic effects
in ovaries and follicles.

The results will be published in full in the
journal Strahlentherapie.

5.100. Cytogenetic Analysis of the Sensitivity of
Plants to Different Kinds of Radiation. V. V.

Khvostova and S. A. Valeva (Moscow,
U.S.S.R.).

1. After irradiation of seeds of resistant varie-
ties of plants, belonging to the same species, with
the same dose of ionizing radiations a lower per
cent of ana- and telophases with chromosome
rearrangements are produced in first mitoses of
seedling roots, than in radiosensitive forms.

2. A comparison of the action on seeds of
related varieties of sparcely and densely ionizing
radiations has revealed the existence of two kinds
of chromosome protection against radiations:
(a) two varieties of peas, markedly different as to
sensitivity to gamma-rays, showed equal sensi-
tivity to fast neutrons; (b) whereas one of two
varieties of maize manifested a parallel resistance
to both gamma-rays and fast neutrons. These
sensitivity differences were manifested at various
stages of the development of the plant: after irra-
diation of dry seeds and seedlings as well.

3. Experiments on the storage of pea seeds
during 6 months after irradiation with gamma-
rays showed, that the sensitive variety exhibits
after storage twice as many per cent of anaphases
with chromosome rearrangements. The suppo-
sition is made that potential chromosome dam-
ages occurring under the action of gamma-rays,
transforms into real breaks under the influence of
the biochemical peculiarties of the cellular me-

dium of the sensitive variety during storage.

4. Studies on the action of 2-4, dinitriphenol
on germinating seeds of two varieties of maize
clearly showed that 2-4, dinitriphenol has no
effect on the number of rearrangements after
neutron irradiation; after irradiation with gam-
ma-rays, dinitriphenol increases the number of
rearrangements, especially in the radiation-sen-
sitive variety. These experiments show that the
damage of chromosomes by the action of neu-
trons is not subject to modifications by the energy
processes in the cell after irradiation; (2) and
that, apparently, the potential damages, occur-
ring under the action of gamma-rays, are not
transformed into real ones under the conditions
of the high energetic level of the cell, and are
transformed only when the energetic level of the
cell becomes lowered.

5.101. Selection for Radioresistance and Resistance
to Chemical Mutagenes in Diploid and Tetra-
ploid Forms of Buckwheat (Fagopyrum esculen-
tum). R. N. Platonova and V. V. Sacharov
(Moscow, U.S.S.R.).

1. Experiments on A-irradiation of four seed
progenies and on the selection of the most
radioresistant plants, grown from these seeds,
produced resistant forms of diploid and tetra-
ploid buckweat populations.

In the usual (2 x =16) buckhweat form one
first selection resulted in an increase of survival
of the irradiated plants. In the autotetraploids
(4 =32) an increase in survival after irradiation
was produced only after two or more selections.

2. Our 2 x and 4 x lines, resistant to A-rays,
were checked for two years following for resist-
ance to densely ionizing radiations (fast neu-
trons). The results obtained were positive for all
characters (survival, power of development and
seed bearing). This was proven by comparative
cytological investigations. In the gamma-resistant
forms there were less chromosome damages,
caused by fast neutrons, than in the usual
populational seeds, receiving the same radiation
dose.

3. Selection for radioresistance caused the
occurrence of forms resistant also to the chemical
mutagene — diethylsulphate.

4. In order to make clear the character of
inheritance in the radioresistant diploid plants,
resistant (selected) plants were crossed with
plants from usual, non-irradiated populations.
The same kind of experiment was carried out on
tetraploid plants. The hybrid plants show a
dominant inheritance, and radioresistance in
2 x hybrids appeared even to be higher, than in

91

Section 5 — Mutagenesis

their radioresistant parents. This increase we
explain as the result of an additional effect of
heterosis. In the 4 x forms, dominant inheritance
of radioresistance cannot be doubted, but it is
somewhat more feebly expressed than in the
diploids. The radioresistance of the 4 hybrids
is somewhat lower than that of their radio-
resistant parents, but it is markedly higher than
the radioresistance of the initial forms (non-
selected).

5.102. On the Possibility of the Modification of the
Ionizing Irradiation Genetical Effect in Barley.

S. I. Yanushkevich (Moscow, U.S.S.R.).

The possibility of the modification of the
A-rays (Co60) effect on barley (Hordeum
sativum L. ssp. distichum) seeds in the dormancy
state under influence of condition preceding and
following the irradiation is discussed. In the
experiments the influence of the agro-climatic
conditions on the growing plants and of the
modification of nutrition conditions of the
barley seed embryos (by means of homo- and
heterogenous transplantation of embryo on the
endosperm) upon the radiosensibility, chromo-
some aberrations frequency and chlorophyll
mutations in M2 was under study.

It was shown that plant growing in different
agro-climatic areas led to the radiosensibility
change of the seeds in the dormancy state. The
differences in radiosensibility could vary in large
limits and their manifestation was correlated to
the moisture level of irradiated seeds. The ad-
ditional data were obtained on the periodical
change of the radiosensibility of the seeds in the
state of dormancy kept under the conditions
of laboratory.

The growing of Mi plants from irradiated
seeds in sharply different agro-climatic areas led
to definite variations of chlorophyll mutations
frequency in the progeny.

The influence of nutrition conditions of
developing embryos of barley seeds on the
irradiation effect was noted. The nutrition of the
irradiated embryo by the non-irradiated en-
dosperm substances led to the change of the
chromosome aberrations total amount. The
irradiated endosperm, in its turn, induced chro-
mosome aberrations in the cells of growth zone
of non-irradiated embryos rootlets.

The results of these experiments are discussed
in the light of the final effect dependence upon
the irradiated organism physiological state and
metabolism in the periods before and after
irradiation.

5.103. Variable Influence of Humidity on Radio-
sensitivity of Seeds from Eight Botanical
Families. Thomas S. Osborne, Milton J.
Constantin, and Allyn O. Lunden (Oak
Ridge, U.S.A.).

Classical studies with the barley grain {Hor-
deum) showed that superdry embryos are
extremely radiosensitive, becoming more re-
sistant by a factor of 4 or 5 as preirradiation
relative humidity (RH) approaches 50 per cent
and staying on a plateau of resistance even when
RH exceeds 90 per cent. We equilibrated various
dormant embryos at RH's of 10 to 85 per cent
then exposed them to gamma rays at doses of
0 to 400 kr. Dry weight of seedlings in controlled
environment rooms was measured. Families
studied were Compositae, Cruciferae, Cucur-
bitaceae, Gramineae, Leguminosae, Linaceae,
Solanaceae, and Umbelliferae.

Embryos of the Gramineae (Hordeum and
Festuca) proved to be exceptional in that they
showed only trivial responses to variations in
preirradiation humidity. All other families gave
immense changes in sensitivity, even more than
200-fold, with changes in RH. There was a peak
RH for maximum resistance, with sensitivity
increasing at RH's both above and below;
the optimum RH differed with species. The
inadequacy of current free radical-water hypothe-
ses to explain increased sensitivity above the
optimum RH is obvious.

Computer techniques with quadratic and
cubic formulae were used and discussed. Parts
of the data have been accepted for publication
by Radiation Botany.

Operated by the University of Tennessee
College of Agriculture for the U.S. Atomic
Energy Commission under Contract No. AT-40-
l-GEN-242.

5.104. Cysteamine and Sensitivity to X-rays in
Barley. A. Moes (Gembloux, Belgium).

In 1951, Bacq, Herve et alS1^ discovered that
cysteamine and cystamine are remarkable pro-
tectors against radiations in mice.

The protective action of cysteamine has been
confirmed in our experiments conducted since
1954 on barley. (2> Our tests are made with
Piroline spring bailey; the X-ray apparatus
works at 30 kV. The inhibition of the develop-
ment of the first leaf issuing from the irradiated
seeds is determined by a growth test made when
the control reaches approximatively 75 mm.(3)

92

Section 5 — Mutagenesis

In the first trials, the seeds were soaked in
cysteamine at 1, 2, 4 per cent during 24 hr at
20 C before irradiation. We observed a marked
protective action in the growth test, confirmed
by the percentage of plants attaining maturity.

In 1957, we reduced the time of soaking to 1 hr
and used only cysteamine 1 per cent; the higher
number of surviving plants observed was
significant.

In 1958 and 1961, the seeds were soaked during
1 hr in cysteamine 1 per cent, and then dessicated ;
the irradiation took place on seeds with normal
water content (13-14 per cent). The growth test
and the percentage of mature plants showed a
marked protective action from 20000 r and
above. The latest experiments proved that
presence of cysteamine is sufficient to account
for its action.

It seems that the effect of cysteamine decreases
when the water content is low (4-5 per cent).

In a number of experiments, (2) the cysteamine
applied before or after irradiation increased the
number of mutants but in other trials made in
1958 and 1959, the protection afforded by
cysteamine against the mutagenic effects of
X-rays is of the same order of magnitude as the
protection against lethality.

1. Z. M. Bacq, et al.; Protection contre le
rayonnement X par la (3 mercaptoethylamine.
Arch. Internet. Physiol. 59, 442, 1951.

2. A. Moes; L'action de la cysteamine chez
Forge. Bulletin de VInstitut Agronomique et des
Stations de Recherches de Gembloux. XXV,
98-107, 1957.

3. A. Moes. Water content, wave-length and
sensitivity to X-rays in barley. Effects of
Ionizing Radiations on Seeds, I.A.E.A.,
Vienna, 631-640., 1961.

5.105. Some Factors Affecting the Action of Chemi-
cal Mutagens. C. F. Konzak, R. A. Nilan,
R. E. Heiner, and Edith E. Froese-Gertzen
(Pullman. U.S.A.).

Diethyl sulfate and ethyl methanesulfonate
induce a remarkably high frequency of chloro-
phyll mutations and a negligible frequency of
gross chromosome aberrations in barley. This
high mutation frequency is induced at a high
rate of plant survival and low levels of seedling
injury. Recent studies provide a basis from
which an understanding of the relationship
between different kinds of biological damage can
be sought. It has become apparent that physical
and chemical factors play a determining role on

the action of mutagens in biological systems.
Hydrolysis of diethyl sulfate in aqueous solution
is 26 times faster than that of ethyl methanesul-
fonate. The Qio for the in vitro hydrolysis of the
mutagen varies from 3.1 for the 0-1 0°C range
to 4.5 for the 30-40°C range, whereas the Qiu
for the in vivo seedling injury response is about
2.7. Our data indicate that the rate-controlling
factor in the in vivo system is duffusion. The
hydrolysis products tend to decrease seedling
growth and plant survival but do not induce
mutations. These injury effects are apparently
due to low pH. Highly concentrated mutagen
solutions reduce mutagenic effectiveness by
increasing undesirable side-effects through mass
action. The mass action is accentuated with
fast-reacting agents. Results from these studies
offer some likely explanations for differences
in the relative mutagenic efficiency of chemical
agents.

Research supported in part by funds from the
U.S. Public Health Service GM 10838-05 and
from the U.S. Atomic Energy Commission
AT (45-l)-353 and conducted under Washington
State Experiment Stations Projects 1435 and
4068.

5.106. The Effects of Some Combinations of Mu-
tagens on Mutation Frequency in Barley.

T. J. Arnason, J. L. Minocha and Laila
Mohammed El-Sadek (Saskatoon, Canada).

Two mutagens acting in sequence may produce
more than additive mutation frequencies if, for
example, one of these exposes previously
protected mutation sites to the second mutagen.
If two mutagens merely compete for the same
sites or if the effects are completely independent
the results should be additive or less than
additive. Combinations of mutagens that have
been tried on barley (Hordeutn vulgare L.), are
ethyl methanesulfonate (EMS) and ethylene
imine (EI); EMS and nucleotides and EI and
nucleotides. The frequencies of chlorophyll
mutations in Mi spikes and M2 seedlings were
recorded.

The treatment with 0.5 per cent EMS solution
for 4 hr yielded 17 per cent Mi spike mutations.
A 4-hr treatment with 0.1 per cent EI solution
yielded 13.8 per cent of mutant spikes. When
grains were treated with both mutagens in
sequence the results were more than additive.
With EI applied first the mutation frequency was
43.8 per cent and when EMS was applied first
the mutation frequency was 36.7 per cent. On an

93

Section 5 — Mutagenesis

additive basis the expected frequency was
30.8 per cent.

Solutions of nucleotides induced very few
mutations. The highest rate (0.54 per cent) was
produced by adenosine-5-monophosphate. When
adenosine-5-monophosphate was applied before
treatment with 2000 r of Co60 A-radiation a
slightly more than additive result was obtained.
Experiments with EI and nucleotides are not yet
completed. Three combinations of nucleotides
and EMS resulted in notable increases in the
mutation rate. Here, it is possible that guanine in
DNA degraded by EMS was sometimes replaced
by the supplied base.

5.107. Mutations Induced by the Action of Metal
Ions in Pisum. Gosta von Rosen (Landskrona,
Sweden).

Radiomimetic effects and gene mutations are
induced by a deficiency or surplus of for a vital
organism important micro elements. The
frequency of mutations induced by the action
of metal ions is lower than the frequency induced
by ionizing radiation or some intense active
chemical substances. The mutation-spectrum in
chlorophyll mechanism induced by treatment
with surplus doses does not differ from the
spectrum at hard ionizing radiations. Treatment
with a simultaneous combination of several
micro metals is more effective than treatment
with single metal ions. Individual metal ions do
not show any trend to specific chlorophyllmuta-
tions spectra at present, but they induce some
other specific types of mutations indeed.

By adding treatments with metal ions and
X-ray the mitoses and cell disturbances are
arrested, and the mutagene activity of the
ionizing particles is somewhat reduced. The
activity of the metal ions seems to be additative
to the effect of the X-rays.

The micro metal elements in a living cell have
functions in specific and very important pro-
cesses, usually in complex protein structures.
Their concentrations and activities in the cell
are regulated by chelating with protein radicals
in co-valent and semi-polar bonds in the protein
molecule, a balance which is very sensitive and
limited. It is that force of the metal ions to form
complexes with protein radicals that causes
their radiomimetic and mutagenic activity. By
oscillating of the dissociation balance destruc-
tions in the cell metabolism occur, which may
induce chromosomal disturbances and gene
mutations. It is suggested that the complex
forming metal ions in the evolution of the plant
and animal kingdom have played a limited role,

partly through their own activities, partly
through an indirect effect on the active radicals
from the natural radiation.

5.108. The Magnitude of the Oxygen Effect in
Irradiated Barley Seeds. R. A. Nilan,
C. F. Konzak, J. R. Harle, and R. R. Le-
gault (Pullman, U.S.A.).

The extent to which radiation-induced damage
in barley seeds can be amplified by oxygen
posttreatment depends on the radiation energy
(LET), the tissue moisture content, and the
temperature and time of storage from the
initiation of irradiation to the initiation of
germination. In very dry seeds ( ^ 2 per cent
moisture) oxygen-effect factors as high as 10 were
demonstrated for high intensity Co60 gamma-
rays, and as high as 7 for 300 kVp X-rays. These
dose-increase factors were obtained for radia-
tion-induced damage measured as Mi seedling
injury, chromosome aberrations in mitotic
cells of Mi seeds, and chlorophyll-deficient
mutations in M2 seedlings. With seeds differing
in moisture from 2.5 to 13 per cent, the oxygen-
effect factors measured by seedling injury
ranged from 9 to 1, respectively. Very dry
irradiated seeds stored at room temperature
appeared to maintain indefinitely their high
sensitivity response to oxygen. Seeds stored at
higher temperatures exhibited a reduced oxygen
sensitivity but developed greater radiation-indu-
ced seedling injury. These results are consistent
with established evidence from electron spin reso-
nance analyses of the influence of temperature,
moisture, and LET on the interaction of oxygen
and radiation products in seed embryos.

Research supported by U.S. A. E.G. Contract
AT (45-l)-353, U.S.P.H.S. Grant GM 10838-05
and funds provided for Medical and Biological
Research by State of Washington Initiative
Measure 171.

5.109. Electron Spin Resonance Studies on Plant
Seeds of Differential Radiosensitivity. S. Bhas-

karan (Karlsruhe, Germany).

Electron spin resonance studies on plant seeds
of differential radiosensitivity were conducted
with a view to evaluate the relationship between
the production and decay of radicals and the
observed differences in the radiation sensitivity.
Dry seeds (moisture content 5 and 2 per cent

94

Section 5 — Mutagenesis

equilibrated over drying agents) of mustard,
tobacco and Agrostis stolonifera were irradiated
with different doses of X-rays and the number of
long lived radicals produced was determined
employing ESR technique. A linear dose effect
relationship was obtained in all the three cases
at different moisture levels. The yield of radicals
in super dry seeds was much higher than in moist
seeds. At 5 per cent level, the yield of radicals
was least in mustard for a given dose as compared
to tobacco and Agrostis. Agrostis yielded a
greater number of radicals than tobacco.
However, at 2 per cent moisture content, this
relationship was altered in so far as mustard
yielded the maximum number of radicals per
gram for a given dose and no difference was
found between tobacco and Agrostis.

The part played by oils and melanin in mustard
will be discussed in explaining the radical yield
at different moisture levels. The results on the
effect of NO, pretreatment with heat and
irradiation at low temperature will be discussed
in relation to the observed biological effects.

5.110. Naturally Occurring and Artificially Induced
Mutations in Maize. Angelo Bianchi (Milano,
Italy).

Analysis of the genetic variability detectable
in open-pollinated populations of maize reveals
that mutations occurring in nature are almost
exclusively point events. On the contrary the
greatest majority of the artificially induced
mutations turn out to be of chromosome type.
So far this picture has not been changed greatly
with physical and chemical treatments performed
under different experimental conditions, even in
experiment designs of high resolving power. In
maize such a requirement has been met, with
increasing reliability, using: (a) endosperm
markers; (b) heterozygotes for suitable plant
characters; (c) pollinator stocks possessing
translocations between A and B chromosomes;
and (d) the waxy locus affecting pollen grain
composition. In the first and in the last case, the
treatments are performed preferably on the
pollen mother cells and/or on the mature pollen,
in the other cases the target is usually the seed.
In all cases, several factors (as temperature,
moisture content, storage time, type of X-rays,
dose fractionation) have been varied with the
consequence of changing at times significantly
the total effect, but hardly the type of response.
In the chemical mutagenesis some compound
(ethylmethansulphonate) turned out to be
highly efficient when administered to seed, much
less when applied to pollen, while the contrary is

true of diepoxibutane. For the latter, moreover,
the isomeric forms show significantly different
mutagenic activity. While chemical mutagenesis
is affecting the hereditary material of maize in
some fundamental aspects similar to those of
physical agents, some difference has also been
detected.

X-ray treatment of very sensitive microsporo-
genesis stages, such as to induce more than 90 per
cent of pollen abortion, have caused an appreci-
able increase of wx ->- Wx events, on the basis
of pollen grains phenotypes. Since this appears
the first case artificially obtained back mutation
in maize, confirmatory data are necessary.

5.111. Radiation Induced Modification of Paramuta-
tion Expression. Duane B. Linden (Mayaguez,
Puerto Rico).

Paramutation as it is expressed at the R locus
in maize involves an interaction between the
gene undergoing a change (Rr) and a source
capable of inducing this alteration (Rst or Rmb).
The components of this system were subjected
to radiation before being brought in contact to
determine their radiosensitivity and to obtain
additional information regarding the nature of
the paramutation phenomenon.

When the site for paramutation change (R1)
was irradiated before crossing to Rst or Rmb the
resulting progeny fall into three categories: one
group in which there is no apparent effect of
the radiation; a second group in which there is
apparent complete inactivation of the para-
mutation process; a third group with reduced
paramutation alteration. The frequency of the
second and third categories suggest that radiation
results in an inactivation type event rather than
inducing a gene mutation alteration at the Rr
locus.

Irradiation of the regulator stocks revealed
differences between the two sources. When the
Rr source was irradiated prior to crossing with
Rr and then testcrossed there was a very low
incidence of complete inactivation of paramuta-
tion but the partial alteration to a less complete
change occurred very frequently. The alteration
was always in one direction toward a less efficient
paramutation alteration. However when the Rmb
source was tested in this manner the results differ-
ed. There was appearent complete inactivation in
some crosses. In other crosses there was an in-
crease in efficiency of inducing paramutation
alterations, as the degree of change was greater
than the controls.

95

Section 5 — Mutagenesis

5.112. Effect of Gamma-radiation on Cytology of
Microsporogenesis in Pinus rigida. Francois
Mergen (New Haven, U.S.A.).

A cesium-137 (9500 curies) source was placed
in a natural pitch pine-oak forest at the Brook-
haven National Laboratory. Beginning on No-
vember 22, 1961, the forest was exposed to gam-
ma-rays 20 hr each day. Pitch pine trees were
exposed to radiation levels ranging from a high
of 960 r/day to a level of normal background
radiation. Microsporangiate strobili were col-
lected at regular intervals to follow the effects of
gamma radiation on the cytology of micro-
sporogenesis.

Trees receiving 82 r/day and higher were un-
able to resume cell division and differentiation
during the spring of 1962, while microspore
mother cells were formed on trees receiving 75
r/day. At 56 r/day meiosis with subsequent for-
mation of tetrads took place, and the highest
level at which mature microspores were collected
was 14 r/day. A slight reduction in the length of
the strobili was observed at 0.4 r/day, and at
3.7 r/day they were significantly shorter than the
controls. The percentage of cells with visible
chromosome aberrations increased with an in-
crease in exposure, e.g. 0.36 r/day = 1 per cent,
7 r/day = 2 per cent, 11 r/day == 12 per cent,
56 r/day == 76 per cent. Illustrations of these
chromosome aberrations are given. The data will
be published in full in Radiation Botany.

5.113. Dependence of the Effect of Dose Rate on the
Physiological State of Irradiated Objects. N. I.

Nuzhdin, R. L. Dozortseva and N. S. Sa-

MOKHVALOVA (MOSCOW, U.S.S.R.).

of forced and organic dormancy. The frequency
of cells with chromosome aberrations (bridges
and fragments) in the first mitoses of rootlet tips
was under study. The rootlets of non-irradiated
seeds of the same generation served as a control.

The cytological study of the rootlets of seeds
irradiated in the state of forced dormancy showed
a definite difference in the frequency of cells
with chromosome aberrations. The irradiation
with high intensity led to the appearance of more
chromosome aberrations than the irradiation
with low intensity (4 1.46 ± 2.38 per cent and 30.79
± 1.43 per cent for one strain and 20.16 d: 1.20
percent and 10.38 ± 0.82 per cent for the other,
respectively).

The results of the irradiation of seeds in the
state of organic dormancy were not the same.
In the cells of the rootlets of seeds germinated
immediately after they were brought out from
the state of organic dormancy the chromosome
aberrations percentage was in fact the same for
the irradiation both with high and low intensity
(9.45 d: 1.56 per cent and 6.21 ± 1.5 per cent for
one strain and 11.97 ± 1.12 per cent and 10.51 :
0.97 per cent for the other, respectively). In the
seeds which were brought out from the state of
organic dormancy at the same time, but grown
after 10 days' storage in air-dry state the influence
of irradiation intensity is distinctly seen. The high
dose rate produces a higher yield of cells with
chromosome aberrations than the same dose but
of lower intensity (23.39 ± 1.36 per cent and
14.39 = 1.09 per cent, respectively).

The data obtained indicate that irradiation-
induced chromosome damage cannot be con-
sidered as a mechanical effect or a direct result of
separate ionization events. They mainly depend
upon the physiological processes in the irradiated
cells.

Sax (1939) made an attempt to explain the
difference in the frequency of exchange-type chro-
mosome aberrations when using the same dose
of different intensity by his hypothesis that this
difference is determined by not-identic possibility
of the broken chromosome ends recombination.
The results obtained by Lane (1951) and Koller
(1956) led to physiological explanation of nature
of the different effect of irradiation intensity.

The hypothesis of Sax can be applied only in
the case of irradiation of dividing cells which
have active metabolism. In view of this peculiar-
ity we irradiated air-dry seeds of the Odessky-17
and Wintering Moskovsky strains of barley by
y-rays Co-&0 with doses of 12 and 10 kr. In the
1st case the dose rate was 423 and 55 r/min, in
the 2nd one 301 and 43 r/min. The irradiated
seeds were in two physiologically different states

5.114. Genetic Advance in the Height of Plants In-
duced by Irradiation in Wheat. Katarina Bo-
rojevic (Novi Sad, Yugoslavia).

The height of plant as a quantitative character
is controlled by polygenic system and it may be
changed by irradiation. For this reason the dry
seed of wheat was treated with the different
dosages of X rays and thermal neutrons.

In the Ri both irradiations reduced the height
of plants from 1 to 32 per cent depending on the
dosage. The variability of this character was in-
creased only in the group treated with high
dosages.

In the R2 and R3 the height of plants remain
reduced in all treated groups. Compared with

96

Section J — Mutagenesis

the untreated material, the heritability has in-
creased in treated material and was higher in the
R.3 than in R4.

The genetic advance in the height of plants is
calculated for all lines which were expected to be
10 per cent shorter than the control.

5.115. Segregation Ratios of Induced Mutations in
Durum Wheat. G. T. Scarascia Mugnozza,
A. Bozzini and F. D'Amato (Pisa, Italy).

Since 1956 experiments were carried out on the
induction of mutations in durum wheat by X-
rays, fast and thermal neutrons, ethylmethane-
sulfonate, diethylsulfate and ethilenimine. So far
more than four hundred independently obtained
cases of viable mutations have been isolated;
moreover several hundreds of lethals are being
maintained as heterozygotes. This material is
the object of genetic and cytogenetic analyses.
Data are presented on segregation ratios in the
progeny of M2 and M3 individuals heterozygous
for induced mutations and of M2 progenies of
the cross of viable mutants to the mother line
(total mutations analyzed: 51). In a total of
28 M2 individuals heterozygous for pigment
mutations (2 albina, 2 xanthalba, 3 chloroalbina,
7 xantha, 2 virido-albina, 7 tigrina, 1 virescens,

2 chlorina, 2 anthocyanic) 19 gave a M3 progeny
with a 3: 1 segregation ratio (mutant condition
recessive). In the remaining 9, the proportion of
mutant individuals was much less; but in M4 al-
ready four out of them attained the 3 : 1 ratio.
This result seems to indicate that the segregation
deficit shown by induced pigment mutations in
the first generations following treatment may be
reduced or eliminated, possibly through pro-
gressive elimination of deleterious genetic chan-
ges accompanying the mutation. F2 analysis of
the crosses between pigment mutants and the
mother line showed that 6 of them behaved as
monogenic recessive (2 tigrina, 1 virescens, 1 viri-
dis, 2 chlorina), whilst 2 tigrina mutations were
inherited as semidominant. As to morphological
mutations, only the progenies of 6 M2 hetero-
zygotes and the F2 of 9 crosses "mutant x mother
line" have been analyzed so far. Out of these 15
mutations, 10 behaved as monogenic recessive
(2 with supernumerary spikelets, 2 semisterile,

3 waxless, 1 with defective endosperm, 1 with an-
thocyanic glumes, 1 with solid stem); in the re-
maining 5 (1 dwarf, 2 waxless, 1 anthocyanic
glumes, 1 brachytic) a strong deficit of indivi-
duals with mutant phenotype was noticed, in
connection with a high degree of sterility.

5.116. Chemical Mutagenesis in Triticum. H. K.

Shama Rao (Bombay, India).

Ethyl-methane sulfonate (EMS), unlike radia-
tions, has been shown to induce chlorophyll
deficient sectors with or without notch or punch
formation and necrotic patches in leaves as well
as speltoid, sub-compactoid, compactoid and
compactum-spikes, during the treated generation,
in the monosomies 2B and 6B of the hexaploid
Chinese-Spring wheat W.

In both mono-2B and 6B, phenotypic changes
during M2 appeared in almost equal numbers
out of a total of 37 affected families amongst
124 progenies raised from all the Mi sectorial and
non-sectorial plants. However, the mutation
spectrum differred. The chlorophyll defectives,
viz. lutescent, chlorina and freckled, were noticed
only in mono-2B. In addition, 2 other families
were with grass-clump types and another with a
rudimentary-spike character.

In mono-6B, lethal dwarfs having short, thick
and dark-green leaves were observed in 2 families.
Another family was with a distinctive type of
spike mutation, viz. "Missing-Spikelets", a
character not observed in any of the nulli-, or
tetrasomics; nor ever reported as appearing
following irradiation.

The types of mutation common to both mono-
2B and 6B were those with increased or decreased
awn development and without hooded character
which could all be accounted for by simple
deficiency of known genes. Two other spike
mutations observed were of semi-speltoid and
base-compact types.

The chlorina, a number of lethals and the
mutants with missing-spikelets and rudimentary-
spikes segregated in approximately 1 : 3 ratio
showing that some of these, at least, involved
single genes. The modes of their inheritance are
under investigation.

1. Shama Rao and Sears, Records of the Genetics
Society of America, 1962.

5.117. Different Mutation Spectra among Related
Strains of Antirrhinum majus, obtained after
Irradiation. Gertrud Linnert (Berlin, Ger-
many).

Two subsequent radiation experiments were
undertaken on normal and mutant strains of the
pure line designated "Sippe 50" by Erwin Baur.
At first pollen was treated with 3000 r; the pro-
genies of 14 plants resulting from this experiment
(= X0 of the following experiment) were once

97

Section 5 — Mutagenesis

more irradiated as seedlings with 300 r. From
every plant 59-158 progenies were grown total-
ling 1488. 237 mutations appeared among them.
The mutation spectra of the 14 X°-plants were
quite different, because 19 phenotypes repeatedly
emerging were not distributed at random, but
accumulated within closely related strains. 6 of
the phenotypes appeared 2-4 times within the
progenies of a single Xo-plant, 9 others were
detected among kindred plants. In addition mu-
tations appeared in mendelian proportions of the
progenies as a result of the first irradiation. These
mutations of course were not included in these
dates. The first radiation experiment yielded
similar results with respect to the mutation spec-
tra as the second one did. Coincidences so fre-
quently occurring cannot be due to mere chance;
therefore the conclusion must be drawn that
some of the loci in different strains excelled above
the others in their particular disposition for
mutations.

5.118. Effects of Ionizing Radiations on Cucumis
sativus L. and Momordica charantia L. Filo-
mena, F. Campos and Walderico M. Gene-
roso (Laguna, Philippines).

Results obtained from subjecting seeds of
Momordica charantia L. and Cucumis sativus L.
to various levels of X-rays and r-rays showed
that germination was relatively unaffected. How-
ever, differences in the relative sensitivity of cuc-
umber seeds to X-rays and r-rays were observed.
Xi and Ri seedlings of both species beyond 15
kr exhibited chlorophyll and morphological
abnormalities. Significant differences between do-
sages were observed when analysis was based
on growth of the plants in terms of length of
the main vine. Regardless of treatment, days to
floweringwas relatively unaffected by irradiations.

In Momordica there was an increase in the
proportions of male to female flowers in the
X-ray series but none in the Co60 series. On the
other hand, sex ratio in cucumber was not altered
by irradiation although reductions in the number
of male and female flowers were noted. Sex
reversion was observed only in Momordica plants
on the treated and segregating generations. Two
types of sex-reversed flower were observed.

Seedling mutations observed in the X2 and R2
generations were mostly chlorophyll abnormali-
ties. However, a few clear-cut morphological
abnormalities were detected. A few mutations
having no conspicuous effect in the seedling stage
but causing distinct variations in the later devel-
opment are also included.

5.119. New Mutations Obtained in Vicia faba L,
through the Injection of Chemicals. Pierre
Bryssine (Rabat, Morocco).

Sundry chemicals were injected by means of a
hypodermic syringe into the hollow inside the
stem of V. faba L.

The chemicals, though extremely toxic and
injected at considerable rates (10-30 ml per stem),
are rapidly absorbed by the tissues. The conse-
cutive depressive effect was evaluated from the
variation of the production of pods (number)
and seeds (weight and number).

Considerable changes were observed on the
treated plants: sterility, growth disorders, fascia-
tion, chlorosis and variegation of the foliage,
dichotomy of branches, anarchic proliferation of
medullary tissues, disorders of the vascular
system, etc.

From Ci onward several types of mutants
could be observed concerning:

(a) The vegetative system (growth habit, bran-
ching type, shape of leaf, chlorophyll defi-
ciency, etc.)

(b) The pods (shape, size, pubescence)

(c) The seeds (size, shape, colour of seed coat
and hilum)

Attention is drawn to the rare or hitherto
unknown forms among these mutants (from C2
and C3 onward):

1. Geant (stems of more than 2 m with long
internodes)

2. Unifoliolata (with a single leaflet)

3. With tendrils (lanceolate leaflets, branched
tendrils)

4. Begonia (prostrate plants with very thick
leaves)

5. Wild (dwarfed erect plants with small seeds)
As to the experienced chemicals, the best re-
sults were obtained with mitoclasic materials (at
high concentrations: colchicin, acenaphthene,
gamma-hexachlorocyclohexane) as well as with
extracts of old seed (tobacco, soya, chick-pea).
Methane-sulfonate-ethyl and acetone equally
proved very interesting.

The advantage of the injection method is dis-
cussed.

5.120. Radiation-induced Reversions to Prototrophy
in Tryptophan-requiring Escherichia coli WP2
B. A. Bridges and R. J. Munson (Berkshire,
Great Britain).

We have attempted to follow gene duplication
in populations of Escherichia coli B/r (strain WP2,
tryptophan-requiring) by irradiating at intervals
during synchronous nuclear replication. A late

98

Section 5 — Mutagenesis

log. phase culture, grown at 25c in glucose-tryp-
tophan-salts medium, was centrifuged and the
cells resuspended in salts medium (l-2xl08/ml)
and incubated for 48 hr at 25° with aeration.
This resulted in a population of largely uninu-
cleate cells.

Upon addition of glucose and tryptophan,
DNA synthesis began and the amount in the
cells had doubled by about 150 min. At this time
the nuclei were seen to divide with a reasonably
high degree of synchrony. No cell division oc-
curred during this time. Aliquots of the suspen-
sion were irradiated (2000 rad 250 kV X-rays)
at intervals during the period of 150 min and
estimates of the number of induced mutants
(revertants to prototrophy) were made immedia-
tely after irradiation.

If the genes for tryptophan requirement re-
plicate in synchrony, a rapid change in the num-
ber and type of induced mutants might be ex-
pected to result. It is hoped to present results
obtained using this technique with WP2 and
other strains of E. coli.

5.121. (D.) Somatic Mutations in Ephestia kiihniella:
Their Dose-relationships in the Low and High
Dose-range of X-irradiation and the Influence
of Pretreatment on the Mutational Spectrum.

Ilse Muller (Koln-Lindenthal, Germany).

After irradiation of young pupae different
types of mutant scales are to be observed on the
hind wings of adult moths, their absolute and
their relative number depending on the dose of
X-rays. From 20 r up to 1000 r the frequencies of
the mutants are proportional to the square of
the dose, approximately. The spontaneous and
the 10 r-frequencies are lower than expected from
the curve in the higher dose-range.

If pupae are kept at different temperatures for
some hours prior to irradiation, the mutational
spectrum at certain doses differs significantly
between the groups which were subjected to low
resp. high temperature treatment. This difference
is brought about by a change in the exponents
of the dose-effect curves, which is alike for all
mutant types, and a change in the sensitivity to
irradiation, the amount of which is larger in the

types with a relative low frequency than in the
more frequent ones.

Graphs and formulas, showing the quantita-
tive relationships, will be presented. The different
mutant scales will be demonstrated under the
microscope.

5.122. Contribution to the Determination of the Rules
of Radioresistance of Agiarian Plants. E. San-

duleac (Bucharest, Rumania).

The Rumanian research works from 1959-
1962, with wet and dry irradiation of the seeds,
in order to determine the critical dose for the
main agrarian plants irradiated with u, x and (3
emitents, have shown that the most radioresistant
plants are the following: Linum ussitatissimum,
Brassica rapa, Sinapis sp., Lallemantia iberica,
which have the critical dose = 100,000 r; after
that Ricinus communis, Sesanum indicum and
Camelina sativa have more than 50,000 r. A few
textile plants like Abut Hon avicenaew ith 1 00,000 r
and Cannabis sativa with 30,000 r are also radio-
resistant. Nicotiana sp. has the critical dose over
50,000 r too. From Gramineae the following are
radioresistant: Oriza sativa > 80,000 r, Sorgum
sp. > 30,000 r, Panicum miliaceum > 25,000 r,
Avena sativa 25,000 r, Hordeum sp. 20,000 r;
relatively resistant are Triticum sp. 15,000 r and
Secale cereale 15,000 r. Radiosensitive are Zea
mays < 10,000 r, and from other families, Helian-
thus annuus < 8000 r, Vicia faba > 5000 r,
Solatium tuberosum > 5000 r, Beta vulgaris 5000
r. The annual Leguminosae have in a majority
the critical dose 15,000 r, Trifolium pratensa
20,000 r, Medicago sativa 25,000 r, Melilotus
albus 15,000 r, Pisum sp. 10,000 r.

We have established the following: (a) the
amplitude of the doses is narrow for cereals about
the dose 15,000 r, but the Leguminosae have a
variable radioresistance ;(b) the oily plants have
the bigest radioresistance; (c) the plant roots are
radiosensitive; (d) the polyploid sorts are more
radioresistant than those with 2 n. The elabo-
ration of a precise method for using the radiations
as action factors which condition the change of
plant heredity must have as a scientific base the
rules of the radioresistance.

99

SECTION 6

CYTOLOGY

6.1. Heteropycnosis and Sex Chromosomes in Mosses.

Lewis E. Anderson (Durham, U.S.A.).

Heterochromosomes have been studied and
figured in a large number of species of mosses.
Invariably these heterochromosomes are hetero-
pycnotic and are represented in interphase
nuclei as conspicuous darkstaining condensed
chromosomes that remain in a tightly coiled
condition. Heterochromosomes have been
postulated to represent sex chromosomes in
mosses, although there has been no direct
evidence that this is the case. The present paper
presents evidence that the heterochromosomes of
mosses are sex-associated. The heterochromo-
somes are represented in interphase nuclei by
heteropycnotic bodies of unequal size. In
sporophytic tissue, a large and a small hetero-
pycnotic body is distinguishable; in gametophytic
tissue of the moss plant, archegonial plants pos-
sess nuclei with the large heteropycnotic body
while antheridial plants possess nuclei with the
smaller heteropycnotic body. It is probable,
therefore, that the heterochromosomes re-
present X and Y chromosomes. Much of the
work reported upon was carried out using several
species of the genus Anomodon.

6.2. A Genetic Test of the Chromosome Replication
Pattern in Maize. G. M. Greenblatt (Shore-
wood, U.S.A.).

Previous studies of twinned red-light variegated
mutations in the pericarp of maize (Greenblatt
and Brink, 1962, 1963) have shown that Modu-
lator {Mp) can transpose from the P locus (cen-
trally located on the short arm of chromosome
one) at the time /'and its conjoined Mp replicate,
and that the receptor sites of Mp may be
either replicated or unreplicated. In the latter
case Mp replicates a second time. Each of these
two possible events appear as distinguishable
twin mutations. An analysis of the sites to which
Mp transposes from the P locus provides a test
of the pattern of replication of this chromosome
arm. By means of a three-point linkage test the
sites to which Mp transposes were measured in
the light variegated sector in both classes of twin

mutations. The results show that the receptor
sites of Mp located on the short arm of chromo-
some one, at which a second replication takes
place, are not randomly distributed between
proximal and distal locations; nine cases of extra
replication were at distal sites while only one was
at a proximal site. In those cases where Mp did
not replicate a second time, four were at proximal
sites and one was at a distal site (the very end of
the chromosome arm). This data strongly sup-
ports the hypothesis that the replication pattern
on the short arm of chromosome one is highly,
but not completely, polarized; the proximal
regions replicate prior to the distal regions.

6.3. Bimitosis. G. Gimenez-Martin, A. Gonzalez-
Fernandez and J. F. Lopez-Saez (Madrid,
Spain).

In this work the mitosis within binucleate cells
of onion root-tip is studied. For this synchron-
ous mitosis the denomination of bimitosis is
proposed. The binucleate cells, an uncommon
condition, are a result of a treatment with
HCCH (y Hexa Chloro Cyclo Hexano). The two
nuclei can be either both diploid or one hypoploid
and the other hyperploid. At the same time, they
can be completely independent or united by
chromosomic bridges.

Many times, these binucleate cells lack com-
pletely the middle lamella between the nuclei.
Other times, the cytoplasmic continuity is main-
tained only through chromosome bridges,
existing in incomplete middle lamella.

Always when a connection existed between the
two nuclei the two mitoses were synchronous. In
this bimitosis both mitoses had either the same
axis or perpendicular directions. The two mitoses
were synchronous even when one nucleus was
hyperploid and the other hypoploid.

The mitotic apparatus showed the same
characteristics as in the normal mitosis. The
separation between the poles is similar to the
normal, although the cytoplasmic space was
sometimes wider.

At last, the interactions between both nuclei
are discussed.

101

Section 6 — Cytology

6.4. Effects of Digitonin on Cellular Division Part I.

L. V. Olah (Carbondale, U.S.A.).

A unique, complex effect of digitonin on
Allium sativum, previously reported by the
author, (1) was analyzed in detail. Digitonin dis-
organizes the spindle, and during nuclear resti-
tution a highly refractive, viscous, hyaline sub-
stance appears. This is always closely associated
with the restituting nucleus. The chromatin
surrounds this substance, entirely trapping it
at times. Simultaneously, synthesis of cell wall
material (PAS and Iodine-H2S04 positive)
originates within the hyaline body, increasing its
size and refringency.

Thus, the shape of the restituting nucleus is
determined by the location, shape, size, and
viscosity of the hyaline body. This mutual
association results in indented, crescent-shaped or
"hollow" nuclei.

Once polysaccharide synthesis begins, it
spreads into the cytoplasm in a haphazard,
ramified manner.

In some cases, semi-recovered cells are able
to form a normal spindle. However, the hyaline
body appears in the same cell, at ana-telophase,
associated with the restituting group of chromat-
ids. PAS testing indicated polysaccharide syn-
thesis in this body, which results in a ramified
cell wall located near the polar region.

The hypothesis is accepted that the phragmo-
plast consists of two categories of elements;
elements of the interzonal area of the spindle and
elements which originate from the polar-nuclear
area. These latter elements were observed in the
area of the restituting nuclei, and according to
Porter etal., <3) ought to migrate to the equator.
Association of both these elements at the equator,
changes the structure of the spindle, resulting in
the appearance of the barrel-shaped phragmo-
plast.

It appears that digitonin hampers the migration
of the "nuclear" elements from the nuclear area.
If a spindle is not present, they appear in the
area of the restituting tetraploid nucleus, and
remain associated with it. In cells able to form
spindles, digitonin blocks the migration of the
polar-nuclear elements to the equator, and they
remain at the polar region, forming a hyaline
body associated with the chromatids of the
daughter nuclei.

Rosette-crystals, formed /'// vivo, similar to
digitonide crystals, were detected in the treated
cells. The role of sterols and the effect digitonin
may have as an anionic detergent is discussed.

Fitogeneticistas e Fitoparasitologistas, p. 10,
1952. Brazil.

2. Olah, L. V. Proceedings of the IX Inter-
national Congress of Genetics. Part II. Caryo-
logia VI, Suppl., pp. 836-838, 1954.

3. Porter, K. R. and Machado, R. D.
J. Biophysical Biochem. Cytology.!, 167-180, 1960.

6.5. Effects of Digitonin on Cellular Division Part II.
Electron Microscope Studies. A. G. Under-
brink and L. V. Olah (Carbondale, U.S.A.).

Allium sativum root tips were treated with digi-
tonin to study the complex effects described by
L. V. Olah.

These root tips were fixed 2 hr in aqueous 2
per cent KMnO-i and treated with 1 per cent
uranium acetate for 1 hr.

The process of the copious and ramifying cell
wall formation and the development of the hya-
line body induced by digitonin were analyzed.
However, using this fixative the spindle substance
was not observable.

Densely packed., circular aggregates of profiles
and vesicular-like elements were found which had
the size and location of the hyaline bodies seen
with the light microscope. In some cells these
aggregates were morphologically identical to the
elements previously termed "phragmoplast" by
Porter et al. (*) Some reconstructing nuclei had
completely enclosed these aggregates and in these
enclosures polysaccharide synthesis was observed.

Most reconstructed nuclei were indented on
one side having one or more branches of the
ramified cell wall system almost always located in
the nuclear indention. Thick and profusely
ramifying cell walls, anastomizing haphazardly
throughout the cytoplasm were observed with
high frequency. Portions of cytoplasm assumed to
be completely encased by cell wall and lacking
chromatin material appeared to be disintegrating.
In other cells the cell wall encircled the nucleus
with a portion of cytoplasm, giving the appear-
ance of formation of a cell within the "mother"
cell.

Many cells were found in which large quanti-
ties of polysaccharide materials occupied large
portions of the volume of the cell.

The identity of the aggregated profiles and
vesicular-like elements with the digitonin-
induced hyaline body described by L. V. Olah
and the relationship of these aggregates to the
synthesis of the unusually large amounts of
polysaccharides is discussed.

1. Olah, L. V. II. Reuniao Latino-Amevicana de

1. Porter, K. R. and Machado, R. D.
/. Biophysical Biochem. Cytology. 7, 167-180, 1960.

102

Section 6 — Cytology

6.6. In Vivo Studies on Mitosis in Neurospora crassa.
J. Weijer, A. Koopmans, and D. L. Weijer
(Edmonton, Canada).

In vivo observation of karyokinesis of the
somatic nucleus of Neurospora crassa reveals
three distinct phases of nuclear division. In the
DNA Replication Phase the continuous genetic
material is oriented along the edge of a disc-like
nuclear structure and replicated as a ring.
During Division Phase the double stranded
filamentous nucleus divides longitudinally.

During Chromosome Phase the divided nucleus
is visible as a structure consisting of seven
chromosomes attached to one another by
thread-like filaments, together with a nucleolus
and a centriole.

6.7. The Communicating Pore between the Nucleus
and the Vacuole in Saccharomyces cerevisiae.

Akira Yuasa (Tokyo, Japan).

By electron-microscopy it has been confirmed
by several investigators that the nucleus of
Saccharomyces cereviseae has a definite mem-
brane which is composed of two-layers (Yuasa,
1958, 1960; Hashimoto and Gerhardt, 1960;
Conti and Naylor, 1960) and also that the
chromatin-threads exist in the nucleus (Yuasa,
1958, 1960; Yotsuyanagi, 1959, 1960).

When fixed with 1 per cent aqueous solution
of osmic acid and stained with aceto-carmine
solution the pore is seen at the contacting place
between the nucleus and the vacuole at the
end of prophase by light microscopy.

By electron-microscopy the ultrathin section of
the cell of Saccharomyces cereviseae also often
shows a figure in which the communicating pore
is seen between the nucleus and the vacuole.

Lindegren (1947, 1947, etc.) already advocated
that the nucleus contacted with the vacuole
and the chromosome appeared in the latter.
The above-mentioned pore is thought to be the
communicating site of the nucleus with the
vacuole at the end of prophase.

6.8. Observational and Experimental Approaches to
Problems of Chromosome Fine Structure.

Helen Gay (Ann Arbor, U.S.A.).

The basic structural component of both the
polytenic giant chromosome and the smaller
mitotic-type chromosome appears to be a fiber
about 100 A in diameter. According to our inter-
pretation, based on observational and experimen-

tal methods, these nucleoprotein microfibrils
constitute a multistranded chromosome. The
mitotic chromosome is composed of a relatively
small number of cytologically separable units,
whereas the polytene chromosome is composed
of many hundreds. Treatment of living, unfixed,
and fixed chromosomes with low concentrations
of pronase, trypsin, or chymotrypsin induces a
separation of the chromosomal strands so that
the subsidiary units can be seen at both the light
and electronmicroscopical levels of resolution.
Analysis, by cytological and cytogenetical
methods, of the effect of deoxyribonuclease and
of 5-bromodeoxyuridine in producing chromo-
somal aberrations reveals that the chromosome
responds as a nucleoprotein complex, the breaks
in some cases extending across the whole chro-
mosome. This response is similar to that evoked
by ionizing radiations. Autoradiographic deter-
minations of the pattern of incorporation of
tritiated nucleosides and amino acids, along
polytene and mitotictype chromosomes supports
the interpretation that DNA in itself does not
constitute the main structural component of the
chromosomes in higher plants and animals.

This research was supported in part by grants
GM- 10499 from the Public Health Service, and
GB-290 from the National Science Foundation.

6.9. The Karyotype of the Domestic Fowl. E. H. New-
comer (Storrs, U.S.A.).

There is now general agreement that the fowl
carries twelve well-defined chromosomes in the
male and eleven in the female. The female is
therefore heterogametic for sex with an XO type
of sex determination, the fifth largest chromo-
some being unpaired.

In addition to these so-called macro-chromo-
somes, there also occurs a variable number
(forty to ninety) of so-called micro-chromosomes
which not only vary in number but range in size
from macro-chromosomal to the limits of mi-
croscopic resolution.

Previous studies from our laboratory have
shown the alleged micro-chromosomes to be
acentric, variably heterochromatic, prone to
fusion and fragmentation and that they disappear
during the second meiotic division of spermato-
genesis. For these reasons they were removed
from the category of chromosomes and named
chromosomoids.

Recent studies by an improved technique of
blood leucocyte culture and autoradiography

103

Section 6 — Cytology

have revealed asynchrony in DNA synthesis
between the chromosomes and chromosomoids,
thus enhancing the grounds for their separation.
Photographs of intact leucocytes at metaphase
show a range in number of the chromosomoids
from sixty to ninety. Karyotype preparations of
these cells confirm previous observations that the
chromosomoids are acentric, variably hetero-
chromatic, morphologically inconstant and as
such do not fulfil any definition of a chromo-
some. These observations together with their DNA
asynchrony indicate that they constitute an
extension to the spectrum of known chromatic
entities in the nucleus. Their genetic properties
can only be surmised at present.
Full details will be published in the Int. Rev. Cyt.

6.10. Cytophotometric Studies on the Spermatogonia
of Nepa cinerea (Hemiptera). Olli Halkka
(Helsinki, Finland).

In the water scorpion, Nepa cinerea, the gonio-
mitotic divisions proceed strictly synchronously
in each cyst. The cyst is the smallest nutritional
unit of the testis. At the 8-cell stage, the mean
nuclear volume is about 430 |i3 but only about
130 u3 at the 128-cell (last gonial) stage. The
corresponding cellular volumes are about 1070
u3 and 340 ia3, respectively. At the 8-cell stage,
each cell has about 250 u3 of cyst surface area for
its metabolism to use while at the 128-cell stage
this area has decreased to about 45 n3. The
nutritional conditions of cells in a 8-cell cyst may
be quite different from those of cells in a cyst
approaching meiosis.

The concentrations of the main chemical com-
ponents of the cell were determined photometric-
ally at different stages of testis development. The
amount of DNA (Feulgen dye — DNAcomplex)
was found to be the same in 8-cell (x = 10.35
arbitrary units at metaphase measured with the
two-wavelength method) and 64-cell (x = 9.87)
cyst nuclei as well as at first maturation division
metaphase (x = 9.30). Thus, the smaller size of
the chromosomes at later gonial generations is
due to increased "condensation" and not to a
change in the degree of polyteny.

During gonial development, no statistically
significant change in the concentration of arginine
(the McLeish modification of the Sakaguchi
test) in the cytoplasm was found. The same is true
of the tyrosine and tryptophane containing
proteins (Millon reaction). The ratio DNA/
cytoplasmic protein therefore was higher at the
64-cell stage than at the 8-cell stage. Highest
interphase concentrations of RNA (Azure B)
were observed at the 8-stage.

6.11. The Formation of Chromosomal Axis in
Mitosis and the Function of Nucleolus. Gon-

pachiro Yasuzumi (Kashihara, Japan).

The present communication is concerned with
electron microscope studies on prophase chromo-
somes of Ehrlich ascites tumour cells and Pelo-
myxa carolinensis, and on nucleoli of sper-
matogonia and spermatocytes of Ascaris megalo-
cephala. Ehrlich ascites tumour cells, amoebae,
and testes of ascaris were fixed in formalin, potas-
sium permanganate or osmium tetroxide, buffered
to pH 7.4-7.8 with Michaelis veronal-acetate
buffer. After fixation, they were dehydrated with
increasing concentrations of ethyl alcohol, and
embedded in Epon epoxy resin or a mixture of
methyl- and n-butyl methacrylates. Sections
were cut with the Porter-Blum microtome
equipped with a glass knife. DNase, RNase and
trypsin were used singly and in combination to
treat formalin-fixed material in block or in
section. The sections were stained with a
slightly modified method of Watson's lead
acetate or uranyl acetate. They were examined
with an electron microscope of the Japan
Electron Company, model JEM-6C, or the
Akashi electron microscope, model TRS, 50E i.

The mitotic early prophase chromosome deve-
lops from the nucleolus towards the nuclear enve-
lope and finally comes to a close association with
the nuclear envelope. An axis has been found in
the early prophase chromosome, consisting of a
pair of tubular elements. In more advanced stages
of prophase the axis is provided with symmetri-
cally disposed processes. In the nuclei of sperma-
togonia and spermatacytes in Ascaris nwgalo-
cephala at least two nucleoli are found, one of
which is composed of numerous dense granules
of varying size an shape, each granule consisting
of an aggregation of dense particles 110 to 150
A in size, but the other is almost compact. The
dense granules are gradually separated to the
composed particles which eventually appear
through the pores of nuclear envelope into the
cytoplasm. Thus the nucleolus seems to have
two functions: the reorganization of chromo-
somes in mitosis and the formation or storage
organ of ribonucleoprotein.

6.12. Effect of Mono- and Divalent Ions on the
Structure of Isolated Metaphase Chromosomes.

Carolyn E. Somers (Houston, U.S.A.).

Recently, methods have been described for
the mass isolation of metaphase chromosomes.
With the isolation of metaphase chromosomes,

104

Section 6 — Cytology

rather than interphase chromosomes, preser-
vation of chromosome morphology can be used
as a significant criterion in the evaluation of the
techniques used. Chinese hamster cells grown
in vitro provide an excellent source of material
for chromosome isolation since 30 to 50 per cent
of the cells can be accumulated at metaphase
with a 12-hr colcemid treatment. Treatment
of metaphase cells for 3 min with 0.1
m sucrose containing 7 x 10~4m CaCl-2 and
3 x 10_4m MgCb serves to hydrate cells sufficient-
ly for chromosome spreading while maintaining
a minimal degree of chromosomal hydration.
Following this hypotonic treatment, cells can be
disrupted in 0.4 m sucrose containing 5 x 10~4 m
each of CaCl2 and MgClo by drawing the cell
suspension into a 30 ml syringe and expelling it
through a 15 gauge hypodermic needle. Divalent
ions are required to preserve chromosome
morphology when chromosomes are isolated in
sucrose. In order to test the effects of ions on
metaphase chromosomes, a drop of 0.4 m
sucrose (5 x 10_4m CaCb and MgCl2) containing
isolated chromosomes was placed on a slide and
covered with a cover slip. Chromosomes were
perfused with 0.4 m sucrose containing desired
test ions. Changes in chromosome morphology
were observed with phase contrast optics.
Chromosomes will swell or uncoil if divalent
ions are replaced by monovalent ions, e.g. NaCl.
Uncoiling can be reversed by subsequent addition
of divalent ions. If extensive swelling or uncoiling
has occurred, the metaphase configuration is
destroyed, and the divalent ions promote a
contraction to the chromonema level only.

6.13. Chromosomes of Normal and Tumour Epithelioid
Derivatives (2n=22) of the Chinese Hamster.

George Yerganian (Boston, U.S.A.).

Normal adult epithelia derived from various
organs of the Chinese hamster, Cricetulus
giiseus, fail to proliferate in vitro beyond several
passages. In contrast, "spontaneous" carcino-
matous derivatives proliferate extensively and
have presently exceeded the 45th passage while
averaging 18 hr generation times. In addition to
the retention of classic diploidy, tumor de-
rivatives lack contact inhibition, display a
metaphase: anaphase ratio of 10 : 1, and an
average single cell plating efficiency of 20 per
cent.

Eighty per cent or more of the metaphases
are classic diploid, and some 5 per cent each are
hypodiploid, hyperdiploid, and tetraploid. Equal-
ly stable are clonal derivatives (XiO quasidiploid
4- trans I: III, and In + Iso LAIII). Each clonal

subline exhibits varying degrees of cytopathic
effect (CPE) caused by an adenovirus (type
undetermined) isolated from one of five original
primary sublines.

Of possible significance is the slight morpho-
logical variation in the short arm of the hetero-
chromatic X in a good portion of normal and
tumor epithelioid forms. In the majority of
fibroblast-like derivatives, this heterochromatic
segment or X2 is slenderer and undergoes late
DNA synthesis. In epithelioids, this portion ap-
pears intermediate in dimensions or X 1-2. If
this morphological feature is substantiated by a
corresponding intermediate type of tritiated
thymidine uptake pattern, epithelioid derivatives
would then have an X1X1-2 status, whereas
fibroblast-like cells feature the moie differenti-
ated or X1X2 status.

These observations suggest that some chromo-
somal segments may reflect patterns of nuclear
and cellular differentiation, possess intermediate
levels of heterochromatization (gene expression),
and govern rates of CPE caused by viruses.

6.14. The Chromosomes of A- and B-spermatogonia
of the Mouse. Eva Hansen-Melander (Lund,
Sweden).

In the tubules of the mouse two types of
spermatogonial cells are found, the A- and
B-spermatogonia, which differ considerably in
their chromosomal behaviour. The chromosomes
of the A-spermatogonia resemble from prophase
to telophase the chromosomes of any other
dividing somatic cell. The A-spermatogonia give
rise to new-A-spermatogonia or to B-spermato-
gonia.

From the onset of prophase the chromosomes
of the B-spermatogonia behave differently from
those of the A-spermatogonia. The differences
are visible already during prophase and become
striking during full metaphase. When the division
of the B-spermatogonia is over the cells enter
meiosis.

The heteropycnosis of the sex chromosomes,
particularly of the Y, is conspicuous during
prophase in both kinds of spermatogonia.

6.15. Origin of Sex Chromatin and Related Phe-
nomena. Yngve Melander (Lund, Sweden).

The sex chromatin of the female rabbit
originates during early embryogenesis. The long
arm of one X chromosome becomes positively
heteiopycnotic during interphase as a result of a

105

Section 6 — Cytology

heavy attenuation during anaphase. The stretch-
ing is due to a delayed separation of the daughter
chromosomes in a terminally located segment.
The formation of sex chromatin in mammals is
not an isolated occurrence as similar events
cause cell differentiation at embiyogenesis of
other animals. Thus, in planarians (Palitdicola,
Tricladida) chromosome segments of the blasto-
meres become allocyclic, forming chromocenters
during interphase, after an attenuation during
anaphase. Similarly, more or less long-lasting
chromocenters originate in the blastodermic
cells of a highei fly {Calliphora erythrocephala).
Another result of chromatid attenuation in
planarians and Calliphora is diminution of small
chromosome fragments, also obviously differ-
entiating the somatic cells.

All these events are initiated by a delayed
separation of daughter chromosomes during
anaphase of embryonic cells as is also the well
known elimination of chromosomes in lower
dipterans (Nematocera).

under the terms of a contract with the
Regents of the University of California.

These observations will be published in full
in Hereditas during 1963.

6.16. The Pattern of DNA Synthesis in the Chromo-
somes of the Marsupial Potorous tridactylis1.

Kirsten H. Walen (Berkeley, U.S.A.).

The marsupial, Potorous tridactylis, has 12
chromosomes in the female, and 13 in the male
(XY1Y2). The chromosomes are large and all
pairs are morphologically distinguishable. Kid-
ney and testis cells can be cultured indefinitely
and the cell population remains largely diploid.
Tritiated thymidine labeling trials have shown
the mitotic cycle to be about 32 hr from meta-
phase to metaphase. Early sampling of labeled fe-
male material showed that silver grains were pre-
sent over all the chromosomes except the short arm
of one X chromosome. The same procedure with
male kidney cells resulted in thesametypeof asyn-
chronous uptake of triated thymidine into the
single X chromosome. In general both chro-
matids were labeled in the first metaphase after
exposure to tritiated thymidine, whereas only
one chromatid was labeled in second- and third-
cycle metaphase figures. Frequently, however, it
was observed that the labeling switched from one
chromatid to the other. No open chromatid breaks
or gross chromosomal aberrations were observed
in any of the division cycles.

6.17. The Ciliate Macronucleus. B. R. Seshachar
(Dehli, India).

The electron microscope has revealed, in the
macronucleus of two Spirotrichous ciliates,
Blepharisma and Spirostomum, a system of
greatly elongated filaments disposed inside it in
a random manner. The thickness of the filaments
is variable and each individual filament shows
varying thickness along its length. Sections of
the filament resemble very strikingly those of the
chromosomes of higher organisms. They are
interpreted as chromosomes of the macronucleus.
Each filament is made up of many microfibrils
about 150 a thick, also disposed in a random
manner but with a slight orientation in the long
axis of the filament. Earlier findings of the
author show that on centrifugation and cyanide
action, the macronucleus of these two ciliates
yield long Feulgen positive filaments, corroborat-
ing the observations with the electron microscope.

These greatly elongated chromosomal filaments
would have to be interpreted in the light of the
special attributes of the ciliate macronucleus: its
origin and differentiation from a diploid micro-
nuclear product, its large size (believed to be
due either to polyploidy or to its compound
nature), its capacity for growth and regeneration,
its high DNA content, its amitotic division and
its genie and metabolic functions. It would also
have to be integrated with our knowledge of
DNA structure and mitotic chromosome gener-
ally.

Sponsored by the Office of Naval Research

6.18. Behaviour of Nucleoli in Isolated Nuclei.

L. F. Lacour (Hertford, Great Britain).

Nuclei have been isolated from HeLa cells
without appreciable loss of nucleic acids (Fisher
and Harris, 1962). In isotonic solutions the
isolated nuclei swell and the nucleoli disappear
but reappear when the nuclei recover normal
size in buffered saline. No nucleolar material
can be detected in swollen nuclei in the electron
microscope (Crawley and Harris, 1963). This
behaviour is being investigated by labelling of the
nucleolar RNA with 3H-cytidine before isolation
of the nuclei.

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Section 6 — Cytology

6.19. The Variable Characters of Chromosomes.

Atif Sengun (Istanbul, Turkey).

The chromosomes carry the hereditary units,
the genes, and are responsible for the continuity
of life and of the characters of an organism.
Therefore, it has been accepted for many years
that the chromosomes should have a static
organization represented by a thread with a
definite number of chromomeres, varying in
size and which occupy a special place on the
chromonema. But a comparison of the homolo-
gous chromosomes shows a variation of chromo-
some morphology, e.g. in corresponding phases
of mitotic and meiotic divisions or in different
tissues of the same animal, etc. The morphologi-
cal or structural differences of the homologous
chromosomes described in relevant literature
are: (a) the degree of spiralization, (b) the
length and (c) the thickness (the diameter) of
the chromosome, (d) the number and the
localization of differentiated, specific, regions
such as the heterochromatic parts, the puffs and
the macrochromomeres, etc., (e) the amount of
the non-hereditary substances including the
metabolic DNA and RNA. All these variable
characters together form the investigated and
visible chromosome.

A survey of the literature reveals: (1) the
above-mentioned characters are controlled by
internal and external factors — including the
microenviromental ones, (2) an alteration of a
character of the chromosome is correlated with
certain, even with definite physiological variation
of chromosome, (3) Some substances are formed
and given off into the surrounding medium by
the chromosome, (4) certain structures of the
chromosomes are responsible for the formation
of these non-genetical, temporary substances.
Therefore, it may be suggested that the visible
structure or the morphology of the chromosomes
represent the phenotype of a chromosome which
is merely a product of non-visible internal and
external factors. According to the above-
mentioned conclusion the question "What is the
genotype of a chromosome?" is still obscure.

6.20. Heterocyclicity of the System of Cell Nucleus.

A. Prokofieva Belgovskaya (Moscow,
U.S.S.R.).

The study of the behaviour of the parent
chromosomes of the nuclei in Drosophila
melanogaster salivary gland cells has shown
that in a number of cases lack of conjugation of
homologous regions is caused by a difference
of the cyclic state of the chromosomes or their

corresponding regions. Heterocyclicity of chro-
mosome regions becomes increased in the cases
of rearrangements. Distal regions IABI brought
near to heterochromatic region 20A in the
X-chromosome of homozygous female sc8 differ
by their states and do not conjugate in 27 per
cent of the nuclei.

The study of the cell nucleus on early stages of
development of Salmo salar, Corregonus baeri
and Cyclops serrulatus showed that parent
chromosomes till to the stage of middle gastrula
remain isolated and up to the late blastula differ
from each other in many respects, such as:
character of spiralization of prophase chromo-
somes, time of their mitotic reorganizations and
sometimes the intensity of the nucleal reaction.
During prophase the parent chromosomes form
two separate skeins of threads oriented towards
opposite cell poles. Parent metaphase chromo-
somes in many blastomeres are oriented in
different plates. In late blastulae parent chromo-
somes distinctly differ in their ability to form the
material of nucleolus.

While investigating chromosomes of ir-
radiated embryos of Salmo salar and Misgurnus
fossilis (X-ray, 800r, stage of 8 blastomeres)
differential damage of parent chromosome
complexes could be stated. This could be
connected with a difference of their cyclic states
at the time of irradiation.

Heterocyclicity of parent chromosome sets is
clearly expressed in a primary culture of human
embryonic fibroblasts. It leads to the formation
in a number of cells at various stages of mitosis
of separate chromosome complexes. With the
aid of thymidin - H3 and autoradiography it
could be shown that homologous parent chro-
mosomes differ in relation to the reduplication
cycle of their DNA as well. This peculiarity is
highly characteristic for the homologues of the
X-chromosome pair, and slightly expressed for
some autosomes in the primary culture of
embryonic fibroblasts and leucocyte cultures of
periferal blood. So far as observations show
the high grade of heterocyclicity of parent
chromosomes present during early stages drops
down in the succession of cell generations. It is
possible that the levelling of parent chromosome-
cycles presents one of the factors of chromosome
conjugation and transformation of mitosis into
meiosis.

The data obtained confirm the assumption of
former cytologists concerning prolonged inde-
pendency of parent chromosome sets (Hacker,
1895; Amma, 1911). These data are considered
in the light of the hypothesis offered by the
author in 1948, according to which hetero-
cyclicity of the system of the cell nucleus is
evolutionary fixed by the peculiarities of the

107

Section 6 — Cytology

development of gamete nuclei and plays a
fundamental role as a factor constituting the
high metabolic properties of the cell.

vacuolae and their chromosomes do not show
any evidence of puff formation in the region.

6.21. Natural and Experimental Modification of
Chromocenters in Interphase Nuclei of Hyme-
nolepis diminuta. L. T. Douglas (Baltimore,
U.S.A.).

The distribution and size of DNA-positive
centers (i.e. chromocenters) within interphase
nuclei of Hymenolepis diminuta appears to be a
function of the tissue in which the nuclei are
located. Chromocenters of nuclei in early blasto-
meres and embryonic tissues are numerous and
small while those in fully differentiated tissues tend
to be larger and less numerous. Intranuclear
contraction centers accentuated in freshly iso-
lated nuclei by 10-2m arginine correspond
morphologically to foci which are positive for
DNA and basic nucleoprotein in fixed material.

It is tempting to conjecture that variation in
distribution and size of intranuclear chromo-
centers in different tissues has physiological signi-
ficance; and, for example, it might be that the
imposition of a confining microscopic geometry
on specific regions of a submicroscopic com-
ponent (e.g. DNA double helices) would alter
the phenotypic expression of a genome in such a
way as to provide, via coding, for different cyto-
plasmic and nuclear enzymes in different tissues.

6.22. Functional Changes in the Polytene Chromo-
somes of Drosophila melanogaster. B. M. Sli-
zynski (Edinburgh, Great Britain).

A recessive autosomal mutant gene in Dro-
sophila melanogaster located in the second chro-
mosome at locus 12.0, called "fat" symbol ft,
produces besides its described effects (short
abdomen, thorax and wings with some abnor-
malities in the morphology of the posterior cross-
vein) also numerous vacuolae in the cells of
salivary gland. The vacuolae are formed at the
end of the second instar. The formation appears
to proceed in a wave starting from the distal end
of the gland. Later the vacuolae disappear and
still later new vacuolae are being formed. The
formation of vacuolae is accompanied by varying
degree of puff formation in the band or bands
corresponding to the cytological location of the
gene, namely in the subdivision 24D/E of the
cytological map of salivary gland chromosome.
The neck cells of the gland do not form any

6.23. Pulse Labeling Studies of Nucleoprotein Syn-
thesis on Drosophila Polytene Chromosomes.

W. Plaut (Madison, U.S.A.).

Drosophila salivary glands excised from ad-
vanced larvae have been incubated in modified
Ringer's medium with tritiated thymidine, ly-
sine, uridine, and cytidine. Results to date indi-
cate that autoradiographically detectable chro-
mosomal labeling can be obtained in 2} min of
incubation. The labeling patterns resulting from
brief incubations in DNA, RNA, and protein
precursors are strikingly different. DNA labeling,
in particular, is frequently discontinuous along
the chromosome. The possible significance of
these synthesis patterns relative to chromosomal
structure and function will be discussed.

6.24. Variability of RNA Synthesis in Polytene
Tissues. Claus Pelling (Tubingen, Germany).

Investigations of RNA synthesis in salivary
gland chromosomes of Chironomus larvae have
demonstrated that the rate of uridine H3 in-
corporation into nuclear structures is highly
variable.

Larvae of the same batch, taken at approxi-
mately the same time under the same experi-
mental conditions, may sometimes differ by
more than 3 orders of magnitude in the amount
of incorporated uridine determined by grain
counts of a given nuclear structure (Balbiani
Ring II).

Different batches, grown up in different
culture vessels, may also differ in the overall
level of uridine uptake.

Salivary glands are not the only polytene tissue
showing variation in RNA synthesis. Autoradio-
graphy of the anterior part of the intestine, the
malpighian tubules, and the rectum also reveal
large alterations in uridine incorporation from
animal to animal. Activities in different tissues
vary independently. Striking differences have
also been observed between cells within the same
tissue. A mosaic-like pattern of nuclear uridine
incorporation is typical for some areas of the
intestine, whereas activity gradients seem to
occur along the malpighian tubules.

In contrast to the polytene tissues, embryonic
diploid nuclei seem to incorporate at a more or
less constant rate.

All observations point to the conclusion that

108

Section 6 — Cytology

variation of RNA synthesis is a characteristic
feature of polytene cells of Chironomus. Attempts
to control the rhythm of synthesis in salivary
glands by modifying light and temperature con-
ditions have so far failed.

6.25. Localization of Deoxyribonucleic Acid exclu-
sively in the Bands of Drosophila Salivary Chro-
mosomes. Dale M. Steffensen (Urbana,
U.S.A.).

Drosophila salivary chromosomes were labeled
with HMhymidine to study the distribution of
radioactive DNA by quantitative autoradio-
graphy. Stretched salivary chromosomes were
examined for the distribution of silver grains
(radioactivity) produced from tritium over bands
and interbands. The evidence leaves little doubt
that DNA is primarily if not exclusively in the
bands. On the other hand the interbands would
appear to contain no more radioactivity than
background and therefore little or no DNA.

A variety of physical considerations were ex-
amined and it was calculated that DNA must be
only in the bands and probably absent in inter-
bands. The theoretical evaluation showed that
radioactivity would have been detectable if
DNA were in interbands. The presence of DNA
in interbands proved to be highly unlikely. All
in all it would appear that Bridges was right all
along in his hypothesis that genes (DNA) are
located only in the bands.

As might be expected there was intense radio-
activity from heterochromatic regions indicating
a high concentration of DNA. Regions with few
bands and "puffs" exhibited little radioactivity.
In some puffs, DNA may be absent altogether.
The temporal inactivity of "heterochromatin" is
discussed in light of recent evidence about
localized RNA synthesis and chromosome differ-
entation.

This study was supported by the National Sci-
ence Foundation (NSF G 17600).

6.26. Structure and Function of the Y-heterochromatin
in Drosophila. G. F. Meyer and O. Hess (Tu-
bingen, Germany).

In spermatocyte nuclei of Drosophila structural
modifications of the Y-chromosome occur which
have been shown to be homologous to the loops
of lampbrush chromosomes. Several pairs of
such structures can be distinguished on a mor-
phological basis (e.g. in D. hydei, "threads",

"clubs", "pseudonucleolus" and "tubuli"). By
analogy with lampbrush loops these structures
have been considered as regions of the Y-heter-
ochromatin whose activity is increased enorm-
ously. This can now be directly demonstrated by
autoradiography and by experiments using
actinomycin C. Evidence for the genetic func-
tions of loop-forming loci comes from a study
of the correlation between the involved regions
of the Y-chromosome and the length of sperma-
tozoa. Spermatozoal length varies between and
within species according to the total mass, the
degree of differentiation, and the number of the
individual types of Y-chromosomal loops. It
seems that almost any alteration in the quantity
of Y-heterochromatin results in changes of the
sperm length. In a X-ray induced Y-autosome
translocation in D. hydei the two translocated
fragments of the Y-chromosome were found to
carry two loop-forming loci each. In the trans-
location stock males occur which are deficient
for one or the other Y fragment and which,
correspondingly, lack two of the four loop pairs.
These Y-deficient males produce immotile sperm
which are shorter than normal sperm. Males
with partial or complete duplications of loops
are also found and show the expected increase in
sperm length. The chromosomal modifications
described here are unique in that they are formed
only by the (heterochromatic) Y-chromosome.

6.27. Amounts of DNA and RNA in the Chromosomal
and Nucleolar Regions of Isolated Plant Nuclei.

John McLeish (Bayfordbury, Great Britain).

Amounts of both DNA and RNA were deter-
mined in nuclei isolated from the root apices of
different species of higher plants. A new method
of isolation was used W by which approximately
50 per cent of the nuclei known to be present in
the roots could be recovered. There was no
significant loss of nucleic acids from these nuclei
during the isolation procedure. The plants chosen
included both closely and distantly related spe-
cies; a polyploid series within one genus; and the
diploid and tetraploid forms of a single species.
The results demonstrated the absence of any
correlation between the amounts of DNA and
total nuclear RNA; the high DNA: RNA ratios
found in most species suggested that, at any given
time, a lot of the DNA is not associated with
RNA.

A new technique for the isolation of nucleoli
from these nuclei has now made it possible to
estimate the amounts of DNA and RNA in both
the chromosomal and nucleolar regions. Al-
though it can be stated that there is no correlation

109

Section 6 — Cytology

between the amounts of DNA and RNA in the
chromosomal regions, the precise quantitative
relationship between DNA and RNA in the
nucleoli themselves is still under investigation.

1. McLeish, J. (1963) Proc. Roy. Soc. B (in the
press).

6.28. Chiasma Distribution at Normal and Elevated
Temperatures in a Locust. S. A. Henderson

(Cambridge, Great Britain).

Using the technique of directly measuring
chiasma position along each chromosome, an
analysis of the normal chiasma distribution has
been made for all members of the complement of
the desert locust, Schistocerca gregaria, where a
wide range of chromosome size is present. On
subjecting this species to high temperature
treatment the pattern of chiasma distribution is
greatly modified. Reduction in chiasma frequency
is accompanied by a change in the position occu-
pied by those chiasmata which do form. This
shift, to the chromosome ends, may culminate in
univalence. Chromosomes of different sizes differ
in their responses to heat-treatment.

very typical configurations ensue whereby the
tips and often also the proximal regions attach,
the more movable parts being strongly displaced
in the centrifugal direction. The more indifferent
parts can be displaced at very low speeds, e.g.
1000 g, especially in interphase and early pro-
phase, less well in later prophase when condens-
ing chromosomes become rigid but in prometa-
phase they are deposited in toto. Breaks of chro-
mosomes with tips still anchored are found and
also indentations in the NM at points where tips,
especially many of them, attach. In most plants
studied there is no bouquet orientation. Just
prior to pairing the chromosomes detach them-
selves and then pair immediately. In early pachy-
tene the "standard" situation is resumed, i.e.
reattachment. The bouquet of Tradescantia and
Liliaceae is complicated: the chromosomes be-
have as if they were totally loose. Yet, there
seems to be a very weak bond — they seem to glide
towards the bouquet arrangement as has been
observed by others before. Pachytene shows
attachment of the tips. Condensed chromosomes
in diakinesis are peripheral in toto, as is well
known. The attachment is firm in pachytene but
in most cases will become strikingly labilized at
diplotene though they seldom detach themselves.
Nonhomologous associations, if present, to some
extent interfere with the pachytene reattachment.

6.29. Some New Principles Governing Chromosome
Pairing — The Spatial Relations of Chromosomes
and Nuclear Membrane. K. Pusa (Helsinki,
Finland).

It has been shown by others that in many
instances in the soma the chromosomes attach
very firmly to the nuclear membrane (NM) by
their (1) heteropycnotic regions or (2) proximal
or (3) distal parts. This has been demonstrated
by micromanipulation or ultracentrifugation
which thereby produces characteristic configura-
tions. There are many observations on mere
attachment by direct inspection of parts (2) and
(3) and the situation of (1) is well known. The
questions are: Can those findings be generalized?
How is it just before and at chromosome pairing?

A study was undertaken on a number of flower-
ing plants, mainly Cruciferae (conspicuous heter-
opycnosis at centromeric regions), Tradescantia,
Liliaceae and Plantago as well as scattered
samples from various families. Centrifugation,
both normal and ultraspeeds (up to 150,000 g)
were employed. In all cases, in the archespore
and early PMC's before pairing the situation is
the same as in the surrounding parenchyma:

6.30. Chromosome Pairing at Meiosis. C. R. Burn-
ham (St. Paul, U.S.A.).

Previous studies (M. Tabata in maize and K.
J. Kasha in barley) indicate that pairing is initia-
ted at or near the ends of chromosomes and not
at the centromeres. This information has come
from hybrids between stocks of interchanges in
which the breaks in both chromosomes were in
opposite arms in the two parental interchanges.
In the previous studies in maize the total length
of the "between-breaks" segments was equal to
or less than the total length of the interchanged
segments. In the combinations now planned the
total length of the "between-breaks" segments
will be greater than that of the interchanged
segments.

Observations (O. L. Miller) on asynaptic (as
as) plants in maize show that the members of
partially synapsed pairs are associated at the
centromeres. The two seemingly divergent obser-
vations can be reconciled if it assumed that in
as as maize plants pairing at the ends of the chro-
mosomes is prevented, but that this may or may
not extend to the remainder of the two homo-
logues.

110

Section 6 — Cytology

6.31. Differential Rates of Meiotic Development in
Desynaptic Hordeum vulgare L. E. B. Wage-
naar (Ottawa, Canada).

Cytological analysis of the first meiotic
division of desynaptic barley (var. Huskey,
2« = 14; homozygous for gene ds) produced
evidence that the developmental rate was not
equal for all metaphase cells. This differential
rate was apparently related to two cellular
characteristics which simultaneously affected
metaphase development. These characteristics
were the degree of irregularity and the chiasma
frequencies per bivalent. Cells with numbers of
bivalents approaching that of the normal con-
dition (7 bivalents), as well as cells with highest
average chiasma frequencies per bivalent, tended
to proceed most rapidly through metaphase I.
Possible underlying factors inducing the observed
developmental pattern are discussed.

6.32. Cytogenetical Studies of Oryza sativa L. and
its Related Species. 5. Differential Conden-
sation and Chromosome Pairing in the Hybrid

0. sativa X O. australiensis. H. W. Li, C. C.

Chen, H. K. Wu and Katherine C. L. Lu
(Taipei, China).

The chromosomes of O. australiensis were
made up partly of heterochromatin whereas
those of O. sativa were almost completely
euchromatic.

In the Fi hybrid of O. sativa O. australiensis,
there was differential condensation in these two
morphologically different types of chromosomes.
The ones with partly heterochromatin and partly
euchromatin condensed early, starting off
presumably from pachynema, on till diakinesis.
The ones with only euchromatin seemingly
started their condensation later but condensed
more complete at first metaphase. Thus before
diakinesis, the australiensis chromosomes were
darker in staining, while at Ml-Al they were
2-4 times the size of the sativa chromosomes.

At either diakinesis or MI-AI, about two
bivalents could be found per PMC. These
bivalents could be separated into two types, at
MI-AI by size, or by difference in having taken
up the stain at diakinesis:

1. Autosyndetic.

2. Allosyndetic (multivalents were also found
but very rarely).

All these bivalents were proved to be authen-
tically true bivalents. The evidences were:

1. There were loosely paired chromosomal
segments observed repeatedly in many PMC's at
pachynema.

2. At diplonema, allosyndetically paired biva-
lents were found to have one chiasma mostly, or
two or more chiasmata in some PMC's.

3. At diakinesis, these allosyndetically paired
bivalents were found to be ring-shaped as well as
end-to-end ones.

4. Closed allosyndetic bivalents with two
chiasmata were frequently observed at MI-AI.

Pairing of the homologous segments in these
allosyndetic pairs was assumed to be carried out
at the euchromatic regions of the two chromo-
somes concerned. Presumably, these euchromatic
regions of the chromosomes from two different
species might have the same rate of condensation
at various stages of meiosis.

6.33. Meiosis of Luzula purpurea. H. Nordenskiold
(Uppsala, Sweden).

Luzula purpurea belongs to the materials, the
chromosomes of which have diffuse or poly-
centric centromeres. Somatically it has six equal-
sized chromosomes. During early prophase the
chromosomes pair and chiasmata are formed.
The chiasmata are easily studied during diakine-
sis, and one or two per bivalent is the most
common number. They may be interstitial or
more or less terminalized at first metaphase.
During that stage each chromosome of the
bivalents is arranged in the equatorial plane, i.e.
is showing auto-orientation.

The stage at which the individual chromatids
can best be studied is the transition between first
anaphase and telophase. At this stage the two
halves of each side of the equatorial plane always
form mirror-images when compared with each
other. This phenomenon illustrates the cytolo-
gically equational mode of the first meiotic
division. Relict chiasmata may occur between the
chromatids of the half-bivalents. This pheno-
menon demonstrates the fact that the two
chiasma-forming chromatids are able to move
towards the same pole. As chiasmata are gener-
ally assumed to be formed between non-sister
chromatids the separation of chromatids in the
points of the original exchange in such cases
ought to have occurred between the sister-chro-
matids. The separating half-bivalents are com-
posed of two chromatids which may be kept
together by the relict chiasmata or their cast
formations, and/or by the ends of the chromatids
belonging to the same original chromosome, the
terminal parts of which seem to separate at a
comparatively late stage in meiosis. At interki-
nesis the homologous chromatids of the half-
bivalents pair again and these pairs form two very

111

Section 6 — Cytology

contracted metaphasic plates before the separa-
tion during second division.

To prove the validity of the described type of
meiosis in a material with a heteromorphic
bivalent, the meiosis of plants with one broken
chromosome have been studied. One hetero-
morphic association is then formed in each
meiotic cell, originating from the pairing between
the two fragments and their homologous un-
broken partner. At first metaphase this asso-
ciation is always an open chain with the large
chromosome in the middle of the association
and the two small ones on each side. This
association separates equationally during first
anaphase giving one long and two short chro-
matids on each side of the equator. At inter-
kinesis the two fragments pair again with their
long partner, the result of which can be studied
at the end of the separation during second
telophase. In the four cells of the tetrad the two
small chromosomes regularly substitute the origi-
nally broken chromosome, giving rise to four
cells, two with three large chromosomes and two
with two large and two small ones.

facilitated by the knowledge that the organisms
are diploid as shown by the cytological evidence,
and not haploid as previously assumed.

6.34. Meiosis in the Sex Organs of the Biflagellatae.

E. Sansome (Zaria, North Nigeria).

Critical cytological evidence showing that
meiosis occurs in the oogonia and antheridia is
given for Pythium debaryanum, Phytophthora
cactorum and an Achlya sp. In the case of
Pythium debaryanum (n = 18 ca.) an association
of four chromosomes was observed in metaphases
in the sex organs. In the case of Phytophthora
cactorum (« = 9 ca.) and Achlya (n = 8 ca.)
multivalents were observed in polyploid nuclei
in the oogonia and antheridia after treatment
with natural camphor. Additional evidence in the
case of Phytophthora cactorum was the observan-
ce of a bridge and fragment at anaphase,
evidently resulting from crossing over in an
inversion.

The fact that two successive divisions occur
and that the size of the nuclei at the end of the
two divisions is approximately half that of the
vegetative nuclei is in full accordance with the
other cytological evidence in indicating that
these two divisions constitute meiosis.

The finding that meiosis occurs immediately
before and not after fertilization has important
genetical implications. A survey of the scanty
genetical data in this group shows that they can
be more readily explained on the basis of the
organisms being diploid rather than haploid.
Further genetical investigations should be

See Sansome, E., Nature, 191, 827 (1961);
Sansome, E., and Harris, B. J., Nature, 196,
29-292 (1962); Sansome, E., Trans. Brit. Myc.
Soc. in the press.

6.35. Which are the Specific Biocatalizers for the
Multiplication of Chromonemata and, conse-
quently, of the Genes or of the DNA Molecules
in Higher Plants? F. Resende (Lisbon, Por-
tugal).

Growth in a pericellular plant is the result
of the growth of its constituent cells. This
growth is till now considered as of 2 types:
(a) cell multiplication and (b) membrane disten-
sion.

In some species of the succulent genera
Bryophyllum and Kalanchoe, growth is extremely
sensitive to photoperiodism, being much greater
under LD (long day) conditions than in SD
(short day). This LD effect results from a much
enhanced growth of the stem internodes, of
petiole length and of leaf area. The amount of
growth biocatalizers is consequently greater in
LD than in SD, IAA (3-indolyl-acetic-acid)
concentration, for instance, may be 20 times
greater (Linskens and Resende).

In plants with arrested growth (i.e. in rosette)
through SD action, nuclei are, however, larger
than in plants under LD and showendopolyploidy,
which may reach 32 x, whereas under LD
nuclei never show more than 8 x (Von Witsch
and Fliigel).

It all seems to function as if biocatalysis govern-
ing cell multiplication and cell membrane
distension does not affect the growth and the
multiplication of the chromonemata and, hence,
the synthesis of genematerial, DNA, and protein.
This synthesis in LD is at most equal (proba-
bly even inferior) to that in SD, and therefore
not related to the general plant growth bio-
catalizers known up to the present. Hence, it is
logical to postulate the existence of specific
biocatalizers for the growth and multiplication of
the chromonemata, and which are independent
from those governing multiplication of the nucleus
and cell and the distension of its membrane. This
hypothesis further explains the cases of ectomi-
tosis (Resende), i.e. nucleus multiplications
without multiplication of chromonemata.

112

Section 6 — Cytology

6.36. Functional Structures in Cytoplasm and
Chromosomes of D. hydei Salivary Gland Cells.

H. D. Berendes (Leiden, The Netherlands).

Each salivary gland of D. hydei contains 100-
150 cells. Larval development at 25°C under
optimal food conditions is completed in 121
hours, both first and second instar lasting
25 hours and the third instar 71 hours. The
development of the gland was observed using
phase contrast microscopy of whole glands
mounted in 0.7 per cent saline at various stages
of all instars, and sectioned as well as squashed
glands at stages of the third instar and prepupa.
Nuclear diameters increase from 4.5 u at hatch-
ing, to 55 u. About 20 hours after beginning of
the third instar, a differentiation can be seen
between proximal and distal part of the glands.
First sign of this differentiation was a faster
enlargement of the distal nuclei, in connection
with a higher polyteny of the chromosomes. The
cytoplasm of these distal cells then produces a
large number of granules with diameters of 1-2 u.
These granules consist of PAS positive material
and are not stained by toluidine blue. At the
end of the third instar these granules disappear
again. At this moment the nuclei of the distal
cells have a diameter of 45-55 u whereas the
proximal nuclei have a diameter of 28-35 u.
During the third instar the cells of the proximal
part never contain PAS positive granules, but
much smaller particles instead. These particles
are positively stained by toluidine blue. They
are also present in the distal cells during the third
instar.

The puffing pattern within the five chromo-
some arms has been studied in relation to the
development of the cells in proximal and distal
part of the gland during the third instar. Dif-
ferences in puffing pattern between both parts
have been found.

6.37. A Technique for Obtaining Chromosome
Preparations of Rat Embryo. M. T. Biola
(Fontenay-aux-Roses, France).

During the examination of chromosome
aberrations of the rat embryo after irradiation
of the mother, we have determined a technique
for obtaining chromosome preparations without
tissue culture.

With this technique we can examine the chro-
mosomes of the cells which were in mitosis when
the animal was killed and observe the real
anomalies of the embryonic cells.

Embryos are taken, minced into small frag-

ments which are digested by the combined action
of both trypsin and hyaluronidase.

After 45 min at 37°C, serum is added to inhibit
the enzymes.

The undigested residues are allowed to settle
and the cells of the supernatant are carefully
removed. The cells are washed with serum and
incubated a few hours at 37°C in a nutritive
medium, supplemented with colchicine.

The mitoses are stopped at metaphase. The
cells are submitted to hypotonic expansion, they
are fixed and spread on slides and air dried.

With this technique we have been able to
obtain metaphase cells with well-spread chro-
mosomes allowing good analysis of the caryotype
of the rat embryos.

6.38. Mode of Action of Metal Blocking Agents in
Nuclear Dissolution. Fernando Silio and
Dolores Angulo (Madrid, Spain).

Versenate and other chelating substances
induce a strong chromatic dissolution and a
remarkable increase of cell volume. From the
beginning we thought that this action might be
due to an indirect process of activation of
proteolytic enzymes. In order to confirm this we
used cyanide that does not have any chelating
action, but is an inhibitor for (heavy metals
containing) terminal oxidases.

The cytological effects obtained with cyanide
were equivalent in some aspects to those induced
by versenate, which confirms our hypothesis
work based on the following facts.

1. Papain, and other papainlike enzymes,
suffer strong activation by cyanide, versenate and
other peroxidase inhibitors. Thyolic compounds
and many other reducing agents, increase this
activation while oxidants inhibit it.

2. The EDTA presents a much greater affinity
for Fe+++ or Cu ++ than for Zn++, which proves
its selectivity at low concentrations. The affinity
for Ca++ and Mg is still lower. Therefore it is to
be supposed that it will react with Fe before it
will do so with Ca and Mg.

3. As the lysis is already verified at very low
concentrations of EDTA or CNK, a very selec-
tive action on trace elements may be assumed,
rather than on macroelements like Ca or Mg.

The enormous increase of cell volume ob-
served means a great imbibition which may be
attributed to a great increase of the osmotic
pressure. All this would confirm an intense des-
polimerization of macromolecules.

With coenzyme A we have obtained similar
effects.

113

Section 6 — Cytology

6.39. Nucleic Acid Profiles of Germ Cells of the
Mouse. John H. D. Bryan (Ames, U.S.A.).

Primordial germ cells may be distinguished
both from their definitive counterparts in the
adult, and from adjacent somatic cells by their
large nuclei and chromosomes. The present work
forms part of an attempt to determine the proba-
ble chemical basis underlying these size diffe-
rences. The results of these experiments should
also contribute to our understanding of chro-
mosome structure. The distribution of DNA and
RNA in sections of acetic-alcohol fixed fetal and
adult gonads and liver tissue has been studied
following simultaneous visualization by means
of azure B staining^).

In certain cases staining was carried out follow-
ing treatment with enzymes (trypsin and/or
RNAase), or chemical extraction of RNA (cold
10 per cent perchloric acid). The results obtained,
though qualitative in nature, suggest that there
may be differences between the nucleic acid-
protein complexes of primordial germ cells and
their definitive counterparts. Somatic cells, on the
other hand, appear to react to the staining experi-
ments in a manner essentially identical with that
of the definitive germ cells.

Supported by grant CA 05591-02 from the
National Cancer Institute, U.S.P.H.S.

1. Flax and Himes, Physiol. Zool. 25, 297-311,
1952.

6.40. (D.) Endomitosis during Megasporogenesis in
Saccharuni. G. Bremer (Wageningen, The
Netherlands).

In many interspecific hybrids of this genus it
was found by Bremer in 1920, and afterwards
also by other investigators, that the somatic
chromosome number of such hybrids equals the
sum of twice the haploid number of the female
parent plus once the haploid number of the male
parent. It appeared to Bremer that this pheno-
menon is based on endo-duplication of chromo-
somes during megasporogenesis after a normal
first meiotic division reducing the chromosome
number to the half.

The endo-duplication will be shown in micro-
scopical slides with drawings to match.

6.41 . Sites of Formation of the Extra Nucleoli during
Early Oocyte Growth in the Freshwater Teleost
Salvelinus fontinalis Mitchill. L. A. Choui-
nard (Quebec City, Canada).

The behaviour of nuclear structures during

early oocyte growth in Salvelinus fontinalis will
be described with special attention focused on
the sites at which the extra nucleoli first appear.
In the species investigated, the process of extra
nucleoli formation begins at about the mid-
pachytene stage of meiotic prophase and pro-
ceeds in an uninterrupted fashion until the late
strepsitene stage is reached; during that period
of time, close to a hundred extra nucleoli form
within the nuclear cavity. From the mid-pachy-
tene up to about the mid-strepsitene stage, our
observations suggest that, while some of the
extra nucleoli arise by successive growth and
detachment from a single large heterochromatic
body adjacent to the oocyte's original nucleolus,
the others develop in contact with minute chro-
mocenters located in the peripheral portion of
the nuclear cavity. The relevant observational
evidence would seem to favour the view that the
heterochromatic body adjacent to the oocyte's
original nucleolus contains one of the four
alleged pairs of nucleolar organizing regions of
the usual nucleolar chromosomes. During the
mid-strepsitene stage, on the other hand, our
observations reveal that, while the heterochro-
matic body and the chromocenters disappear
from view, additional extra nucleoli develop at
various points along the length of a number of
chromosomes. These observations are taken to
indicate that non-heterochromatic chromosomal
regions distinct from the nucleolar organizing
regions of the usual nucleolar chromosomes are,
during that period of meiotic prophase, instru-
mental in the formation of extra nucleoli. The
significance of the above findings will be dis-
cussed in relation to the observations of othei
workers on the sites of formation of nucleoli in
general.

6.42. An Investigation of DNA Replication in Bovine
Sex Chromosomes. Stanley M. Gartler and
Barbara E. Burt (Seattle, U.S.A.).

Both the X and Y chromosomes in cattle can
be unambiguously distinguished from the auto-
somes. The X is a large sub-metacentric chromo-
some, the Y a small metacentric and all the
autosomes are acrocentric. DNA replication
studies using tritiated thymidine in cell cultures
have shown the following: in the male the Y is
the last chromosome in the complement to
replicate while the short arm of the X replicates
shortly before it. The long arm of the X replicates
at a distinctly earlier period. In the female one
X replicates as in the male while the other X

114

Section 6 — Cytology

begins replication a little earlier but both arms
replicate in a relatively short period. Thus, as in
man the X chromosomes in the female replicate
asynchronously and the chromatin body is ap-
parently derived from the X in which the long
arm replicates last.

6.43. (D.) Effects of Digitonin on Dividing Meriste-
matic Cells and Cell Wall Formation. L. V.

Olah (Carbondale, U.S.A.).

6.45. (F.) Mitosis with Undivided Chromosomes in
Irradiated Cells. Gunnar Ostergren and
Jadwiga Mole-Bajer (Lund, Sweden).

In irradiated endosperm of Haemanthus va-
rious disturbances of mitosis occur. An interest-
ing deviation consists in nuclei entering into
mitosis with undivided chromosomes each con-
sisting of a single chromatid only. These are
often associated by means of a presumably un-
specific pairing mechanism that suddenly stops
working with the start of anaphase.

Demonstration of the mitotic cycle of "digi-
tonin" mitosis is represented by a series of micro-
photographs. The ultrastructure of "digitonin"
mitosis is shown by a series of electron micro-
photographs. Alteration of the geometry of mi-
tosis and the origin and function of the phrag-
moplast is shown by a series of schematic
drawings. Phases of "digitonin" mitosis are
demonstrated by microscopical slides.

6.44. Reciprocal Translocation obtained after Col-
chicine Treatment in Phlox drummondii.

T. S. Dhillon (Hong Kong).

Seedlings of Phlox Drummondii were raised in
clay pots. After two to three leaf pairs had
developed, the growing points of seedlings were
smeared with 0.5 per cent emulsion of colchicine
in lanolin. Twenty out of fifty treated seedlings
survived and produced main stem from the
colchicine treated growing point, as well as
lateral branches from nodes below the level of
colchicine application. First meiotic division was
studied in the PMCs of buds taken from the
main stem as well as the lateral branches. Eight-
een treated plants and fifty untreated plants were
found to be meiotically normal and showed
seven bivalents at M.I. Main stem of one of the
treated plants was found to be autotetraploid
whilst the laterals originating from first two
nodes of this plant were diploid. In the remain-
ing treated plant, main stem showed an inter-
change complex involving four chromosomes
and five bivalents. The first and second node
lateral branches of this plant were meiotically
normal, showing seven bivalents at M.I. Whilst
the lateral branches of this plant were completely
fertile, the main stem and lateral branches origi-
nating from fourth node and above, showed 40
to 50 per cent aborted pollen grains and had
reduced seed set.

6.46. (D.) Cytological Studies of Nothoscordon, Al-
lium and Molium. S. E. Eid (Shatby, Egypt).

I. Centromere Structure: Following hydroxy-
quinoline treatment of chromosomes of Notho-
scordon inodorum (Ait) Asch et Gr., the median
centromere could show up to 6 or more pairs of
chromomeres. The terminal centromere, being
a free end, may have a greater chance for ex-
tension, showing up to 8 or more pairs of chro-
momeres. At anaphase I the centromere may
show two structures directed towards the near
pole. These two structures can be seen trans-
versely connected. (To appear in Bull, of Faculty
of Science, Alexandria University, 1962.)

II. Effect of the Environment on Meiosis, in
Nothoscordon inodorum {Ait.) Asch et Gr. and
Some Allium Species:

A. High temperature treatment of flower
buds on Nothoscordon inodorum, leads, in
certain cases, to increase of the chromosome
number from 19 to 32, in the pollen mother cells.
This is attributed to duplication of the 13 meta-
centrics but not the 6 telocentrics, of the com-
plement.

B. Cold leads to asyndesis and high tempera-
ture may lead to asyndesis or equational division
with transitional conditions. (To appear in
Journal of Botany of the United Arab Republic,
1962.)

III. Cytological studies in section Molium of
the genus Allium.

6.47. (D.) Chromosome Reconstruction for Studies on
Mutagenesis and Other Cytogenetical Problems.
I. I. Oster, R. Schwarz and R. Binnard
(Philadelphia, U.S.A.).

Several new stocks and techniques have been
devised in order to facilitate studies on muta-
genesis and other cytogenetical investigations by

115

Section 6 — Cytology

combining mutations currently maintained at
the National Science Foundation-supported
Drosophila Stock Center in Philadelphia with
specific chromosomal disarrangements induced
by radiation in experiments precisely designed
for such purposes. These include: "multi-pur-
pose'* strains for detecting chromosome loss,
sex-linked recessive lethal mutations, trans-
locations, and mutations at 8 specific loci in the
offspring of the same treated flies; improved
methods for detecting mutations arising in the
female germ line; strains for determining the
genetic components of somatic damage induced
by low doses of radiation; stocks for detecting
mutations arising at 20 specific loci in the female
germ line and at 37 specific loci arising in the
male germ line; a method for determining how
often homologous loci mutate simultaneously
following various types of treatment ; several ring-
shaped X chromosomes containing portions of
the Y chromosome; several ring-shaped Y
chromosomes with and without visible markers;
new marker-inversion stocks for "isogenizing"
the whole (or parts of the) genome; and combi-
nations with visible effects in the immature
stages to facilitate recognition of different ge-
notypes during very early life for developmental
studies.

Diagrams of these stocks and techniques, the
manner in which they can be utilized, and the
applicability of the principles in methodology
involved to other organisms will be shown.

This work has been supported by grant
G14148 from the U.S. National Science Foun-
dation.

Information Service ("■) has been utilized for the
following projects:

1. Additional evidence was obtained that
ring-shaped chromosomes in Drosophila are
highly susceptible to loss following irradiation
as shown by their breakage and frequent involve-
ment in anaphase bridges. This is in agreement
with our previous results derived from breeding
tests and confirms our preliminary cytological
observations. These effects can be demonstrated
even after the cells have been exposed to relatively
low doses of rays.

2. Examination of the somatic cells of Dro-
sophila larvae which had been fed chemical mu-
tagens (e.g. urethane, nitrogen mustard) or
compounds currently under investigation as
"chemosterilants"(e.g. "Mapo":tris(l-(2-methyl
aziridinyl) phosphine oxide) by the U.S. Depart-
ment of Agriculture have revealed the presence
of fairly large numbers of broken chromosomes.
In line with the theoretical considerations con-
cerning the mode of action of such alkylating
agents as the lattei, these results tend to indicate
that chemicals which are capable of causing
sterility in insects act primarily via the induction
of genetical damage.

3. Analyses of structurally altered X and Y
chromosomes, particularly newly-synthesized
ring-shaped chromosomes, in Drosophila.

4. Karyotypic analyses of the housefly (Musca
domestica) and the honey bee (Apis melliferd).

5. Detection of the Barr body in human buccal
smears (by the use of some portions of the
technique only). This method yields preparations
which are relatively less distorted than slides
made by the techniques currently in use (e.g.
thionine, cresyl violet).

Data, slides, and photomicrographs illustrating
these areas of research will be shown.

6.48. (D.) Cytological Observations on Drosophila
and Human Material. I. I. Oster, G. Balaban
and R. Binnard (Philadelphia, U.S.A.).

A modified method for observing somatic
chromosomes recently described in Drosophila

This work has been supported by grant
G 24261 from the U.S. National Science Foun-
dation and grant AT(30-1)-2618 from the U.S.
Atomic Energy Commission.
1. Oster. I. I. and G. Balaban No. 37,
142-144 (1963).

116

SECTION 7

CYTOGENETICS

7.1 . Tomato Chromosomes and Their Relationship to
the Linkage Maps. B. Snoad (Hertford, Great
Britain).

The tomato is a rather exceptional plant since
it has been for many years a subject for intensive
genetical research and also it has chromosomes
which are morphologically distinguishable at
pachytene. It is only recently, however, that
attempts have been made to correlate the linkage
maps with the chromosomes themselves. Each
chromosome is composed of distinct hetero-
chromatic and euchromatic regions and it is
therefore possible to compare the genetic
activity of these two regions.

Such a comparison is being made by the use of
X-ray induced reciprocal translocations. The
broken chromosomes are identified at pachytene
and test-crosses are made with appropriate
genetic marker stocks. In this way linkage
between the visible points of chromosome
breakage and the selected genes can be estimated.

The evidence so far indicates that crossing-
over does not occur in the heterochromatin and
that the active genes studied are located in the
euchromatin. The great majority of chromosome
breakage occurs in the heterochromatin so that
linkage between the genes and such points of
breakage is really a measure of the amount of
euchromatin separating them rather than the
total amount of euchromatin and heterochro-
matin.

7.2. Localizing Genes by means of Induced Chromo-
somal Deficiencies in Tomatoes. Gurdev
S. Khush and Charles M. Rick (Davis,
U.S.A.).

The induced deficiency method, previously
applied {Genetics, 46, 1389-1393) to genes on
chromosome 11, has been extended successfully
to localize other seedling marker genes. Stocks
homozygous for unlocated recessive genes are
pollinated with X-rayed pollen of a normal, non-
mutant parent, and all mutant progenies are
searched cytologically at pachytene for chromo-
somal alterations. More than 95 per cent of
such individuals are deficient for that part of the

chromosome in which the gene in question
resides. In this fashion /// of chromosome 11,
dl of 8, and fid and clau of 4 were assigned to
the achromatic regions of the short arms (S) of
their respective chromosomes; likewise a\ to
the achromatic of 11L and 7i to 8L. No unequi-
vocal evidence has been obtained for genes in the
chromatic regions. More than half the deficient
plants are monosomic, but simple monosomies
have been found only for chromosome 11, the
remainder being translocated monosomies, in
which one element of a reciprocal translocation
has been lost. Of ten such deficiencies, the lost
element nearly always comprises the shorter
arms of the respective chromosomes, longer
deficiencies doubtless being less viable. Ex-
ceptional was a case in which centromeric
breaks led to translocation and loss of 8L and
9S. Since this plant had h phenotype and ap-
peared in the progeny of dl-bu-h X-irradiated
+ + + pollen, 7i clearly lies in 8L and both bu
and dl in 8S. No deficiencies were transmitted to
any of 1429 progenies of female parents deficient
for arms of 1 1 and to 1 3 1 offspring of haplo- 11$.

Research partly supported by Grant GM
06209 of the U.S. Public Health Service.

7.3.Cytogenetic Correlations in Tetraploid Tomatoes.

Peter B. Moens (Toronto, Canada).

Five markers (dv, d, m, aw and wv) on chromo-
some no. 2 of the tomato, the satellited chromo-
some, were found to segregate 1 : 30 in the
progeny of duplex tetraploids. The individual
ratios did not differ significantly from each
other (x2=0.40, d.f. 4) and an average ratio of
1: 30.257 was established for the markers
(3.20 per cent recessives +0.05 percent, based on
110,000 observations). The fact that the ratios
do not vary with the distance from marker to
centromere, as is normally the case in tetraploids,
results from the specialized structure and be-
haviour of chromosome no. 2.

Recessive zygotes in excess of 1 : 35 are the
result of genetic non-disjunction in quadrivalents.
The frequency of chromosome no. 2 quadri-

117

Section 7 — Cytogenetics

valents was found to be 0.30. Random pairing
of homologues was ascertained through the use
of plants having two homologues with long
satellites and two homologues with short
satellites. Quadrivalent formation was found to
be of a specialized type: only the long arm of
chromosome no. 2 participates. For example,
in a ring quadrivalent each long arm must have at
least two chiasmata and one partner exchange.
Due to the space requirements the proximal
chiasma always lies near the centromere.
Consequently, all genetic markers on chromo-
some no. 2 will segregate independently of the
centromere.

The predicted frequency of recessive zygotes
is 1/29.8 for markers separated by one chiasma
from the centromere when the frequency of
quadrivalents is 0.3.

7.4. A Cytogenetic Study of the Tumour-bearing
Hybrid Nicotiana glauca X N. langsdorffii.

M. R. Ahuja (Philadelphia, U.S.A.).

It is well known that the cross N. glauca X
N. langsdorffii gives 100 per cent tumors in the
Fi progeny. The triploid and the tetraploid
combinations also yield an entirely tumorous
progeny. Kehr and Smith (Brookhaven Symp.
Biol. 6, 55-78, 1954) and Smith and Stevenson
(Z. Vererb. 92, 100-118, 1961) have shown that
tumor formation depends on the interaction of
many genes in the hybrid. Furthermore they
found that with one or several glauca chromo-
somes on diploid langsdorffii, tumors do not
form. On a different hybrid between An N. deb-
neyi tabacum x N. longiflora (Ahuja, Genetics,
47, 865-880, 1962) tumor production was found
to be a function of an alien longiflora chromosome
or a fragment of it on the debneyi-tabacum
background. Since Kehr and Smith did not
examine the cytology in the glauca-langsdorffii
hybrid combination it was considered worthwhile
to check the cytology in the triploid generation
(having two langsdorffii and one glauca genomes).
A study of meiosis revealed that in 15 plants not
all plants in the triploid generation carried a full
complement of 30 chromosomes (9n + 12i).
Three plants were found with 29 chromosomes
(9n + Hi). Somatic elimination of a chromo-
some is ruled out as buds on different branches of
the same plant had the same chromosome
number. All the three deficient plants are tumor-
ous indicating that tumor production in this
hybrid may not be function of the complete
genomes of the parental species. This opens
the way to a more complete analysis of the role
of chromosomes in tumor forming process.

This work was supported by Grant DRG 622
from the Damon Runyon Memorial Fund for
Cancer Research.

7.5. A Pseudo-supernumerary Chromosomein Col-
linsia heterophylla. E. D. Garber (Chicago,
U.S.A.).

Progeny of plants of C. heterophylla (n = l)
but not of six other species in the genus treated
with colchicine included individuals either with
an extra chromosome (trisomy) or with a hetero-
zygous reciprocal translocation. Thirteen inde-
pendently obtained trisomies had the same extra
chromosome which behaved cytogenetically as
did supernumerary chromosomes in two other
species. Primary trisomes from an autotriploid
plant of C. heterophylla, however, were orthodox
in their cytogenetic behavior. Although a trisome
for one of the seven chromosomes in this species
simulates a supernumerary chromosome, this
chromosome cannot be lost (monosomy) from
the complement. The pseudo-supernumerary
chromosome presumably responds to colchicine
by undergoing non-disjunction during mitosis
and may be responsible for the unexpected
chromosome breakage induced in this species by
colchicine.

Research supported by the National Science
Foundation.

7.6. Genetics of B-chromosomes. A. Rutishauser
(Zurich, Switserland).

Most accessory or B-chromosomes of higher
plants show some kind of boosting-mechanisms :
the B-chromosomes are preferentially included
in the generative or sperm nucleus (directed non-
disjunction in pollen grain-mitosis) or in the
functioning megaspore (preferential segregation).
In Crepis capillaris, where plants with one B-
chromosome rarely occur in nature, a new type
of boosting-mechanism has been found: the
number of B's is doubled in the inflorescences.

The B-chromosomes of Crepis capillaris are
by no means genetically inactive. Although
partly heterochromatic they exert a variety of
effects on the carrier plants. The flowering time
is retarded, the shape of inflorescences changed,
the pollen fertility lowered and, depending on
the number of B's, seed setting reduced to \
(IB) or 1/4 (2B's). Even more striking is their
effect upon bivalents of the A-chromosomes:

118

Section 7 — Cytogenetics

the number of chromatid-bridges accompanied
by acentric fragments of first divisions of
PMC's, and accordingly the number of tetrads
with micronuclei and persisting bridges, rises
considerably if B-chromosomes are present.
In 2B-plants B-chromosomes themselves form
bridges.

From these findings it may be inferred that
B-chromosomes of C. capillaris enhance vari-
ability and therefore may well have an evolution-
ary effect.

markers in linkage groups II, III, and VI has
given genetic evidence of nuclear passage ; coinci-
dent second division segregation for all markers
is observed in one type of ascus. No such coinci-
dent segregations were found in other types of
asci from this cross nor in asci from control
crosses.

* Deceased.

7.7.Frequency and Geographical Distribution of
Rye (Secale cereale) with Accessory Chromo-
somes in Korea. Woong-Jik Lee (Seoul, Korea).

Frequency of accessory chromosomes in po-
pulation of rye from Korea was known to be
exceedingly high. Extensive present work for
frequency and geographical distribution of rye
with accessory chromosomes in Korea has been
carried out and the result has revealed that the
frequency is ranged from 39 to 53 per cent. This
figure, smaller than the result in previous report,
is still higher than any other region in the world.
Correlation between frequency of accessory
chromosomes and edaphic factor (type and
pH of soil) will be discussed.

7.8. The Cytogenetic Effects of a Paracentric Inversion
in Neurospora crassa. Patricia St. Lawrence
and Jesse R. Singleton* (Berkeley and La-
fayette, U.S.A.).

The mutant SI 325 carries a paracentric
inversion which is inseparable from a niacin
requirement and involves about 70 per cent of
the known genetic length of the right arm of
linkage group I. Recombination between the
nic-2 and lys-3 loci is much reduced in viable
progeny from crosses of SI 325 to normal stocks.
In immature asci from such crosses a conspicuous
bridge and fragment is seen at anaphase I.
Persistence of the bridge is occasionally
observed leading to fusion of the daughter
nuclei or to aberrant chromosome segregations
in subsequent divisions. Migration of the nuclei
is irregular and non-identical sister spores may
be formed. It has not been possible to determine
whether a breakage-fusion-bridge cycle is
operative. At maturity most of the asci are
completely abortive; the major class of asci
with ripe spores has one ripe pair in each half of
the ascus. Analysis of isolates from asci of
crosses of SI 325 to a stock carrying centromere

7.9. Polyploidy and the Intel change-heterozygote.

Keith Jones (Kew, Great Britain).

The nature of chromosome pairing and its
genetic consequences are well understood in
diploid interchange-heterozygotes. Permanent
hybrids of the Oenothera type have maintained
heterozygosity by control of chromosome
segregation and the elimination of homozygotes.
Polyploidy is also an efficient means of maintain-
ing heterozygosity and its role in the evolution
of Anthoxanthum odoratum (2w = 20) is discussed.

7.10. Genically Controlled Variability of Chromosome
Number in Pennisetum Hybrids. P. Gilden-
huys and K. Brix (Pietermaritzburg, South
Africa).

Crosses between diploid Pennisetum typhoides
(2« = 14) and tetraploid P. purpureum (2« = 28)
yield sterile triploids (2«=21) with regular
mitotic behaviour. The colchicine induced
hexaploids (amphidiploids) are fertile but show
instability in somatic chromosome number, even
in cells of the same root, as do the C2 plants
(2» = 36 to 49 with 2«=42 occurring most
frequently). These hexaploids produce pollen
grains which contain from 12 to 21 chromo-
somes. When back-crossed to P. typhoides the
resultant progenies, as expected, have 2m = 21 to
2« = 28 chromosomes, but, in addition, the
crosses also yield offspring with chromosome
numbers in excess of 2n = 28. These arise from
unreduced egg cells in P. typhoides.

Whilst CI and C2 plants all show similar
ranges of intraplant variability in somatic
chrosome number, this is not so in the back-
crosses, where the range of variation is highly
significantly different from plant to plant. For
example, in one plant numbers range from
18 to 29 (28 occurring most frequently), in
another from 32 to 38 (35 most frequent), where-
as in another plant the range is only 34 to 36

119

Section 7 — Cytogenetics

(35 most frequent) whilst in yet another the
number is constant at 2« = 35. It is suggested
that intraplant aneuploidy, known to exist in
other intergeneric and interspecific hybrids, is
in this case under genetic control, probably
with similar gene action to that which we have
demonstrated in the amphidiploid of the cross
between P. typhoides and the mitotically unstable
P. dubium.

To be published in the South African Journal
of Agricultural Science.

7.11. Colchicine-induced Somatic Chromosome Re-
duction in Sorghum. J. G. Ross, C. H. Chen
and G. M. Simantel (Brookings, U.S.A.).

As a result of colchicine treatment of diploid
sorghum seedlings of certain genotypes, true-
breeding and segregating diploid mutants have
been observed. A laboratory method by which
light, temperature and humidity are controlled
was developed to ensure the appearance of the
phenomenon in "colchicine reactive" genotypes.
To explain the appearance of true breeding
mutants a mutational effect was postulated in
conjunction with somatic chromosome reduction
followed by restoration to the original chromo-
some number. This hypothesis was tested by
treating tetraploid seedlings of "colchicine
reactive" genotypes. Out of 80 treated seedlings
there were 9 diploids (one a chimera of diploid
and tetraploid tissue) among the 17 survivors,
while no diploids occurred among the 118
control tetraploid plants. Histological study
indicated that diploid cells occurred at the base
of the apical dome 2 to 3 days after colchicine
application and that these may proliferate to
take over the growing point at the 5th to 6th day.
As an additional test, 441 seedlings containing
structural chromosome markers (reciprocal
translocations) in the heterozygous condition
were treated. Of 258 survivors 6 were true-
breeding mutants homozygous for the structural
markers and 4 appeared to be chimeras of mu-
tant and original tissue and still heterozygous.
No homozygosity occurred in the 108 surviving
untreated controls. These tests indicate that after
colchicine treatment of sorghum seedlings of a
reactive genotype under certain environmental
conditions somatic chromosome reduction oc-
curs. Cells so formed may take over the growing
point to form a plant containing half the original
chromosomes or homozygous if restored to the
original number.

7.12. Cytogenetic Studies of Perennialism in Deriva-
tives of Interspecific Hybrids of Zea*. Donald
L. Shaver (Upton, U.S.A.).

Perennialism is recovered in 4x derivatives
of hybrids between 4x maize and 4x perennial
teosinte by sib-mating among the most perennial
segregates of each generation. Similarly, only 3
generations of selection at the 75 per cent maize
level have resulted in a progressive recovery of
the perennial expression. A high degree of maize-
likeness therefore appears compatible with the
perennial expression at the 4x level.

Diploid derivatives of maize and perennial
teosinte are produced by crossing the 30 chromo-
some triploid hybrid of 4x perennial teosinte
x2x maize back to 2x maize, and then inter-
crossing among the resulting array of euploid
and aneuploid plants. The average chromosome
number in the first post-triploid generation is
24.6, and in the second post-triploid generation
is 20.7. The third post-triploid generation is
almost entirely euploid. The study of pollen
abortion and seedling lethality in euploids of the
post-triploid generations indicates the presence
of genetic lesions which are functionally viable
in 4x teosinte but which act as diploid-lethals.
In spite of the high degree of preferential segre-
gation of markers in the triploid indicating
that post-triploid material should carry a large
proportion of perennial teosinte genes, derived
diploids are very maize-like. The apparent
tendency to lose teosinte genes is probably
connected with the elimination of diploid-lethal
factors. However, even among euploids and
aneuploids of the first post-triploid generation
which have not yet suffered elimination of
teosinte genes, it has not been possible to demon-
strate true perennialism. One exceptional 21
chromosome plant has been found which
appears truly perennial. It has been cloned up
through the fourth generation of culms. Data
on its cytology and genetic progeny ratios will
be presented.

* Research carried out at Brookhaven National
Laboratory under the auspices of the U.S.
Atomic Energy Commission.

7.13. The Use of Hypoploids in Identifying [Natu-
rally Occurring Duplications in Maize. J. R.

Laughnan (Urbana, U.S.A.).

We have commenced using various hypoploids
in an arm-by-arm search for naturally occurring
duplications in the maize genome. Assuming that

120

Section 7 — Cytogenetics

such duplications occur (there is some indepen-
dent genetic evidence for this) it is argued that
during meiosis in the hypoploid individual, the
chromosome arm that is in haplo condition
should synapse occasionally with segments of
other chromosomes representing duplications of
chromatin in the haplo arm. Crossing over in
such "illegitimately" paired regions should yield
gametes carrying reciprocal translocations; the
identification and analysis of interchanges origi-
nating in this way might be expected to reveal
the nature and extent of naturally occurring
duplications.

In our scheme, hypoploid individuals among
the progeny of pollen parents carrying an A-B
translocation, are used as pollen (or egg)
parents in crosses with vigorous single-crosses.
The progeny of such crosses are grown on a large
scale and at harvest are searched for the occur-
rence of scatter-grain ears, an indication of
heterozygosity for a reciprocal translocation.
These are routinely analyzed to confirm the
presence of an aberration and to identify it
cytologically.

Preliminary results based on an analysis of
progeny of TB-9a and TB-9b hypoploids indicate
the occurrence of reciprocal translocations which
appear to involve preferentially the haplo arm
of the hypoploid as one member of the inter-
change. On the other hand, the haplo arm is not
always involved in the identified interchange, but
even in these cases it appears that certain arms
may be involved more frequently than others.
It may be that the haplo arm in the hypoploid
plant leads to secondary "illegitimate" associa-
tions, or that chromosomes are more prone to
break in hypoploids and that in this respect some
regions are more unstable than others. Prelimi-
nary results indicate that certain hypoploids
yield higher frequencies of interchanged progeny
than do others.

7.14. Inversion Polymorphisms in Teosinte. Y. C.

Ting (Chestnut Hill, U.S.A.).

During last few years, five varieties of Guate-
malan teosinte and eight varieties of Mexican
teosinte have been cytologically investigated.
Chromosome inversions were identified in most
of these varieties. In9 in the short arm of chromo-
some 9 was present in the Guatemalan varieties,
Florida, Jutiapa, El Valle and Lake retene; but
not in the variety Huixta of northern Guatemala.
Measurements at pachytene showed the length of
this inversion to be about 60 per cent of the
length of the short arm. Bridges and fragments
were found at both anaphase I and anaphase II

of the microsporocyte divisions. Among Mexican
teosinte, the varieties, Chalco, Xochimilco,
Durango, Nobogame and perennial teosinte
were observed to have the same In9. In addition,
In8 was also present in these Mexican varieties;
but not in perennial teosinte. The average
length of this In8 at pachytene occupied about
50 per cent of the total length of the short arm
of chromosome 8. It was further found that the
variety Xochimilco had, in addition to In8 and
In9, an In3 in the long arm of chromosome 3.
This In3 being equivalent to about half of the
length of the long arm, manifested inter-
chromosomal effect on the frequency of crossing
over within the inverted segments of both ln8
and In9. The convincing evidence of this effect
was that at pachytene the frequency of loop for-
mations of both In8 and In9 was higher than this
in in plants having only In8 and In9 but not In3.
(Detailed report of this study will be published
in a monograph under preparation.)

7.15. An Evolutionary Significance of a Pericentric
Inversion in a Barley Trisomic for Chro-
mosome 6. Takumi Tsuchiya (Yokohama
Japan).

The seven primary trisomic types have been
produced in Hordeum spontaneum var. transcas-
picum. A new trisomic type has been found in
the progeny of Purple, the trisomic for chromo-
some 6. The extra chromosome of the new
trisomic type had two constrictions located at
subterminal positions of opposite sides. The
study of mitotic chromosomes showed that the
one of the two constrictions was the kinetochore
and the other the secondary constriction of the
chromosome 6. Thus the changed chromosome
was assumed to be derived from the chromosome
6. The length of the changed chromosome was
about 18 per cent shorter than that of the normal
chromosome 6. The tiivalent chromosomes
associated with the nucleolus at diakinesis.
Thereby it has been confirmed that the changed
extra chromosome might be derived from
chromosome 6. Meiotic behaviour was almost
normal resembling that of the primary Purple
trisomies. From the somatic chromosome mor-
phology and the meiotic behaviour it has been
assumed that the structural change in the new
Purple trisomic would be a pericentric inversion
accompanied by a deletion. The new Purple
trisomicd showed vigorous growth with inter-
mediate characteristics between normal diploid
and primary Purple trisomies and almost normal
fertility. Tetrasomic and some other chromoso-
mal types have been obtained in the sibling and/

121

Section 7 — Cytogenetic:,

or progeny of the new trisomic plants. Some
problems in the linkage studies using trisomies
are considered in connection with the occurrence
of such pericentric inversions accompanied by a
deletion as has been reported here in this paper.

7.16. Genetic Recombination and Chromosome
Disjunction in a Balanced Tertiary Trisomic of
Barley. R. T. Ramage (Tucson, U.S.A.).

The interchanged chromosome 2"'A is broken
close to the centromere in the short arm of
chromosome 2. The MS2UIS2 locus is tightly
linked with the breakpoint of 27( and is carried
on chromosome 2J (arm not known). The Vv
locus is carried on the long arm of chromosome
2 and, in normal stocks, exhibits about 26 per
cent recombination with the Ms2ms2 locus. A
balanced tertiary trisomic was set up so that the
two normal chromosomes carried the v and ms2
alleles and the interchanged chromosome 2\
carried the V and Ms2 alleles. In the balanced
tertiary trisomic the frequency of recombination
between the genes V and ms2 was measured by
determining the frequency of male gametes
carrying the V allele. Chromosome disjunction
types, which are dependent upon the type of
pachytene pairing, were obtained by determining
the frequencies of microspore abortion and of
primary trisomies in the progeny of the balanced
tertiary trisomic. Microspore abortion and the
production of primary trisomies are results of
the same pachytene pairing event and should be
found with the same frequency. If genetic re-
combination is dependent upon an exchange of
chromosome parts at pachytene, the frequency
of microspore abortion, or of the production of
primary trisomies, should equal one-half the
frequency of male gamete carrying the V allele.
In these studies, much more genetic recom-
bination was observed than can be accounted
for by an exchange of chromosome parts at
pachytene.

7.17. Chromosome Segregation in a Tertiary Trisome
of Secale cereale in its Three Karyological
Backgrounds. J. Sybenga (Wageningen, The
Netherlands).

Tertiary trisomes originate from reciprocal
translocation heterozygotes. The extra chromo-
some can occur in three "backgrounds": the
translocation homozygote, the translocation
heterozygote and the normal type. All three
forms may be found in the progeny of a selfed

translocation heterozygote, that is trisomic for a
translocation chromosome.

Meiotic configurations depend on the "back-
ground", and chromosome segregation depends
on these configurations. In the translocation
homozygote background the trisome is in fact a
primary trisome. In the normal background as
largest configuration a chain of five can be for-
med. In the heterozygous background this is
again a chain of five that, with zigzag orientation,
segregates into the translocation complement
with the extra chromosome against the normal
complementwithout it, or the reverse. With equally
complete pairinga bivalent and a chain of three
may be formed, segregating into the translocation
complement without the extra chromosome
against the normal complement with it. The third
possibility is a ring of four with a univalent, that
leads to loss of the extra chromosome, or inclu-
sion in either group with equal probability. Less
complete pairing or non-zigzag orientation
leads to increased loss of the extra chromosome,
increased randomness of its distribution and
increased sterility.

In the homozygous backgrounds no segrega-
tion in regard to the translocation occurs. The
type of configuration affects the frequeny of the
extra chromosome in the progeny and the
sterility. Meiosis and chromosome segregation
were studied in one tertiary trisome in all three
backgrounds in Secale cereale. Orientation of
the multivalents was predominantly zigzag.
Preference for type of segregation in the hetero-
zygote could not be demonstrated. Apparent-
ly the translocation did not tend to fa-
vour pairing between completely homologous
chromosomes only. This agrees with the results of
Ahloowalia on the autotetraploid translocation
heterozygote. Data on meiosis and chromosome
segregations will be presented.

7.18. Transmission of Rye Chromosomes in Mono-
somic Addition Lines of Rye Chromosomes in
Wheat. Shams-ul-Islam Khan (Lahore,
Pakistan).

In the monosomic addition lines of rye
(Secale cereale L.; "Swat Rye", In = 14)
chromosomes in wheat (Triticum aestivum L. ;
C271, 2« = 42), transmission of individual rye
chromosomes has been studied, both through
male and female gametes. Ratios (42/43)
through male and female gametes are:
I (4.78 : 1/5.0 : 1), II (2.8 : 1/3.0 : 1), III
(2.0 : 1/2.25 : 1), IV (12.0 : 1/10.0 : 1), V
(3.0 : 1/2.9 : 1), VI (8.0 : 1/6.0 : 1), VII

122

Section 7 — Cytogenetics

(6.0 : 1/8.0 : 1) and total (5.5 : 1/5.3 : 1). These
ratios lead to the following conclusions:

(a) Transmission is almost the same through
male and female gametes when all seven
chromosomes are considered together.

(b) In spite of differences in transmission these
seven chromosomes fall into five groups:
(1), I; (2), II and V; (3) III; (4) IV; and
(5), VI and VII.

(c) Groups (1), (3) and (4) consist of single
chromosomes, and show almost the same
transmission through male and female
gametes.

(d) In groups (2) and (5), each consists of two
chromosomes, and the transmission ratio
of one through the male gamete is the
same as that of the other through the
female gamete.

From the above transmission data of rye
chromosomes it is tempting to infer that the rye
progenitor may have had 5 and not 7 pairs of
chromosomes and that the evolution of genera
and species in the Triticeae has been from an
ancestral type with 5 pairs of chromosomes:
undoubtedly through alterations in the physical
constitution of certain chromosomes only.

7.19. The Nature of a Spontaneous Transfer of Hairy
Neck fiom Rye to Wheat. C. J. Driscoll and
E. R. Sears (Columbia, U.S.A.).

A series of backcrosses transferred the rye
character "hairy neck" (pubescent peduncle) to
Chinese Spring wheat from the Cornell wheat
selection 82al-2-4-7, a derivative of a wheat-rye
hybrid. This dominant character exhibited mono-
genic inheritance. By means of monosomic
analysis it was located on chromosome 4A (IV).

When heterozygous plants, with one entire
and one telocentric chromosome 4A, were selfed,
non-hairy offspring were obtained that possessed
an entire 4A. Since the hairy-neck gene was
carried by the entire chromosome in the parent
plant, this result demonstrated that the gene is
located on the arm homologous to the telocentric.
In the plants heterozygous for hairy neck and the
telocentric, pairing (to form a heteromorphic
bivalent) was observed in only about 30 per cent
of pollen mother cells. When unpaired, the two
chromosomes often exhibited co-orientation,
suggesting that synapsis had occurred but that
crossing over and chiasma formation had not
ensued. As regular pairing occurs between
normal 4A and this telocentric, it may be as-
sumed that the hairy-neck gene is located in a
rye segment of substantial length in which
crossing over with the telocentric does not occur.

Heterozygotes with the heteromorphic bivalent
were backcrossed as males to Chinese for deter-
mination of the distance of the hairy-neck gene
(or rye segment) from the centromere. As desyn-
apsis leads to fewer functioning non-cross over
gametes, a correction factor, calculated as
0.56, will presumably need to be applied to the
obtained value.

7.20. Cytogenetic Studies Bearing on the Nature of
the Centromere. L. M. Steinitz-Sears (Colum-
bia, U.S.A.).

Telocentric chromosomes resulting from mis-
division were studied in hexaploid wheat to
determine whether their somatic instability might
be due to deficient centromere regions. Among
3134 offspring of monosomic 3B, which carried
the hemizygous-ineffective gene v (virescent)
as a marker of the short arm, 28 telocentrics and
7 isochromosomes for the short arm of 3B were
found. Eighty additional short-arm telocentrics
were presumably present in the virescent
seedling but were lost before meiosis. A few of
these plants, virescent as seedlings though green
and monosomic at the time of meiosis, had 41
normal plus 1 telocentric chromosomes in their
root tips.

In order to prevent meiotic loss, a telocentric
for the long arm of chromosome 3B, which
carries a gene for synapsis, was combined with
the different telocentrics for the short arm. Thus
lines were established possessing 20 normal and
2 telocentric pairs, in which the very stable
pair for the long arm of 3B was identical but the
pairs for the short arm differed in origin.
Observations of leaf sectors suggest differences
in the degree of stability of the short-arm
telocentrics.

In the meiosis of individuals with only one of
each of the telocentrics, varying amounts of
co-orientation were found at metaphase I. The
most stable short-arm telocentric was co-oriented
with the long-arm telocentric in approximately
half of the analysable cells. Co-orientation is
interpreted to be a consequence of an overlap of
centromeric regions of the two different stable
telocentrics. Centromeric regions are believed
to be complex and specific for each chromosome.

7.21. Gene Evolution in Polyploid Wheat. E. R. Sears
(Columbia, U.S.A.).

Where genes are duplicated, as in polyploids,
mutations to a different level of activity-even

123

Section 7 — Cytogenetics

to the zero level — may not be detectable, because
of the masking action of the duplicates at other
loci. Harland W suggested that a duplicated gene
could escape this masking effect by mutating to
an allele with a divergent function. An example
of this kind of mutant may be Neatby's virescent
(v) on chromosome 3B (III) of common wheat,
a hexaploid.

Increase in dosage of v from 2 to 3 results in
greater abnormality (less chlorophyll), whereas
increased dosage of the normal allele For either
of the homologous (related) chromosomes 3A
(XII) and 3D (XVI) shifts the phenotype toward
normal. This suggests that v is antimorphic to V
and that F(or VI) has duplicates, V2 and V3, on
chromosomes 3A and 3D.

That V3, and therefore presumably VI and V2,
are involved in chlorophyll production was
shown by inducing a deficiency for vl and com-
bining this with nullisomes 3A and 3D, respec-
tively, thus reducing the dosage of V from the
normal 6 to only 2. Nullisomic-3D plants (V2V2)
had normal chlorophyll, whereas nullisomic-3A
plants (V3V3) were of reduced chlorophyll
content. Thus V2 is more potent than V3 in
promoting chlorophyll development. On the
other hand, V3 is more effective than V2 in
reducing the expression of v. Although v may be
a mutant with a divergent function, another ex-
planation is possible; namely, that it is less
efficient than V in producing chlorophyll but
more efficient in competing for substrate.

1. Biol. Rev. 11, 1936.

7.22. Induction of Autosomal Crossing Over by cc-
heterochromatin and Demonstration of Chro-
mosome Interference in Phryne cincta. B. E.

Wolf (Berlin, Germany).

Investigating the internal causes of crossing
over in the euchromatic chromosomes of Phryne
cincta the compact- or cc-heterochromatin was
recognized to be a decisive factor. Obviously the
so-called "a-quantum difference", i.e. the differ-
ence in the amount of cc-heterochromatin (or
DNA) between the homologues of the X-chro-
mosome, is causally related to the crossing-over-
process ("cc-differential-effect" ; Wolf, Chromo-
soma, 13, 646, 1963). It was supposed, that by
way of a potential function it effects crossing
over not only within the "a-differential" tetrad
but also in nonhomologous and unrelated pairs
of chromosomes in the same meiotic cell.

This hypothesis could be ascertained in cyto-
logical crossover studies by means of either two

independent inversions coupled in autosome 2
and in autosome 3. Both pairs of inversions per-
mitted to establish the influence of the "oc-quan-
tum-difference" in the X on crossing-over-
frequency in the autosomes. Brother and sister
males and females heterozygous for one pair of
the inversions or for both together, furthermore
characterized by particular X-chromosomes or
X-chromosome-combinations (a/Y, b/Y, c/Y in
the male and a/b, b/b, c/b in the female: a and c
with much cc-heterochromatin, b with less), have
been backcrossed to homozygous animals of the
standard stock (b/b resp. b/Y, free from in-
versions).

In the progeny of the males only the parental
inversion-combinations in the autosomes 2 and
3 were found (n = 356 and 270 resp.). In the test
cross progeny of the females the expected types
of recombination appeared: with only one in-
version in autosome 2 resp. 3 or simultaneously
in both autosomes. There appeared a very high
difference in linkage dependent on the present
X-chromosome-combination in the mother. Fe-
males homozygous for the X-chromosomes (type
b/b) exhibited only little recombination in chro-
mosome 2 (3.0 per cent, n = 430) and no in chro-
mosome 3 (« = 318) though the tested segments
approximately involve a third of the total length
in both chromosomes. Sister-individuals due to
the presence of "a-differential" X-chromosomes
(type a/b or c/b) gave a high rate of recombina-
tion in chromosome 2 (31.0 per cent, /; = 434)
as well as in chromosome 3 (11.4 per cent,
n = 784).

Interchromosomal effects have been detected
by observation of crossing over in the autosomes
2 and/or 3 and in the X-chromosome simultane-
ously. The frequency of contemporary crossing
over in both autosomes as well as in the X and in
one of the autosomes, based on independent
occurrence, is significantly reduced. The results
represent a cytological parallelism to genetic
data on Drosophila of Schultz (1933) and Schultz
and Redfield (1951).

7.23. Inherited Partial Sterility in Habrobracon.

R. C. von Borstel (Oak Ridge, U.S.A.).

Inherited partial sterility has been shown to be
the consequence of meiotic segregation of normal ,
translocated, and duplication-deficiency chro-
mosomes in individuals heterozygous for a trans-
location. In the wasp Habrobracon, these can be
detected by hatchability methods and confirmed
by outcrossing and progeny-testing surviving
haploid males from females giving low hatch-
ability. One-half of the surviving males should

124

Section 7 — Cytogenetics

carry the translocation. After X-irradiation,
twenty-seven cases of inherited partial sterility
were found in Habrobracon. Of these, twelve of
the females had hatchabilities between 30 and
40 per cent. Six were below 30 per cent, four
were above 60 per cent, and five were between
40 and 55 per cent. By classic theory, it can be
assumed that the 40-55 per cent class involved
adjacent I segregation and the 30-40 per cent
class involved mostly adjacent I and adjacent II
segregation. The other classes require additional
interpretations such as multiple chromosome in-
volvements for those with hatchabilities lower than
30 per cent, and translocations involvingchromo-
some ends for those with hatchabilities above 60
per cent. The time of death of the duplication-
deficiency products is extremely regular among all
of the induced cases, occurring at midem-
bryonic stages of development. On the other
hand, embryonic recessive lethal mutations are
expressed at all stages of embryonic develop-
ment from the blast ula stage to hatching. Very
few cases of inherited partial sterility are found
in progeny from females irradiated in prophase I
or metaphase I; inherited partial sterility is com-
monly induced after irradiation of males with
mature sperm.

7.24. Crossing-over in the Sex Bivalent of Male
Mammals. J. Wahrman and U. Ritte (Jeru-
salem, Israel).

A knowledge of the pairing relationships
between the X and Y chromosomes of mammals
and man is essential for an understanding of the
heredity of X-borne genes, and especially of
partial sex-linkage.

In the meiosis of Apodemus mystacinus, a
murine species of Israel, pre- and post reductions
occur normally side by side. Chiasmata can be
directly demonstrated at first meiotic prophase.
The occurrence of two disjunctional types may
be observed at first anaphase and is confirmed
by the inspection of secondary spermatocytes.
Some of these contain X-dyads or Y-dyads
resulting from pre-reduction, while others clearly
exhibit the heteromorphic X-Y dyad awaiting
post-reduction.

In Apodemus sylvaticus the sex bivalent is less
favourable for analysis but a large proportion
of secondary spermatocytes displays the X-Y
dyad, which suggests the previous existence of a
chiasma. Thus the examination of secondary
spermatocytes may furnish critical evidence for
the existence of chiasmata in species where these
cannot be directly observed.

In certain mammalian species a small fraction

of primary spermatocytes exhibit a chiasma
between X and Y, although none can be seen in
the majority of cells. Finally, in most mammalian
species, including man, the X and Y appear in
end-to-end configuration, which is generally
attributed to non-chiasmic association. This
point requires re-examination.

The occurrence of chiasmata in the male sex-
bivalent, and therefore probably of genetic re-
combination, is firmly established in at least
certain mammalian species.

7.25. Interchromosomal Effects on Crossing-over in
Drosophila melanogaster. David T. Suzuki
(Edmonton, Canada).

Crossing-over was measured in chromosome 3
of females heterozygous and homozygous for
the X-chromosome inversions v3p, j>4, sc4, sc8,
sc9, dl-49, wm4, and rst3. Crossover frequencies
were increased in all inversion heterozygotes and
in all but the sc9 and dl-49 homozygotes. The
lack of effect in sc9 and dl-49 homozygotes in-
dicates that the interchromosomal effects of in-
version heterozygotes are not due to the intrinsic
properties of the rearrangement per se. Since the
inversions which had effects when homozygous
have the X-tip apposed to heterochromatin (y4)
or heterochromatin located distally, it was
suggested that the position of heterochromatin
adjacent to the tip region was responsible for the
interchromosomal effects of the inversions.
Fragment-1 (YSX-), a normal X chromosome
with the short arm of the Y chromosome attached
to its tip, was tested and crossing-over was shown
to be significantly increased in chromosome 3 of
Fragment-1 homozygotes and heterozygotes.
Since crossing-over was not changed in females
homozygous and heterozygous for a normal X
with Y-short attached to its centromere (X'YS),
the distal position of the Y heterochromatin
appears to be important for the interchromoso-
mal effects of a Y-arm attachment.

7.26. The Mode of Interchromosomal Effect of In-
version Heterozygosity on Crossing-over Fre-
quency in Drosophila melanogaster. Tarvo
Oksala (Turku, Finland).

The mechanism by which inversion hetero-
zygosity affects crossing over in heterologous
chromosomes is still unknown. In order to eluci-
date this problem the author has carried out a
few experiments in which the (structurally nor-

125

Section 7 — Cytogenetics

mal) X chromosome is the affected chromosome,
the large autosomes representing different types
of inversion heterozygosity. The X was divided
into nine regions, each of which was tested se-
parately in four parallel experiments: the stand-
ard autosomal homozygote as a control, the
In(2L + 2R)Cy heterozygote, the In(3L + 3R)P
heterozygote, and the combination of these two
inversions. It turned out that in all regions the
Curly + Payne combination is very much more
effective in increasing the crossover value than
these two inversions used separately. When the
effect curves of Curly-, Payne-, and combined
experiments are computed, all three prove to be
nicely parallel, showing conspicuous peaks at
both ends of the X and in the very middle of this
one-armed chromosome (more or less around
vermilion). This result suggests that all these
types of inversion heterozygosity affect the X in
basically the same way: they simply enable the
X to produce more crossover points than nor-
mally. As there are now -instead of one or two
crossovers -quite often three in the same tetrad,
it is only natural that, owing to interference, they
should be situated as far from each other as
possible at regular intervals, i.e. at both ends and
in the middle. This state of affairs is reflected in
the three-peaked curves obtained.

Research supported by PHS Grant RG-6780.

7.27. The Centromere Effect on the Distribution of
Exchanges in Drosophila Females. Peter E.
Thompson (Ames, U.S.A.).

Tests of crossing-over with homozygous trans-
locations have shown that a distal region attached
near a centromere undergoes a reduction in ex-
changes. Conversely, a region normally lying
near a centromere shows an increase in crossing-
over when translocated to a distal position. This
phenomenon is widely known as "the centromere
effect on crossing-over".

In studies with these same translocations made
heterozygous, the finding has generally been that
crossing-over is reduced adjacent to the break,
possibly as a result of synaptic ambiguities. The
present investigation has shown, however, that
marked regions very near the centromere pro-
duce more crossing-over if one of the homologues
has a translocation break between the centro-
mere and the markers. This has been strikingly
true for chromosome 4, which normally does not
undergo exchange in diploids, but which has
produced appreciable frequencies of crossing-

over in the presence of a translocation having a
break near the centromere of 4.

This increased crossing-over in translocation
heterozygotes applies only to those centric inter-
vals which show a great disparity in cytological
and genetic (crossover) length. Since the trans-
location has in effect removed only one of the
adjacent centromeres, it is suggested that the
normal distribution of exchanges is strongly in-
fluenced by some condition arising out of actual
centromere pairing. The credibility of a repulsion
of homologous centromeres prior to exchange is
being examined.

These findings suggest a new interpretation of
the "interchromosomal effect", based on switch
pairing of homologous and non-homologous
elements when rearrangements are present

7.28. Distal Crossing-over in Free-X and in Reversed
Acrocentric Attached- X Triploid Drosophila
melanogaster. J. D. Mohler and John
C. Neeley (Corvallis, U.S.A.).

Free-X triploids having normal chromosome
structure give an estimated 6.0 per cent crossing-
over for all X-chromosomes in the y-w region.
The increase over the diploid frequency of
1 per cent accounts for nearly all of the distal
increase reported for the longer y-rb region
(Bridges and Anderson, 1925). In free-X triploids
having crossing-over suppressed in one chromo-
some by Ins (1) sc8, dl-49, the frequency of
crossing-over in the y-w region between the
normal chromosomes is 9.3 per cent, which is
equivalent to a mean recombination frequency of
6.1 per cent for all X-chromosomes. Apparently
two of the three chromosomes pair distally and
yield cross-overs in roughly 9 per cent of the
disjunction products. In uninverted triploids the
non-pairing, non-crossover chromosome may
be any of the three randomly; but in the inversion
heterozygote the inverted X is usually the ex-
cluded chromosome.

The possibility of new investigation of this
distal increase is opened by studies involving a
reversed acrocentric attached-X triploid strain.
The distal normal member of the attached-X
probably pairs with the normal, free homologue,
leaving the inverted (Ins (1) scs, dl-49), proximal
chromosome unpaired. In this case the recom-
bination frequency in the y-w region is 16.5 per
cent. This secondary increase may be causally
related to the initial increase over the diploid;
but since our attached-X is deficient for some
large part of the X-heterochromatin we are not
yet able to distinguish between the various
mechanical and physiological hypotheses that

126

Section 7 — Cytogenetics

have been offered previously (Schultz and
Redfield, 1951).

The work of the senior author was done while
receiving the support of a National Science
Foundation Grant.

7.29. Crossing-over in Irradiated Females and Males
of Megaselia (Phoridae). F. Mainx and

E. Doschek (Vienna, Austria).

Megaselia scalaris has become an interesting
object for genetic studies in many respects. A
new type of sex determination was discovered
in this fly and reported on recently. Crossing-over
occurs also in males. The crossing-over values
are considerably reduced in males. The differing
rates of this reduction depend on the localization
of a given zone in the chromosome. Crossing-
over values increase after irradiation of the
pupae to a certain extent. This increase again
differs for particular chromosomal zones in
female and in male. Established on these results
a new theory of crossing-over could be con-
sidered.

7.30. Experimental Analysis of Interchromosome
Distribution and Genetic Significance of
Chiasmata. H. K. Jain and S. L. Basak (New
Delhi, India).

Observations on chiasma formation in
structurally homozygous and heterozygous
plants of Delphinium ajacis and other species,
in which two or more groups of chromosomes
could be recognized on the basis of size differ-
ences, have indicated the factors which control
interchromosome distribution of chiasmata. An
intraplant analysis first showed that chiasma
frequency in an organism like Delphinium can
be divided into an autonomous fraction which
the different chromosome pairs claim indepen-
dently of one another and a correlated fraction for
which they show interrelationships. It has been
found that the relative magnitudes of these two
fractions depend primarily on the karyotype,
particularly its symmetry. A highly asymmetrical
karyotype such as in Delphinium, favours a
relatively large interrelated fraction, which
generates negatively correlated interchromo-
some distribution. A symmetrical karyotype on
the other hand, like the one in Chrysanthemum,
favours a disproportionately large autonomous
fraction which is associated with absence of

interchromosomal effects, or with positively
correlated chiasma formation in different pairs.
It has been found possible to induce an inter-
chromosome redistribution of chiasmata in
Delphinium following elimination of one or two
of the chromosome pairs from forming chias-
mata— a condition brought about in a number of
plants, raised from X-irradiated seeds. The inter-
chromosomal effects for chiasma formation in
the normal and the Xi plants find a close
parallel in the "Schultz-Redfield" effect for
genetic recombination and the present analysis,
thus, provides further evidence for a corres-
pondence between chiasmata and genetic
recombination. A one to one relationship
between chiasma formation and chromatid
exchange has also been demonstrated in two
interchange heterozygotes of Delphinium, in
which it was found possible to record obser-
vations on chiasma formation in the interstitial
segments of the interchange multiple and on
anaphase separation of the chromatid parts
involved.

7.31. Autosomal Behavior in Claret-nondisjunctional
Drosophila Females. Gale Davis (Oak Ridge,
U.S.A.).

High frequencies of exceptional behavior of
chromosomes X and 4 were found by Lewis and
Gencarella (1952) in progenies of female
Drosophila melanogaster homozygous for caIld.
Studies reported here were undertaken to
determine the extent of chromosome-4 nondis-
junction and to clarify patterns of chromosome
behavior under the influence of cand.

Mating systems were devised to test nondis-
junction and loss of chromosomes X and 4
simultaneously. Females having y/y; cand/cand;
ciD/spaCat were mated to males having a com-
pound chromosome 4. The resultant progeny
reflected 63.4 per cent nullo-4, 3.2 per cent
diplo-4, and 33.4 per cent mono-4 egg types, and
18.3 per cent nullo-X, 2.7 per cent diplo-X, and
45.5 per cent mono-X egg types among recovered
offspring. The X-chromosome classes contained
an apparently nonrandom distribution of
chromosome-4 types, leading to the conclusion
that X and 4 were involved coincidentally in
nondisjunction as well as loss. Nondisjunction
and loss of chromosome 2 was studied by
mating cand females to males having a compound
chromosome 2. Estimates of frequency of
chromosome-2 nondisjunction were not possible
because behavior of compound autosomes in
the male was poorly understood. The ratio of
recovered nullo-2: diplo-2 egg types (2:1) con-

127

Section 7 — Cytogenetics

trasted to those observed independently for
nullo-X: diplo-X (6:1) and nullo-4: diplo-4
(20:1), yet these same ratios of X and 4 egg types
concomitant to chromosome-2 nondisjunction
were essentially unchanged.

This investigation was supported by U.S.
Public Health Service Predoctoral Fellowship
No. GPM-13,926-C1 from the National
Institutes of Health.

7.32. Nondisjunction in Drosophila hydei.
T. G. Gregg (Oxford, U.S.A.).

A previous report on nondisjunction in
Drosophila hydei^ indicated that the rate of
primary nondisjunction in hydei females was
somewhat higher than the rate in Drosophila
melanogaster females. On the basis of recent
work (2) it has become apparent that primary
nondisjunction in melanogaster females results
from the failure of two x-chromosomes from a no
exchange tetrad to engage in distributive
pairing during meiosis. It also seems that dis-
tributive pairing depends largely, although not
exclusively, on the centric heterochromatin.
On the basis of these latter observations one
might expect the rate of primary nondisjunction
in hydei females to be much lower than the rate
in melanogaster females, provided the mecha-
nisms involved in meiosis are similar in the two
species. This expectation is based on the unusual-
ly large amount of heterochromatin in the
hydei X which should facilitate distributive
pairing, and on the fact that the genetic length
of the hydei X is twice as great as the melano-
gaster X which should result in fewer no exchange
tetrads. Data have been collected that indicate
that the rate of primary nondisjunction in hydei
females actually is markedly and significantly
lower than the rate in melanogaster females. The
difference in rate between this report and Spen-
cer's report probably lies in Spencer's use of the
markers bobbed and Notch, which he himself
suggested might increase the rate of nondis-
junction. Data in this report also indicate that
in hydei females the rate of nondisjunction does
not increase with age.

1. spencer, J. Ohio Acad. Sci. 1930.

2. See especially grell, Proc. Natl. Acad. Sci.
U.S. 1962.

7.33. Evidence from Some Unusual Mutants Con-
cerning the Nature of t-alleles in the Mouse.

M. F. Lyon (Harwell, Great Britain).

"Mutation" of ^-alleles in the mouse results
usually from crossing-over in an abnormal
chromosome region. Study of the "mutants"
should therefore reveal whether the different
properties of r-alleles can be ascribed to
linearly arranged factors or depend on the
whole length of abnormal chromosome. The
properties of the allele t6 include: modification of
expression of dominant alleles at the T-locus,
homozygous lethality, male sterility, abnormal
male segregation ratio, and crossover sup-
pression. Mutants derived from t6 show that the
lethal factor is not at the T-locus but is close to
the nearby locus of tf, while the T-modifying
factor is very close to the T-locus. The allele
th7, derived from /6, decreases the expression of
T rather than enhancing it as /6 does, and can
mutate back, by crossing-over, to the /"-en-
hancing form. This suggests that it carries a
duplication of the T-modifying factor, and
hence that this factor has a gene activity which
is reduced, but greater than half normal.
Another mutant, thl5, also derived from th7 by
crossing-over, has a new lethal factor, comple-
mentary to the t6 lethal factor.

These facts suggest the hypothesis that the
fundamental chromosome abnormality in t-
alleles is reduced gene activity combined with
loss of specific pairing. This would lead to
crossover suppression, unequal crossing-over,
and hence to duplications and deficiencies. A
functional change in nucleic acid (e.g. to hetero-
chromatin) would provide a better explanation
than a structural one (e.g. inversion).

7.34, Cytological Investigation on the T-locus in Mus
musculus L. Irene Geyer-Duszynska (New
York, U.S.A.).

The T-locus in mice is known as a most complex
and odd one. Cytological analysis has revealed,
that this complexity is due to the fact that
lethal t-alleles are not point mutations but
deficiencies located in various regions of the
chromosome. The size and the position of the
deficiency is for a given t-allele constant. The
analysis of compounds and translocations T190
and T138 gave additional evidence that the defic-
iencies found are located in the chromosome of
linkage IX group, and connected with t-alleles.
Cytogenetically this chromosome can be de-
scribed as follows: centromere — T — tf — Ki —
Fu—H2— small deficiencies (twl8 and tw6)—

128

Section 7 — Cytogenetics

large middle deficiencies (t° and t"1 groups) of
terminal deficiency (t12). The spatial order —
deficiencies on the chromosome fits well with
the pattern of action of t-alleles which, when
homozygous are embryonic lethals, and indicates
that genes involved in different embryonic pro-
cesses are grouped in different regions of the
chromosome of.

Lethal t-alleles suppress recombination in the
region of 8-10 crossover units between T and

tf; it is suggested that this effect is due to
asynapsis accompanying a deficiency and spread
on other chromosomal regions. The possibility
that in the origin of a new t-allele which is
connected with the presence of an old t-allele
and with exceptional recombination within the
region where recombination usually is suppressed
by this allele, effective pairing occurring in the
time of chromosome reduplication can be
involved, will be discussed.

129

SECTION 8

CYTOTAXONOMY AND EXPERIMENTAL TAXONOMY

8.1. Cytology, Morphology, and Evolution in the
Proteaceae. L. A. S. Johnson and Barbara
G. Briggs (Sydney, Australia).

The Proteaceae have a predominantly South-
ern Hemisphere distribution, in Australia, Africa
and South America, extending into both eastern
and western tropics. A fairly complete survey,
chiefly at the generic level, suggests that the
"Proto-Proteaceae" had 7 pairs of large chromo-
somes, as found today in Placospermum, Persoo-
nia, and Garnieria. Reduction to 5 pairs of
moderate size is represented in Bellendena,
with subsequent tetraploidy to 10 pairs in
Symphionema. In the remainder of the family,
ancestral doubling of the original 7 is indicated;
all of these genera have very much smaller
chromosomes, n = 14 occurs in both subfamilies
Proteoideae and Grevilleoideae, but repeated and
independent reductions to « = 13, 12, 11 and 10
appear to have taken place. So far as known,
numbers within genera are constant, except for
one case of tetraploidy (Persoonia) and one of
aneuploid increase (Orites, n = 14, 15). The
changes in chromosome number and size appear
to have occurred at early stages in the evolution
of the family.

Comparative study of all morphological fea-
tures, together with cytology, has made it possible
to reconstruct the probable ancestral conditions
for the family and for its various subgroups -a
condition most nearly represented today in the
relict Placospermum (N.E. Queensland). A scheme
of relationships and evolutionary trends has
been worked out, taking into consideration
adaptive changes in relation to habitat, polli-
nators, and distribution. A tropical origin is
probable, with the temperate members in Austra-
lia and South Africa independently derived from
tropical sources, although the phytogeographic
history is complex.

See Austr. Journ. Bot. 11, No. 1, in press.

8.2. The Role of the Plastome of Oenothera in Evolu-
tion. W. Stubbe (Cologne, Germany).

Cleland (1957, 1958) has elaborated the out-
lines of the phylogenetic relationship between the
complex heterozygotic races of the subgenus
Euoenothera and their homozygotic ancestors.
The diversity of species was derived from three
fundamental groups of complexes carrying,
respectively, the genes for (a) the hookeri-
strigosa-phenotype, (b) the grandiflora-biennis-
phenotype and (c) the argillicola-(3 parvi-flora-
phenotype.

Independent investigations of Stubbe (1959)
demonstrated that the same major groups of
genome complexes stand out when attention is
confined to the co-operation between genomes
and plastomes. Five plastid types can be identified,
their distribution being of systematic importance.
On the basis of recent results the following facts
can be established about the role of the plastome
in evolution:

1. All known kinds of damage to the normal
plastid development lead to a negative selection.
Sometimes these damages arise by spontaneous
mutation, but more frequently we find them after
interspecific hybridization. Since the pollen
contributes plastids to the zygote, though less
than the egg cell, we get a somatic segregation-
pattern of different plastid phenotypes, these
being green or chlorophyll-deficient, depending
on a good or bad compatibility between genome
and plastome. The viability of such chimaeras
depends upon the size of their green foliage
portion.

If not only one but both kinds of parental
plastids are incompatible with the hybrid
genotype, the plastid differences result in a barrier
for hybridization, i.e. a factor establishing
isolation. Such plastome-dependent incompatibil-
ity has been demonstrated in crosses between the
homozygous species of the hookeri-, elata- and
grandiflora-group on one hand and the argilli-
cola-group on the other.

2. In hybrids in which the genetically different
plastids of both parents develop normally, there
will be an invisible somatic segregation-pattern.
Concerning evolution, another factor brings its
influence to bear, namely plastid competition
(Schotz, 1954). This is due to differences in the
multiplication rates of the plastid types taking
effect in the "mixed" cells. If outcrossing is
guaranteed, repeated mixing of the different

131

Section 8 — Cytatoxonomy and Experimental Taxonomy

plastids occurs in the zygotes, and the subse-
quent plastid competition will lead to a gradual
elimination of the plastid type which multiplies
more slowly. It is a proven fact that this process
did happen during phylogeny, since wild species
which have been shown to be compatible with
more than one plastid type, now possess the
faster multiplying plastid type.

3. Sometimes the incompatability between
certain genomes and plastomes is expressed even
in the microspores by inhibited germination of
living pollen grains or by early cessation of
pollentube growth. As this character shows up
mainly after hybridization, Renner (1919)
regarded it as the mechanism responsible for the
elimination of one complex in the pollen. This
mechanism would tend to make the complex-
heterozygotic species heterogamous. Species of
the parviflora-groups would seem to offer
examples for this mechanism. Furthermore,
based on experimental results about plastid com-
petition and about the compatibility correlations
between the five wild type plastids and the
genotypes of the subgenus Euoenothera, the
phylogeny of the five plastomes of this subgenus
are being inferred step by step in an unambiguous
manner.

8.3. Chromosome Differentiation in Genomes of
Gossypium. M. S. Brown (College Station,
U.S.A.).

Chromosome studies in several leguminous
genera made by various investigators suggest
heterogeneity within the genera as to base num-
ber, chromosome dimensions and shape.

Investigations in Indigofera from West and
East Africa, and in a few Himalayan and East
Asiatic species, reveal differences in base number,
shape and size between the various species, and
a few cases of polyploidy.

Recent data for the genus Vigna show two
base numbers, 10 and 1 1, this in accordance with
earlier reports, and considerable differences in
chromosome dimensions within the Vigna
unguiculata complex. Grafting experiments be-
tween some cultivars of the cultivated cowpeas
resulted in a success of 80-90 per cent when
using types with equal chromosome dimensions,
and a success of below 50 per cent in the case of
deviating dimensions.

There exist several other genera, e.g. Trifolium,
Astragalus, Lespedeza, in which either ranges of
base numbers or entirely different base numbers
are present. Sometimes such a difference agrees
with different gene centres. In some case loss or
increase of chromosomes has been suggested.

Another line of thought next to those men-
tioned above may be the acceptance of poly-
phyletic evolution: the same lines of develop-
ment acting on different original substrates under
similar circumstances, but at various points of
time may result in convergent morphological
types brought under an equal denomination by
the taxonomists.

Chromosomes of species and Fi hybrids of
Gossypium are compared at pachytene, diplotene,
diakinesis and metaphase. At pachytene, all
chromosomes pair closely, even in sterile hybrids
between species with chromosomes of unequal
size and low bivalent number at metaphase. At
diplotene, genome differences become apparent;
and at diakinesis and metaphase, size differences
in chromosomes are shown to be correlated with
amount and distribution of chromatic material.
In monosomies of Gossypium hirsutum, an allo-
tetraploid, chromosomes of A and D genomes
can be distinguished in like manner. These
differences in chromosome size and composition
can be correlated with variation in the cytosine/5-
methylcytosine ratios as determined in A, B and
D genomes by Ergle and Katterman.

8.4. Signification of Cytological Data for the Tax-
onomic Conception of the Leguminosae. J. A.

Frahm-Leliveld (Wageningen, The Nether-
lands).

8.5. Desynapsis in Lotus Hybrids. William F. Grant
(Quebec, Canada).

In meiotic analyses of several embryo cultured
interspecific hybrids between six diploid (2n =
12) species of Lotus (Leguminosae), closely
related to L. corniculatus, univalents, resulting
from precocious separation of the bivalents, were
observed at diakinesis and metaphase I. In some
cells up to 50 per cent of the chromosomes were
observed as univalents. Univalents, however,
were present in only 40 per cent of the cells
examined. Desynapsis is considered to be pri-
marily of the "weak" type as defined by Prakken.
In some cases, at metaphase I, the univalents were
observed to be loosely connected. At anaphase
I, lagging chromosomes were observed, most of
which consisted of univalents. Occasionally
lagging chromosomes were observed to remain
behind in the cytoplasm at telophase I and to fail
to be incorporated within the restitution nuclei.
In some cases the telophase I chromosome com-
plements consisted of more, or less, than the

132

Section 8 — Cytato.xonomy and Experimental Taxonomy

normal chromosome number as a result of une-
qual distribution of the chromosomes at anaphase
I. The loose bivalents observed in a number of
cells at metaphase I signify some lack of specific-
ity in pairing of the homologous chromosomes
which may represent the visible remnants of the
effects of earlier translocations through which
species differentiation has occurred. The high
percentage of pollen sterility and the low seed set
in the hybrids may partly be caused by extensive
intrachromosomal rearrangements, since it is
difficult to reconcile the very low fertility with the
relatively low frequencies of visible cytological
anomalies only. The precocious division of the
bivalents is considered, therefore, to be the
result of segmental and genetic differences
between the parental species. The hybrids exhib-
iting this desynaptic behavior were found in
crosses between L. japonicus x L. schoelleri, L.
krylovii X L. filicaulis, L. japonicus X L. a I pin us,
and L. schoelleri x L. krylovii.

8.6. The Phylogenetic Riddle of Astragalus (Legumi-
nosae). G. F. Ledingham (Regina, Canada).

Chromosome counts of 252 species of Astra-
galus have been made. The species counted fall
into two distinct groups; the 163 Old World
species are based on an 8-chromosome series with
23 per cent polyploidy while the 89 New World
species counted form an "aneuploid" series with
n = 11 (39 species), n = 12 (34), n = 13 (12), or
« = 14 (3) and n = 22 (1), the last species being
the only polyploid. Ten Old World species
(6 with In = 16 and 4 with 2n = 32) have
spread recently, probably during the Pleistocene,
from N.E. Asia into N.W. America, but these
are still easily recognized by their morphological
similarity to other Old World species. There is
no indication of an aneuploid series among Old
World species of Astragalus and no deviation
from the 8-chromosome number series except
for A. boeticus (In = 30), A. pentaglottis {In =
28) and A. somalensis (In = 20) and these occur
in the Mediterranean area or in Africa. Thirty
species of Oxytropis have been counted and these
all have n = 8 or some multiple of 8, 36 per cent
polyploidy. Oxytropis seems to be an integral
part of Old World Astragalus. The origin and
significance of the consistent differences between
New and Old World phylogenetic lines of
Astragalus is discussed.

8.7. Cytogenetics of Vicia cracca and V. tenuifolia.

Arne Rousi (Piikkio, Finland).

Vicia cracca L. consists of three chromosome

races (In = 12, In = 14 and In = 28). The two
latter ones are morphologically close to each
other and have basically similar karyotypes. The
karyotype of the 12-chromosome race suggests an
origin from the 14-chromosome race as a result
of one or probably two interchanges and the
subsequent loss of one chromosome. Systemati-
cally it seems to tally with the taxon V. cracca
subsp. Geraldi (All.) Gaudin. Spontaneously
arisen albinotic and inviable seedlings with 13
chromosomes were interpreted as hybrids
between the 12- and 14-chromosome races on
the basis of their karyotypes and hemagglutin-
ating properties. This suggests that these races
are effectively isolated by means of hybrid in-
viability. Hybridization between the self-in-
compatible 14- and 28-chromosome races was
attempted by placing solitary potted individuals
of the former among populations of the latter.
The seed set of the diploids was very low, but one
viable triploid and one tetraploid arose, the
latter being apparently a result of an unreduced
egg cell. Although there is a selection against
triploids the isolation between the 14- and 28-
chromosome races is therefore not complete.
V. tenuifolia Roth occurs as a diploid (2n = 12)
and a tetraploid (In = 24) race, the diploid
being rare and somewhat distinct. The karyo-
types of both are basically similar to the 12
chromosome race of V. cracca. Certain morpho-
logical resemblance between this race and
tetraploid V. tenuifolia suggests a close evolu-
tionary relationship.

8.8. Cytological Studies in Melilotus Polyploids.

J. Jaranowski (Poznari, Poland).

Inducement of artificial polyploids in the genus
Melilotus was mainly intended to utilize them
in the attempts to overcome the barriers of re-
productive isolation in species hybridization.
Owing to their peculiar properties these poly-
ploids were closely examined and cytologically
analysed to obtain some information on the
relationship of species.

The basic number of chromosomes for the
genus Melilotus is n = 8. Fourteen species, 7
varieties 3 species hybrids (M. alba x M. offi-
cinalis), and 33 [different forms of backcrosses of
the species hybrids were treated with colchicine
(0-2-0-7 percentage cone.) to induce polyploidy.

The number of polyploids obtained in the
different species varies greatly. Biennials (be-
longing to the section Eumelilotus) and annuals
(belonging to the section Micromelilotus) re-
sponded quite differently to colchicine treatment

133

Section 8 — Cytatoxonomy and Experimental Taxonomy

The latter forms gave a much larger proportion
of polyploids. In the biennials it was more difficult
to induce polyploidy among others because — in
cases when the action of colchicine on the hypoco-
tyl was not strong enough — their second-year
spring growths were diploids. In addition, there of-
ten occurred mixoploid forms. Some of the species
are exceptionally resistant to the mutagenic
action of colchicine (e.g. M. suaveolens, M.
volgica, M. taurica). A 0.7 per cent solution of
colchicine failed to bring about changes in
these species. Progressive rises of the colchicine
concentrations up to a strength, resulting in a
total destruction of plants, did not induce
polyploidy.

In regarding pollen viability as a criterion of
fertility, the induced polyploids greatly varied in
this respect. Some autotetraploids like M. offi-
cinales and M. alba yielded nearly 100 per cent
of viable pollen, while others like M. messanensis
only 10-15 per cent; M. hirsuta, M. segetalis and
M. italica about 50 per cent. Frequency in the
occurrence of quadrivalents, univalents and
polyvalents in meioses accounts partly for this
situation.

Analyses of pollen viability in vitro show that
polyploidization changes the physiologic reaction
of pollen grains to the medium and to the optimal
temperature of germination. Differences between
species are great.

Inducement of polyploidy failed to overcome
self-incompatibility in M. officinalis.

8.9. (D). Evolution in the Genus Mentha from a Genetic
Viewpoint. M. J. Murray (Kalamazoo,
U.S.A.).

The fertile species in the spicate-flowered
section of the genus are Mentha rotundifolia
{2n — 24), Mentha longifolia (2/7 = 24), Mentha
spicata {In = 48) and Mentha aquatica (2« =
96). The sterile species are M. spicata (In = 36),
M. niliaca (2n = 36), and M. piperita (2n = 72).
All colchicine-induced 4ns are fertile. Fi and F2
hybrids between the basic species are intermedi-
ate in appearance and do not resemble M. spicata;
but hybrids between M. spicata and An M.
rotundifolia or An M. longifolia are perfectly
fertile. M. spicata strains are highly variable in
leaf size, leaf shape, crispness of leaves and plant
habit. Contrary to popular belief, M. spicata
individuals do not invariably have a spearmint
odor with the ketones carvone and dihydro-
carvone. Some strains have the ketones menthone
and pulegone as also found in the species M.
aquatica and M. piperita (genotypes ccAA of
ccAa), while others have piperitone and piper-

itenone as found in the basic species (genotype
ccaa). Selfed heterozygous strains (CcAa) give
a ratio of 12:3:1 for the 3 ketone types. M.
aquatica with 40 per cent menthofuran and the
menthone-pulegone ketones can be hybridized
to M. spicata (menthone-pulegone or piperitone-
piperitenone genotypes) to resynthesize M. pipe-
rita (2n = 72). These peppermint-odored hy-
brids are of substandard commercial quality in
having too low menthol, too high ester, and
tremendous differences in minor constitutents
(terpene hydrocarbons of which there are at
least 20). They differ greatly in plant habit, leaf
size, leaf shape, leaf crispness depending upon
the M. spicata parent used in the cross. Spear-
mint-odored hybrids occur in all crosses where
the M. spicata parent is spearmint-odored.
Diploid chromosome numbers 60, 72, 84 and 96
may occur in other peppermint-odored inter-
specific combinations.

8.10. Cytological Studies in Section Molium of the
Genus Allium. S. E. Eid (Shatby, Egypt).

The following results have been obtained:
I. A. blomfieldianum Asch. & Schweinf. has
In = 14 chromosomes with median (m) or
submedian (sm) centromeres.
II. A. neapolitanum Cir. shows two forms, an
allotriploid with 2n = 21 m and sm chro-
mosomes, and an allotetraploid with 2n =
28 m and sm chromosomes. The triploid
shows nonpairing of one set of seven chro-
mosomes and is sterile.

III. A. erdelli Zucc. (including its v. roseum
Boiss.) has 2n = 16 m and sm chromosome1,
with four telocentrics.

IV. A. roseum v. tourneuxiii Boiss. shows two
cytological forms, a diploid with 2n = 16 m
and sm chromosomes with two telocentrics;
and an autotriploid with 2n == 24 m and sm
chromosomes with three telocentrics. A.
roseum v. bulbilliferum Vis. is found to be
an allopentaploid with 2n = 40 m and sm
chromosomes with five telocentrics. It is
sterile and viviparous.

The basic number in the present species is
shown to be seven, with centric breakage of
metacentrics into telocentrics in A. erdelli and
A. roseum.

8.11. Species Relationship in the Genus Avena. J. H.

W. Holden (Durham, Great Britain).

The diploid species Avena longiglumis shows

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Section 8 — Cytatoxonomy and Experimental Taxonomy

extensive chromosomal differentiation from all
other diploid species. The nature of this differ-
entiation and its significance in the study of
species relationships in diploid, tetraploid and
hexaploid Avenae will be discussed.

8.12. Some Experimental Data on the Range of the
Species Hordeum spontaneum C. Koch emend.
Bacht. F.Kh. BAKHTEYEv(Leningrad,U.S.S.R.).

Until lately it was thought that Hordeum spon-
taneum includes only two-rowed forms. How-
ever, the discovery in 1958 in the Turkmenian
Soviet Republic territory of a six-rowed wild-
growing barley, conditionally called Hordeum
lagunculiforme Bacht., made it necessary to re-
consider this question, especially as somewhat
later similar six-rowed wild barleys were found
in the Azerbaijan and Tadjik Republics, as well
as in some other localities of the U.S.S.R.

The author'sown large original materials ena-
bled the conclusion to be drawn that H. lagunculi-
forme is actually not an independent species, as
one could suppose up to the present time, but
represents another extreme link in the system
of the species Hordeum spontaneum C. Koch
emend. Bacht. The latter includes four strains:
(1) var. spontaneum (= H. spontaneum var. itha-
burense [Boiss.] Nabelek); (2) var. ischnatherum
(Cosson) Thell.; (3) var. proskowetzii Nabelek;
(4) var. lagunculiforme Bacht. Thus, we may
consider it proved that Hordeum spontaneum
C. Koch emend. Bacht. includes not only two-
rowed individuals, as was thought since the first
description of this species by C. Koch in 1848,
but that it involves also six-rowed forms.

The above data should be regarded not only
as a new illustration to the splended theoretical
conception of N. I. Vavilov, "The Linnean
species as a system", but as a real basis for fur-
ther experimental investigations into the origin,
phylogeny, and evolution of cultivated barley.

In principle one may probably agree with the
conception, that the initial six-rowed and two-
rowed cultivated barley forms originated from
the genetically nearly-related Hordeum sponta-
neum C. Koch emend. Bacht.

Hordeum halophilum and H. muticum are two
diploid sympatric species distributed along the
mountains from Peru to Argentina. The mor-
phology, chromosomal meiotic behaviour and
karyotypes of these two species and of their
natural sterile hybrid have been studied.

Meiosis in the parental strains is regular or
nearly so. H. halophilum had the following mean
chromosome associations: 6-99 II, 002 I, and
H. muticum 6*81 II, 0-38 I, while the hybrid had
002 IV, 001 III, 6-63 II, 0-62 I. The meiotic be-
haviour of the chromosomes in the hybrid as
well as the karyotypes indicate that the genomes
of both species are largely homologous. They
differ, however, in at least one reciprocal trans-
location and one paracentric inversion.

The main isolating mechanisms between both
species seem to be the slightly different edaphic
requirements and the absolute sterility of the
hybrids that makes gene interchange impossible.

At anaphase II 8.5 per cent of the cells had 1-3
"dineocentric" bridges. These originated from
activation of usually terminal or moie rarely
interstitial neocentromeres, each stretching the
laggard and causing the formation of a bridge,
with the centric region attenuated and stretched.
This phenomenon has already been observed in
other grass hybrids (Hunziker and Covas, Rev.
Investig. Agric. 9 (3), 155-175, 1955; Walters,
M. S., Univ. of Calif. Public, in Bot. 28 (6), 335-
447, 1957, etc.).

8.13. Evolutionary Relationships between Two South
American Species of Hordeum. Juan H. Hun-
ziker and Leonor Maumus (Buenos Aires,
Argentina).

8.14. Genome Construction Within the Triticinae.

L. E. Evans (Winnipeg, Canada).

The two synthetic amphiploids Triticum durum
var. Carleton — Aegilops squarrosa (AABBDD)
and Triticum durum var. Stewart — Agropyron
elongatum (AABBEE) when crossed produced a
partially fertile F- having an average meiotic
metaphase configuration of 14- + 1411. By means
of cytological selection in the F2 to F7 generations
32 moderately stable, fertile, hexaploid derivates
were obtained from this cross. These individuals
should possess the 14 pairs of T. durum chromo-
somes (AABB) plus a mixture of Aegilops (DD)
and Agropyron (EE) chromosomes in the third
genome. Several of these lines were backcrossed
to the original hexaploid parents and the back-
cross Fi plants were cytologically studied in
order to determine the number of Aegilops and
Agropyron chromosomes in the third genome.
The combinations that theoretically could be
produced range from 7D+0E to 7E+0D chro-
mosomes and the results indicate that the entire
range may be produced. The identity of the spe-

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Section 8 — Cytatoxonomy and experimental taxonomy

cific D and E chromosomes in each derivative
has not been determined.

If this phenomenon has occurred in nature it
may explain the difficulty encountered in deter-
mining the exact parentage of such naturally
occurring allopolyploids as Triticum aestivum.

8.15. DNA and Wheat Ancestry. H. Rees and W. I. C.

Davies (Aberystwyth, Great Britain).

It is generally agreed that the AA chromo-
somes found in the AABB tetraploid and
AABBDD hexaploid wheats are derived from
Triticum monococcum or a closely related diploid
species, and that the DD chromosomes are de-
rived from Aegilops squarrosa. There is some
doubt concerning the source of the BB chromo-
somes. The chief candidates are Ae. speltoides,
Ae. bicornis and Agropyron triticeum. From an
analysis of the DNA content of nuclei in the
polyploid and diploid species our evidence shows
that Ae. speltoides is the most likely contributor
of the BB chromosomes. Its nuclear DNA con-
tent added to that of T. monococcum (AA) agrees
with the values found in AABB (T. durum), and
in combination with AA and DD values agrees
exactly with the DNA value for AABBDD (T.
aestivum). The DNA values for Ae. bicornis and
A. triticeum are, compared with that for Ae. spel-
toides, too high and too low respectively.

8.16. Cytotaxonomic Studies in the Genus Agropyron
Gaertn. Jurgen Schulz-Schaeffer, Peter
Jurasits, Helmut Lorenz and Penelope W.
Allderdice (Montana, U.S.A.).

Within the framework of a biosystematic in-
vestigation in the genus Agropyron studies of
cytological characteristics are being carried out
for taxonomic purposes. Karyotypes of 33 Agro-
pyron species were determined from root tip
cells and were compared. In general, centro-
meres are median or submedian. Agropyron
triticeum (2/7 = 14) and A. orientate (2n = 28)
are exceptions to this rule. Nuclear organizer
chromosomes were compared. According to an
evolutionary concept, one nucleolar organizer
chromosome should be present for each basic
genome. This was almost averaged in previous
studies of the genus Bromus. Only about half of
the expected number were observed in Agro-
pyron. There seems to be a considerable difficulty
involved in detecting certain nucleolar organizer

chromosomes in mitotic metaphase. Investiga-
tions in meiosis are performed to supplement.
Eighty-eight nucleolar organizer chromosomes
were observed in 160 genomes. They can be
classified as 11 different types. One type (F-l,
F-2) occurs in 19 of the 33 species. Type F seems
to represent one basic genome which probably is
common to a great number of Agropyron species.
The nucleoli as a possible measure of nucleolar
organizer chromosome number were determined
by the Rattenbury-method for 5 species. The
average number was, in general, corresponding
with the number of basic genomes. An average
of 4 nucleoli was found for tetraploid A. deser-
torum. This may confirm unpublished evidence
from coworkers that A. desertorum has a certain
number of nucleolar organizer chromosomes.

8.17. Karyotypic Studies of South American Snakes.
W. Becak:, M. L. Becak: and H. R. S. Naza-
reth (Sao Paulo, Brazil).

The sex elements can be readily identified by
cytological means in the chromosome comple-
ments of most mammalian and avian species. In
the lower vertebrates, however, the Z and W, or
X and Y, may still be so undifferentiated as to be
morphologically indistinguishable.

Karyotypic studies have been made of two
species of South American snakes, Boa constric-
tor amarali and Bothrops jararaca. Both have 36
chromosomes: 16 macrochromosomes and 20
microchromosomes.

In Boa constrictor amarali the centromere is
almost median in the first, third and fourth
pairs, submedian in the second pair and sub-
terminal in the fifth, sixth, seventh and eighth
pairs. Neither quantitative evaluation nor direct
observation of the karyotypes revealed the pres-
ence of a heteromorphic sex pair in either the
male or female of this species.

In the male of Bothrops jararaca, the first,
third and fourth pairs have submedian centro-
meres and the sixth and eighth pairs have sub-
terminal centromeres. In the female, however,
the fourth pair is distinctly heteromorphic. One
element is much smaller than the other and has
a subterminal centromere as well. Thus in this
species the fourth largest pair of chromosomes
appears to be the sex pair; the female is the
heterogametic sex, and the sex-determining mech-
anism is the ZZ-ZW type. The fact that the W
chromosome appearing only in the heterogametic
female is seven-tenths the size of the Z, permits
cytological identification of the sex chromosomes
in Bothrops jararaca.

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Section 8 — Cytatoxonomy and Experimental Taxonomy

8.18. Chromosome Studies of Domestic Mammals.

J. F. Lopez-Saez and G. Gimenez-Martin
(Madrid, Spain).

In this work the chromosome complement(s)
of the horse (Equus caballus), ass (Equus asinus),
mule {Equus caballus X Equus asinus), bull (Bos
taurus), sheep (Ovis aries), goat (Capra hircus)
and domestic pig (Sus scrofa domesticus) are
studied. The karyotype of each species is as
follows:

The horse {Equus caballus) 2n = 64. The X
chromosome has submedian centromere and is
in size the second of the complement ; the Y chro-
mosome is telocentric or apparently telocentric,
being in the complement two pairs smaller than it.

The ass (E. asinus) In = 62. The X chromo-
some has subterminal centromere and is in length
the fourth pair of the complement; the Y chromo-
somes, with submedian centromere, is the small-
est chromosome.

The mule (E. caballus, E. asinus) 2« = 63.
The male has the X chromosome of the horse and
the Y chromosome of the ass; the female has the
X chromosome of the horse and the X chromo-
some of the ass.

The bull (Bos taurus) 2n = 60. The sex chro-
mosomes are the only ones with metacentric or
submetacentric centromere. The X chromosome
is the longest of the idiogram and the Y chro-
mosome is among the five shortest chromosomes,
but it has median centromere.

The sheep (Ovis aries) 2n = 54. The X chro-
mosome is the longest of the telocentric or ap-
parently telocentric chromosomes and the Y,
also telocentric, is the smallest of the idiogram.

The goat (Capra hircus) 2n = 60. All chromo-
somes are telocentric or apparently telocentric.
The X chromosome is the second in the haploid
idiogram and the Y is the smallest of the comple-
ment

The pig (Sus scrofa domesticus) 2n = 38. The
X chromosome is metacentric and the Y chro-
mosome is the smallest of the karyotype. This is
characterized by a median centromere.

8.19. The Karyotype of Normal Pigs and of One
Intersex. B. Henricson and L. Backstrom
(Stockholm, Sweden).

The karyotype of a swine intersex has been
analyzed in blood leucocytes grown in tissue
culture. Also external and internal genitalia have
been investigated.

As the karyotype of normal swine has been
described in only a few earlier works, this is also
presented here with suggestions for grouping and
numerical classification.

The intersex investigated had a karyotype
which contained 36 apparently normal auto-
somes with sex chromosomes of the XX type.

The external genitalia consisted of a vagina
with a relatively big clitoris. There were two
testicles of about normal size. One was situated
in the scrotum, the other was abdominal. No
germ cells were present, only Sertoli cells in well-
defined tubuli and Leydig cells in the intersti-
tium. No ovarial tissue could be detected. Pri-
itive ductuli deferentes from the testicles went
over in a rather well-developed uterus.

Pictures are presented.

8.20. Classification of the Chromosomes of the Mouse
Karyotype. Marco Crippa (Rome, Italy).

Actively proliferating cultures of cells obtained
by trypsinization from embryonic kidneys of
C3H and albino Swiss mice were submitted to
the techniques usually employed for chromosome
studies: colchicine and hypotonic treatment,
air-drying and orcein staining. By arranging the
40 chromosomes of the normal mouse diploid
number according to the criterion of decreasing
length, it was possible to subdivide them into
five distinct groups, each composed by chromo-
somes of similar length. The first group includes
4 chromosomes, the second 7 chromosomes in
the male and 8 in the female, the third 20 chromo-
somes, the fourth 6 chromosomes and the fifth
3 chromosomes in the male and 2 in the female.
According to this classification it seems reason-
able to assume that the X chromosome belongs
to the second group and the Y chromosome to
the fifth group. The X chiomosome was never
observed to have any peculiarity like hetero-
chromatic traits or secondary constrictions which
could differentiate it from the autosomes of the
same group. Nor was it possible to identify the
Y chromosome within the small chromosomes of
the last group. The length of each chromosome
was measured and the data obtained were statis-
tically evaluated by using the analysis of variance.
This analysis showed that the differences in
chromosome length among the five groups are
highly significant, although a variability among
different mitoses is present to a certain extent.
Further details concerning the data obtained by
the statistical analysis will be discussed.

8.21. On the Chromosomes of the Geometrid Moths
Cidaria. E. Suomalainen (Helsinki, Finland).

In the species-rich Geometrid moth genus.

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Section 8 — Cytatoxonomy and Experimental Taxonomy

Cidaria, the chromosome number of 40 Finnish
species has been determined, i.e. of about half
the number of species found in Finland. The
chromosome number has been determined from
oogenesis, but in some species spermatogenesis
has also been studied. The commonest (haploid)
number among the Cidaria species so far investi-
gated is 31, which is likewise the next commonest
number among the Lepidoptera as a whole.
Thirteen species had this number. Most (32, or
80 per cent) of the species had a chromosome
number between 28 and 32, like most of the
other groups of Lepidoptera. No species had
more than 32 chromosomes, whereas eight had
less than 28. The smallest chromosome numbers
found were 25 (two species), 19, 17 and 13
(three species and probably also a fourth one).
The great differences in chromosome number
between closely related species is of interest.
Such discrepancies are shown by the subgenera
Thera (variata and obeliscata 13, firmata 19,
and juniperata 30), Lampropteryx (minna 1 7 and
suffumata 32), and Hydrelia (testaceata 13 and
flammeolaria 30). The chromosomes are clearly
bigger in the species with a low chromosome
number than in those with a high one. Photo-
metric measurements revealed that the DNA-
content of closely related species is almost the
same, in spite of great differences in chromosome
number. This indicates that one chromosome of
a species with a low number corresponds to two
or more chromosomes of another one with a
high number. Consequently, we are not con-
cerned with polyploidy, although the higher
number is close to a multiple of the lower one.
It is probable that this kind of chromosomal
evolution — found among the Lepidoptera — is
rendered possible by the fact that they obviously
have a diffuse kinetochore. Contrary to earlier
assumptions, chiasmata are not formed in the
bivalents during oogenesis in the Lepidoptera.
This is especially evident in preparations stained
with Feulgen, when the elimination chromatin
contained by the bivalents in the female remains
unstained.

8.22. Cytotaxonomy of Syrphid Flies. Janny M. van
Brink and J. W. Boyes (Utrecht, The Nether-
lands).

A Xanthogramma species has five pairs of
chromosomes; six species of Syrphus have four
pairs and three others have five pairs, but in
one species counts of four pairs and five pairs
were obtained. Two Chrysotoxum species in the
subfamily Chrysotoxinae had five pairs. In the
subfamily Cheilosiinae, species in the genera
Neoascia, Pipizella and Cnemodon have four
pairs; in Rhingia, Myolepta and Cheilosia five
pairs; and in Chrysogaster and Liogaster six
pairs. One Vohtcella species of the subfamily
Volucellinae has six pairs but another has five
pairs plus a few microchromosomes. In the
subfamily Eristalinae, seven species of Eristalis
have karyotypes consisting of five metacentric-
subtelocentric pairs, apparently autosomal, plus
one or two additional pairs, or plus a peculiar
complex of small elements which will require
detailed analysis for accurate interpretation.
Six pairs were found regularly in species of
Eristalinus, Merodon, Eurinomyia, Helophilus and
Parhelophilus but only five pairs in Myiatropa
ftorea. In the Xylotinae single representative
species of Tropidia, Syritta, and Zelima have
five pairs. Many variations in chromosome
morphology were also found and will be men-
tioned briefly. This is a preliminary report on an
extensive analysis of karyotypic variation in
this family.

8.23. The Anal Plates of Drosophila Larvae in Differ-
ent Species. Suavi Yalvac (Erzurum, Turkey).

Exposing 3rd instar larvae of Drosophila to
70 per cent alcohol causes a pigmentation which
begins on the posterior end of the larval body
and spreads forward. This occurrence helps to
make two symmetrical plates appear around the
anus of the larva. Otherwise it is too difficult to
notice these plates because of their transparency.

After comparing about 30 species of Droso-
philidae it has been established that different
types of anal plates exist in the larvae of these
flies. Some of them, like D. victoria, D. buskii,
D. mulleri, Chymomyza procnemius and others,
have anal plates so typical for their own species
that one can easily identify their larvae by
means of these plates.

It seems that anal plates of Drosophila larvae
might be considered as a taxonomic factor.

The chromosomes of 56 species of Syrphidae
from the Netherlands have been examined.
Species of most genera in the subfamily Syr-
phinae have four pairs of chromosomes, includ-
ing a small heteromorphic pair and three larger
metacentric to subtelocentric pairs (Platychirus,
Melanostoma, Sphaerophoria, Doros and Scaeva).

8.24. Levels of Speciation and Reproductive
Affinities in the Willistoni Cryptic Group of
Drosophila. Helga Winge (Porto Alegre,
Brazil).

The intercrossing of strains from different

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Section 8 — Cytatoxonomy and Experimental Taxonomy

localities, belonging to D. Willistoni and sibling
species, disclosed several levels in the speciation
process. A total of 90 different intercrosses, with
five repetitions, each with 10 pairs of flies, gave
450 replications, which were periodically trans-
ferred to new vials until the death of the females
(about 60 days).

Amongst the most significant results is the
production of Fi, fertile inter se from paulistorum
males x willistoni females strains from Southern
Brazil, edge of D. paulistorum distribution. The
reciprocal cross yielded few sterile individuals.

The offspring of paulistorum males x equi-
noxialis females showed a large excess of fertile
females and rare, sterile and always mutant
males (eyes, wings or bristles). The reciprocal
crosses yielded few descendants, the sex-ratio
being normal, but all males sterile and mutants
(eyes, wings and bristles).

The degrees of affinities in regard to the
cytoplasm allowing the development of inter-
specific hybrids, at least until larvae, can be
stated very briefly as follows: insularis (all
other tested) > willistoni {tropicalis, paulistorum,
equinoxialis and rarely with insularis) > paulis-
torum (equinoxialis, tropicalis, some strains of
willistoni and rarely with insularis) > equino-
xialis (paulistorum, tropicalis and rarely with
willistoni) > tropicalis tropicalis from Palma
(other tropicalis and paulistorum) > tropicalis
cubana and tropicalis from Tefe and Trinidad
(inter se only).

D. tropicalis cubana (Townsend, 1954) can be
ranked to D. cubana with sub-species distributed
over the Antilles, Trinidad and the Amazon
basin (Tefe to Belem). Males of D. cubana from
Cuba, Trinidad or Tefe crossed with D. tropicalis
tropicalis from the type locality, Palma, yielded
few descendants, the males being sterile and
females fertile with paternal strains only. The
reciprocal crosses produced no descendants.

The D. cubana "complex" of sub-species
from Cuba, Tefe and Trinidad produced, inter
se abundant Fi with fertile females and sterile
males in both ways.

8.25. Cytological Studies in the Cardini Superspecies
of Drosophila. William B. Heed and Jean
S. Russell (Tucson, U.S.A.).

In order to determine the phylogenetic re-
lationships among the 15 species in the cardini
superspecies of Drosophila, a salivary gland
chromosome map has been constructed from a
homozygous population of D. acutilabella from
Cuba. Nine species have been analysed thus far

and their inversions have been compared with
the standard sequence from Cuba. Eight of the
nine species form a natural subgroup in that
their population structures are dependent upon
the position and size of the islands in the Carib-
bean Sea. A total of 26 inversions were found
among 34 separate laboratory populations and
strains. Fifteen inversions are homozygous and
11 inversions are heterozygous. D. acutilabella
is homozygous for the standard gene sequence
in Florida (4 localities), Jamaica (6 localities)
and Cuba (1 locality). It is highly polymorphic,
however, in Haiti (3 localities) with 8 hetero-
zygous inversions. Five separate populations
of D. dunni from 3 localities in Puerto Rico are
homozygous, and they are all fixed for 3 in-
version differences from standard in the X chro-
mosome. Analysis of the polymorphism in the
Haitian acutilabella is in progress.

8.26. Cytotaxonomy in the Drosophila melanica
Species Group. Harrison D. Stalker (St.
Louis, U.S.A.).

On the basis of morphology and reproductive
isolation the six New World species of this
predominantly Nearctic group may be arranged
in a sub-group of four sibling species: D. mela-
nica, D. paramelanica, D. euronotus and D. mela-
nura; and two more distinct species: D. micro-
melanica and D. nigromelanica. D. micromelanica,
considered to be the most nearly ancestral, has
a rod-shaped, single-element X in most strains;
the other five species have V-shaped, two-
element X chromosomes. Salivary chromosome
differences in banding sequence were studied by
hybridization and by the comparison of photo-
graphs and composite photomaps. In all six
species chromosome II is the most variable, show-
ing 57 inversion differences within species. Chro-
mosomes XR, XL, III and IV show 10, 6, 1 and 2
intraspecific inversion differences respectively.
Chromosomes showing the largest number of
heterozygous inversions within species also show
the most homozygous inversion differences
between species. Chromosome comparisons
indicate the following phylogenetic sequence
for the group: D. micromelanica — D. euronotus
and D. melanura — D. paramelania — D. mela-
nica. (D. nigromelanica is apparently derived
from D. micromelanica independently of the
D. euronotus, D. melanura pair.) Within the group
of four sibling species, the ratio of heterozygous
inversions within species to homozygous inversion
differences between species is approximately
3.5: 1, suggesting a rapid isolation of the four
sibling species, followed by an extensive period

139

Section 8 — Cytatoxonomy and Experimental Taxonomy

of chromosomal diversification within each of
them.

8.27. Attempt on the Biological Significance of the
Chromosome Numbers. Mihai Serban and
Cantemir Riscutia (Bucharest, Rumania).

In a paper read at the XVI International
Congress of Zoology, Washington, 20-27
August 1963, we analyzed the distribution of
the chromosomes, from the grandparents (P2)
in the genome of a certain grandchild (F) using
the "indicators of stability" (I.S.), calculated
with the formula (1) for even, and (2) for odd
numbers of chromosomes.

Nj

2 -1

m

N/2

2/2N £ min. C— + 1/2AT. C— ;(1) in which
m=o N N

m + n=N
[N/2] m

2/2N X '«/"■ C — \ (2) in which (N/2) means

m=o N

whole part of N/2

A table of the I.S. values is given calculated
for the numbers N=2-25. We analyzed the
distribution of frequency of the haploid chro-
mosome numbers (TV) for 2374 species of flower-
ing plants (Dobzhansky, 1937) using the I.S.
and the values of I.S. generalized for the
generations P4 and P6. We found a signifi-
cantly greater frequency of numbers N
divisible by 8 than by 4, divisible by 4 than by 2,
and generally a greater frequency of even
numbers compared to odd ones.

The species in which the interactions between
organism and environment are more complex
have generally a greater number N, of chromo-
somes, which assures a greater stability. The
growth of the stability can be explained by the
I.S. values, which augment with the increase of
N.

8.28. Chromosomes of Swiss Ants. E. Hauschteck
(Zurich, Switzerland).

Chromosome sets of a number of ant species
from the subfamilies Myrmicinae, Formicinae
and Dolichoderinae have been investigated.
Diploid numbers between 8 and 40 to 50 have
been found. The diploid numbers of the Formi-
cinae are usually higher than those of the Myrmi-
cinae. There is only one genus of the Dolicho-
derinae studied which has 22 chromosomes.
Species with low chromosome numbers have

longer chromosomes than species with high
chromosome numbers. For instance, Lasius niger
has 30 small chromosomes in metaphase, each
one to two microns. In Stenamma brevicorne
there are only 8 chromosomes but these are five
microns or longer. Leptothorax is intermediate.
It has 18 chromosomes, each two to six microns.
The karyotype of Leptothorax exhibits two
striking constrictions on one pair of chromo-
somes. In the genus Lasius four species have a
diploid number of 30, and one a diploid number
of 28 chromosomes.

8.29. Hybrid Population Analysis by means of
Differential Staining of Chromosomes. Humihi-
ko Ono, and Bunzo Sakai (Tokyo, Japan).

Crepidiastrum keiskeanum (Compositae) is
distributed along the Pacific coast of southern
Japan. In Izu Peninsula, the northern limit of its
distribution, many hybrids of this species and
some allied ones are found. To analyse the
karyotypes of the hybrids, the differential
staining method using cold treatment was ap-
plied. This treatment reveals special beaded
patterns in the chromosomes of Lactuca squar-
rosa and faintly staining segments in the chromo-
somes of Paraixeris denticulata. In the hybrids
each chromosome or its segments are clearly
identified. Analysis of minor karyotypes of
these hybrids revealed the following extraordi-
nary characteristics:

1. The karyotypes of the hybrids are not
always the sum of the genomes of their presumed
parents. The maternal chromosomes remain
intact in the hybrid karyotypes. Marked dimi-
nutions in number and length are often observed
in the paternal chromosomes. Many hvbrids of
Crepidiastrum keiskeanum (/? = 5) and Lactuca
squarrosa (n=9) had 10 chromosomes. But
minor karyotypes revealed by the differential
staining showed individual differences.

2. Some of these hybrids were observed to be
mixtures of the cells with different karyotypes.
Fragmentation and elimination of paternal
chromosomes cause the alteration of karyotypes.
Such alterations occur among the somatic
cells of a single individual. With the lapse of
time, cells with the most balanced karyotype
overgrow less balanced ones. This phenomenon
is ascertained by observing the karyotype of a
single plant at different ages and by measuring
the pollen fertility of each stem of an individual.
The pollen fertility is remarkably divergent
according to the position of the stem even in a
single plant, although it recovers considerably
in successive years.

140

Section 8 — Cytatoxonomy and Experimental Taxonomy

8.30. Cytogenetics of South American Amphibians.

Francisco A. Saez and Brum de Nadir
Zorrilla (Montevideo, Uruguay).

South American species of amphibia offer a
wide scope for investigation due to their a-
bundance and their distribution in different
regions of the continent. Up to present we have
investigated more than 80 species belonging to
eight families.

BUFONIDAE; In eleven species of the genus
Bufo the diploid number is 2/7 = 22. CERATO-
PHRYDAE; in the genus Odontophrynus numeri-
cal diversity was found, six species with 2/7 =22
and seven species with 24, 30, 42, 44, 50 and 60
somatic chromosomes. The genus Ceratophrys
is unique because in the same species C. ornata
were found four different numbers, 26, 92, 96 and
108 chromosomes. The genus Lepidobatrachus
presents 2/? = 30.

LEPTODACTYLIDAE; eight species of the
genus Leptodactylus have 2n=22; three species
of Pleurodema, 2« = 22; two species of Physalae-

mus, 2« = 22; two species of Elogia, 2n = 26; two
species of Crossodactylus, 2n=22; three species
of Eleutherodactylus, 2/7 = 22; three species of
Telmatobius, 2n = 22; Calytocephalella, 2/7 = 26;
Throropa, 2/7 = 26; Eusophus,2n = 22; Cycloram-
phus, 2/2 = 26; and Pseudopaludicola, 2n = 22.

PSEUDIDAE; one species of the genus
Pseudis has 2/7 = 22.

HYL/DAE; Fifteen species with 2/z = 24 and
one with 30.

MICROHYLIDAE; one species of Elachis-
tocleis with 22 chromosomes.

BRACHYCEPHALIDAE; two species with
2/z = 22 and one with 26.

RANI DAE; R. pipiens and R. palmipes from
Costa Rica and Peru respectively with 26
chromosomes. None of the studied species shows
cytologically differenciated sex chromosomes.
Important problems of evolution, geographical
distribution and taxonomy will be dealt with by
cytogenetical analysis in a future publication that
will appear elsewhere.

141

SECTION 9

POPULATION GENETICS

9.1. The Diversity and Complexity of Ecotypic
Differentiation within Plant Species in Response
to Soil Factors. R. W. Snaydon (Reading,
Great Britain).

The Park Grass experimental plots at Rot-
hamsted provide a unique opportunity for
studying ecotypic differentiation in response to
controlled differences in soil factors over short
distances and relatively short periods of time.
The plots, which measure approximately 20 m
X 35 m, have continuously received different
fertilizer treatments since 1856 and different
liming treatments since 1903. The various treat-
ments have led to wide differences in the physi-
ognomy and botanical composition of the
vegetation, yet the grass species Anthoxanthum
odoratum is present on most plots.

Populations samples of A. odoratum from six
plots were grown in spaced plant trials and
greenhouse experiments. The results indicate
that the populations differ significantly in a
number of morphological and physiological
characters. The response of populations to
contrasting soils was closely correlated with
native soil pH. Vegetative height, inflorescence
height, inflorescence posture and rust suscepti-
bility of the populations were closely correlated
with vegetation height on the Park Grass plots.
Data of flowering, number of panicles and
mildew susceptibility were partially correlated
with vegetation height but also with fertilizer
treatment. Yield, panicle size and leaf size were
to some extent correlated with vegetation height,
but the relationship was evidently complex.
These results, together with results of the
physiological analysis of ecotypic differentiation
in response to soil factors in Trifolium repens,
demonstrate the complexity of ecotypic response
to initially simple soil differences, both through
the interactions of soil factors and through the
secondary and tertiary effects of the changing
physiognomy and botanical composition of the
vegetation.

9.2. The Analysis of Evolutionary Processes involved
in the Divergence of Plant Populations.

A. D. Bradshaw (Bangor, Great Britain).

It is perhaps usual to imagine that evolutionary
divergence of distinct populations within a species
can only occur overc onsiderable differences. How-
ever, in a number of plant species this can be
shown not to be true. In two species, Agrostis
tenuis and Anthoxanthum odoratum, populations
occurring on toxic soils associated with old mine
workings show a tolerance to the toxicities
which is not present in normal populations.
This characteristic shows a remarkable localized
distribution. Populations only a few feet away
from the toxic areas fail to show the charac-
teristic. In the same manner, in Agrostis stoloni-
fera, populations occurring in exposed maritime
habitats are found on cultivation to possess a
dwarf growth form not found in populations
occurring in protected habitats a very short
distance away.

In such situations it can be demonstrated
that there is considerable gene flow between
contrasting populations tending to obliterate
the differences between the populations. How-
ever, since the differences between the popu-
lations are retained, strong selection must be
occurring to remove the effects of gene flow.
Evidence for this is available. It appears that the
manner and timing of the selection may be very
different in the adjoining populations, but in
general it occurs in the seedling stage.

Gene flow between populations appears to
occur even between populations much farther
apart. From this it may be argued that the
continuous interplay of strong selection pressures
and gene flow are a normal part of the evolution-
ary processes determining the occurrence of
distinctive -populations within species.

9.3. Variability of Growth of Ryegrass (Lolium spp.)
Progenies under Artificial Environments.

S. O. Fejer (Ottawa, Canada).

Responses of inbred and outcrossed New
Zealand ryegrass progenies to some factors
of artificial environments in the phytotron of
the California Institute of Technology, Pasadena,
are reported. Growth was relatively little
affected by night temperatures, except in some

143

Section 9 — Population Genetics

L. perenne inbreds, and in a hybrid with L. peren-
ne cytoplasm, which were strongly depressed by
high night temperatures. Responses to day
temperature depended on the genotype, sup-
porting earlier results on such interactions in
ryegrass. Long days (16hr) produced more
growth in all material than did short days
(8 hr). Unnatural conditions, such as very short
days (4 hr), cold days and warm nights, or
changes in temperature at noon, caused de-
pressions. Stress conditions caused by water
and nutrient shortage, excessive defoliation, and
very low light intensity also depressed growth,
and interacted with the genotypes in various
measurements. Heterosis and inbreeding de-
pression were enhanced by such conditions.
Under any one condition, variation within clones
was relatively low, depending on plant type but
not on heterozygosity, and variation between
progenies was generally higher than within
progeny variation. The latter conformed to
expectations of the homeostasis theory as the
mean coefficients of variation over all environ-
ments increased linearly with decreasing heter-
ozygosity, but it also depended on genotype
and environment. Conversely, growth of indi-
vidual clones under different environments was
in some cases most uniform in the low producing
inbred lines.

'9.4. Ecotypic Variation in Response to Photo - and
Thermoperiod of Seedlings of Douglas - fir
(Pseudotsuga menziesii (Mirb.) Franco).

H. Irgens-Moller (Corvallis, U.S.A.).

The response of young Douglas fir seedlings to
long and short photoperiods varies with the
geographic origin of the seed. Seedlings from
maritime areas show less sensitivity to changes
in photoperiod than do seedlings from conti-
nental areas. Similarly, the response to differ-
ent thermoperiods also varies with seed origin.
At a constant temperature of 20 C and 8-hr
photoperiods a large percentage of seedlings
from Arizona go through 2-3 periods of dor-
mancy as opposed to only one in seedlings from
Vancouver Island, B. C. Low night temperature
(3°C) causes not only depression of growth for
all sources tested but an earlier induction of
dormancy in the seedlings from continental
areas than in those from coastal areas.

After dormancy is induced and the seedlings
are exposed to 16-hr photoperiods and green-
house temperatures, growth is resumed consider-
ably earlier in the seedlings fiom continental
areas than in those from coastal areas. However,

the seedlings previously exposed to a constant
20°C remain dormant for a considerably longer
period than those exposed to a low night temper-
ature. It is suggested that ecotypic differentiation
in long-lived species such as forest trees may
profitably be studied by short-term studies of
differences in seedling responses to photo- and
thermoperiod.

Supported by grants from National Science
Foundation, Washington, D.C.

To be published in Forest Science, 1964-65.

9.5. Genetics of Drosophila subobscura Populations
in Greece. C. B. Krimbas (Athens, Greece).

Data concerning the type of inversions and
the indexes of free recombination (IFR) of
several populations of D. subobscura in Greece
are reported. An ecological description of the
sites in given. Some of these data are the follow-
ing:

In Mount Parnes a population living in a
fir forest (altitude 1100m, rainfall 1000mm) had
an IRF of 85-6 ±1-2 and of 85-2 ±1-2 in
surveys of two successive recent years.

In Mount Ainos, Cephallonia, a fir forest the
population (alt. 1200m, rainfall 1200mm) had
an IFR of 84-3 ±1-2.

Three populations living in citrus orchards,
in Athens (alt: 100m, rainfall 400 mm), in
Alikianou, Creta (alt. 100m, rainfall 1040mm)
and in Poros (alt. 50m, rainfall 400 mm) had
IFR's of 83-8 ±1-5, 850 ±1-2 and 770 ±1-6
respectively in 1962. The previous year a small
sample of Poros population had an IFR of
80.1 ±3.8.

Attempts to correlate IFR with ecological
characteristics of these and other European and
Middle East populations are reported.

Results are also given of experiments, now in
progress, concerning the measurement of body
weight, wing length, developmental time,
dessication resistance, longevity and fertility of
the individuals of these populations, their inter-
populations Fi's and F2's.

These data are discussed in relation to

(a) the Dobzhansky-Carson hypothesis of the
pattern distribution of the inversion polymorph-
ism within the area of geographical expansion
of the species,

(b) the existence of an integrated gene pool in
each population as far as the measured fitness
components are concerned and

(c) the natural selection of the complex group

144

Section 9 — Population Genetics

of related characters body size, developmental
time, dessication resistance and fertility.

This reseaich is supported by a grant of the
Royal Hellenic Institute of Research.

may be expected. This finding demands the
formulation of a new set of principles for the
design of experiments on rice.

It is not yet possible to assess the exact
economic value of this finding but the cumulative
results seem indicate a hopeful future possibility.

9.6. Polymorphism in the Leopard Frog (Rana
pipiens). David J. Merrell (Minneapolis,
U.S.A.).

The dominant burnsi mutation is shown to be
present in natural populations of the leopard
frog in an area of about 100,000 square miles
centering on the Anoka Sand Plain of Minnesota.
This distribution represents only about 2 per cent
of the total range of Rana pipiens. The highest
frequencies of the mutant type reach only 5-10 per
cent. Evidence will be presented suggesting that
the present distribution of this mutation is related
both to the glacial history of the area and to the
fitness of the mutant in its present environment.

9.7. Intra-specific Interaction in Rice. Subodh
Kumar Roy (Calcutta, India).

When two varieties of rice are grown together,
each may influence the yield of the other. The
nature of interaction may differ with the kind of
mixture, the genotype mixed, the proportion of
the components and the ecological conditions.
The effect is as often favourable as unfavourable.
However, cooperation occurs, and certain pairs
outyield the means of the respective components
when grown in monoculture and occasionally
the better components.

The factors involved in such genotype inter-
actions seem to be very complex and are not
fully understood. However, experimental evi-
dences suggest that biologically active chemical
substance or substances produced by the roots
may be responsible for stimulation or inhibition
at least in certain pairs. The two varieties which
cooperate may make different demands on the
soil or the water and may suffer from comple-
mentary metabolic deficiencies. Diverse nature
of roots, and of lodging and disease resistance
of the components, may have considerable
advantage in a mixture over the pure stand.

The usual breeder's -practice of growing
several varieties of rice experimentally side by
side without any bond of separation between
them may lead to quite false conclusions as to
the yields of the varieties in question in pure
stand because under such a situation interactions

9.8. Distribution, Numbers and Natural Selection in
a Butterfly Population. M. S. B. Seiger and
R. H. T. Mattoni (Lafayette, U.S.A.).

The Lycaenid butterfly, Philotes sonorensis,
tends to form isolated or semi-isolated colonies
throughout its distribution range. We selected
a relatively isolated population in the San
Gabriel Canyon, California, to study population
size, ecology, movement and phenotypic vari-
ability in the six component colonies of this
population. The colonies are no more than 80 m
in diameter and are restricted to those areas
where the larval food plant, Dudleya lanceolata
(Crassulaceae), grows. The two closest and two
farthest colonies are 136 and 473 m apart
respectively. For three years the population was
sampled regularly throughout the flight period
by a capture-mark-release-recapture system. The
captives were classified according to sex, spot
pattern, area and dates of previous captures if
the specimen had been recaptured.

Very few individuals moved from their original
capture sites. Most of the interchange of genetic
material among the colonies was due to the
movement of the males. Thus gene interchange
was facilitated while distribution was localized.
Small ecological barriers such as rocks and
small streams acted as strong deterrents to the
movement of Philotes. Evidence indicating iso-
lation by minor ecological barriers was derived
from differences in sex ratio and spot pattern
frequencies among the six colonies. The effects of
birth and death rates and rates of incoming and
departing individuals on fluctuations in daily
colony sizes weie determined for each of the
colonies. The effects of natural selection were
determined by comparing the population para-
meters for the three years.

9.9. Ecological Genetics of Tribolium and Houseflies.

Robert R. Sokal (Lawrence, U.S.A.).

The ecological components of natural selection
in Tribolium castaneum populations are examined
under two different population systems. (1) The
(more natural) overlapping of generations and

145

Section 9 — Population Genetics

concurrent competition of all life history stages.
(2) Competition among developing larvae and
pupae only, with the adults separated from the
flour at regular intervals. This model simulates
natural selection in simpler univoltine species.
The fate of two alleles {sooty and black) under
both systems of selection seems deterministic
and analytical experiments are reported which
try to isolate the separate components of selec-
tion. Among the findings of interest are: Proof of
gene frequency dependent changes of adaptive
value in the sooty and black locus, proof of
gene frequency and zygotic frequency dependent
facilitation of growth processes by the sooty
allelomorph, genetic differences in response to
environmental conditioning and differences in
medium conditioning produced by different
genotypes (in the case of the black locus). Density
dependent differences in adaptive values are
apparently responsible for changes in the
direction of selection in population system (2).
Related experiments have been carried out with
houseflies and are contrasted with the Tribolium
findings.

9.10. Mimetic Multilocus Polymorphism in South
American Butterflies (Heliconius spp.) (Lepi-
doptera, Nymphalidae). John R. G. Turner
(Oxford, Great Britain).

Heliconius melpomene and H. erato have about
thirty morphs each — though they are mono-
morphic over large areas — and are mutually
mimetic. Experiments (Brower, Brower and
Collins) showing that birds find the insects dis-
tasteful and confuse the two species, and the
existence of beak-marked specimens, showing
that birds attack the butterflies in the wild, show
that this similarity involves synaposematism ; that
theory predicts that aposematic species become
monomorphic, that some "mimetic" pairs are
allopatric, and that some morphs are non-mimetic,
shows that other factors affect the polymorphism.
The only one whose action has been shown is
visual sexual selection. Features advantageous
in courtship are usually dominant, and butter-
flies lacking red (the chief releaser) are rare; all-
black insects are very scarce. There are several
autosomal loci, most of them linked; two
constitute a supergene. Population studies show
strong epistasis and linkage disequilibrium at
this superlocus; never are all four superalleles
found together, and one of them is very restricted
geographically in melpomene and extremely rare
in erato. In erato a third locus is included in the
supergene. One cannot tell what maintains the
polymorphism, although visual predation alone

can produce strong epistasis, and there is some
correlation between gene distribution and cli-
mate; note the similarity to the Rhesus blood-
groups, also supergenic and also maintaining
their polymorphism despite an unstable equili-
brium.

Some results were obtained jointly with
Jocelyn Crane and P. M. Sheppard. Papers on
Heliconius from the New York Zoological
Society's Tropical Research Station appear in
Zoologica (N.Y.).

9.11. The Evolutionary Potential, as Measured by
Seed Germination, of Chromosome Races of
Mimulus(Scrophulariaceae). Robert K. Vick-
ery Jr. (Salt Lake City, U.S.A.).

The purpose of this investigation was to explore
one facet of the norm of reaction of Mimulus,
specifically, the range of temperatures under
which seed germination could occur and to see
whether there were different responses in races of
different chromosome numbers. Seeds of over
20 races were tested in various combinations of
temperatures ranging from minus 7 to 50°C.
They were placed in one temperature for 16 hr
and in another for 8 hr to simulate a daily
periodicity of temperature changes.

In the Mimulus glabratus complex, the diploid
(/? = 15) and tetraploid (n = 30) races germi-
nated in combinations of cooler temperatures,
from 4° to 21°, and the hexaploids (n = 45) from
4° to 25°. Aneuploids of the diploid and tetra-
ploid races exhibited ranges that extended 5° to
10° higher than their respective euploid forms.
The aneuploids of the hexaploid level regularly
tolerated combinations involving 35° and one
race even grew well in a combination involving
-7°. The M. glabratus complex aneuploids ger-
minated and grew under a wider range of temper-
atures than their euploid relatives and the ones
with the highest numbers showed the widest
range of tolerance.

The results for the closely related M. guttatus
complex suggest a note of caution to too easy
generalizations. In this group the diploids ex-
hibited a far wider range of temperature tolerance
than did the tetraploids or aneuploids. However,
in both groups the range of temperature tolerance
of the races was often wide and not necessarily
correlated with the temperature ranges of their
native environments which suggests that Mimulus
has a wide norm of reaction and much unexploi-
ted evolutionary potential at each chromosome
number level.

146

Section 9 — Population Genetics

9.12. An Instance of Interaction of Genotype and
Environment at the Population-level. F. E.

Binet (Werribee, Australia).

The interaction of genotype and enviionment
is usually considered on the level of the individu-
al; it denotes the phenomenon of different en-
vironmental effects on the phenotypic expression
of different genotypes.

In the terminology of Analysis of Variance this
may be expressed as the "non-additivity" of the
"average phenotypic value", and the "environ-
mental contribution".

Considering a population as a whole, another
influence of its environment on its phenotypic
composition needs consideration. Even without
any difference in overall allele-composition the
zygotic genotype-composition of two populations
may be different. In presence of any dominance
and/or epistasis different "average phenotypic
values" follow from that difference.

Environmentally conditioned mating-systems
assemble identical gene-material into different
zygotic genotype-collections. The conditioning
factor is ecological (in wild populations) or
human action (in domesticated populations).

This lecture attempts to account for findings
(Clifford and Binet, 1954), contrary to antici-
pations, based on Anderson's (1949) models.

We measured certain characters of trees in
three stands; their estimated dispersion-matrices
define quadratic forms, whose cartesian images
are hyperellipsoids, with centroids, representing
(as position-vectors) the estimated mean-vectors.
In this representation (cf. Pearson, 1901) Ander-
son's model leads to anticipating the major axis
of the hybrid-stand's hyperellipsoid to lie on the
line connecting the parent-stands' centroids.
Contrariwise, we found the major axes of the
three hyperellipsoids nearly parallel.

It is submitted that these findings do not
contradict Anderson's theory on the formation
of the "recombination-spindle" and on subse-
quent "introgression"; rather, it appears that in
the "hybridized habitat" natural selection may
favour the hybrid individuals so strongly over
the pure offspring of both parents that the hybrid
stand consists almost exclusively of hybrids. From
those hybrid stands whose representative hyper-
ellipsoids are such recombination-spindles, such
pure offspring have not been eliminated by natu-
ral selection. We assume that in our case the
effect of natural selection resulted in a hybrid
stand of a very similar constitution to one at
which a breeder, crossing individuals from
different parent strains only, would aim.

Anderson, E. Introgressive Hybridization,
New York, 1949: Wiley.

Clifford, H. T., and F. E. Binet. A quantita-
tive study of a presumed hybrid swarm between
Eucalyptus elaeophora and E. goniocalyx. Aust.
J. Bot. 2, 325-36 (1954).

Pearson, K. The lines of closest fit to a system
of points. Phil. Mag. 2, 559 (1901).

9.13. Selection ton aid an Optimum and Linkage
Disequilibrium. S. Wright (Madison, U.S.A.).

The author (1935, 1937) gave an approximate
formula for change of gene frequency from
selection under random mating, assuming ran-
dom combination among multiple interacting

factors (/\q cj(\-q) 2"' where the Ws

are selective values of total genotypes, assumed
constant). This is only approximate because
selection with interaction usually maintains
deviations for randomness. Exact treatment
(1944, 1952) of an extreme two locus case
(W7 falling off symmetrically from an optimum:
W = 1 for two plus factors, ( \-s) for one or three,
( M4s) for none or four, recombination c), showed
considerable linkage disequilibrium at (1/2, 1/2)
\{ s = c (0.1465 AB or ab 0.3535 Ab or aB) but
little if s/2c is small (gamete frequencies about

1/4(1

2c

) ). This paper considers three linked

loci, ABC, both recombination fractions c, com-
plete interference, W = 1 for three plus factors
and (\-s), (l-4s), (l-9.s) for increasing deviations.
If s = c, and sc is small, the equilibrium fre-
quencies are 0.0495 ABC (and abc), 0.1387 A Be,
etc., 0.1731 AbC (and aBc) but 0.1097, 0.1278
and 0.1347 respectively if s = 0.1c. If s = c,
the disequilibrium (lAbiaB-^ABiab), for loci A,B
in presence of C,c is 59.7 per cent of that with
no C,c segregation while disequilibrium of loci
A,C in presence of B,b is only 26.5 per cent of
that with BB or bb. The approximate formula
for /\ q may be considered reasonably satisfac-
tory, in spite of the extreme interactions of the
optimum model, with respect to selection co-
efficients of lower order than the recombination
fractions.

9.14. Monte Carlo Investigation of Interaction
between Linkage and Selection under Dominance
Model. N. R. Bohidar, D. G. Patel and
R. L. Hurst (Logan, U.S.A.).

The mathematics of simulation of genetic

147

Section 9 — Population Genetics

systems associated with the problem at hand have
been developed and a fairly general computer
program has been written for the high speed
computer IBM 1620. The experiment consisted
of three levels of selection intensity, eight levels
of linkage condition, four replicates and three
dominance models, no dominance, complete
dominance and over-dominance. Each combi-
nation of the levels of the five factors was sub-
jected to 15 generations of selection with an
arbitrary initial population in linkage equili-
brium. The chief interest of the study was the
progressive changes in mean and variance of the
phenotypes at each generation. The statistical
analyses consisted of fitting a quadratic regres-
sion to the curves to evaluate the rate of genetic-
gain per generation. Analysis of variance proce-
dure was adopted to gain information on the
main effects and particularly the interaction
of linkage and selection. Results showed that
there was a strong indication of presence of inter-
action under no dominance model. No significant
interaction was detected for the other models.
The optimum combinations for the maximum
selection gain per generation were determined
under all models. A full account of the study will
be published in Biometrics.

9.15. The Theoretical Study of Gene Flow between
Sub-species, using a Digital Computer. J. L.

Crosby (Durham, Great Britain).

The representation of plant or animal popu-
lations by abstract models within an electronic
computer allows the theoretical investigation of
evolutionary systems which are too complex for
mathematical treatment.

One problem which has been studied in this
way concerns gene flow between two related sub-
species which come together again and interbreed,
after a period of genetical divergence during
geographical isolation. It has long been held that
where such genetical divergence is great, there
should be selection of factors tending to inhibit
interbreeding (and thus accelerate speciation),
although the theoretical demonstration of the
course of such selection is not easy.

The present series of experiments has consider-
ed one aspect of this problem — the selection of
pre-fertilization barriers to interbreeding in
plants. The example chosen was that of flowering
season in two sub-species which flower simul-
taneously and interbreed, with partial sterility of
the hybrids. Slight differences in the time of
flowering arise in the first place by random
fluctuations, but where the hybrid sterility is high
enough these differences become subject to

selection; eventually, the flowering periods of the
two sub-species hardly coincide, and at the same
time each becomes shorter in duration. Inter-
breeding is thus very greatly reduced, and gene
flow between the two sub-species is almost
eliminated; there has thus been a significant step
in the direction of speciation.

The problem of the selection of post-fertiliza-
tion barriers to gene flow is a much more difficult
one, but it is certainly amenable to the same
kind of theoretical treatment.

9.16. Biometrical Parameters of Self-fertilizing
Diploid Populations. Dewey L. Harris
(Ames, U.S.A.).

The mechanism of inheritance in diploid
populations is examined for the situation where
all reproduction is by self-fertilization. This
study suggests certain parameters for a general
representation of the means, variances and
covariances involved. Letting M(k) represent the
population mean in generation k and letting
Co\(k;r,s) represent the covariance of the rth
generation progeny means of generation k
individuals with the sth generation means of the
same individuals, we have

n 1

\xfc)= Z (5 -v)

X - 0 2kx
and

n n-xiz n-w n-w

Cov(Ar; r, s)

A'12 = 0 w= 0 X\
w X,0W

Xl2 X2 = X12

where n is the total number of segregating loci
which influence the quantitative trait under consi-
deration and Qxuv and u are the basic parameters

u

of this parametrization. The meaning of these
parameters and their relation to the types of gene
action occurring in the population is discussed.
The relation of this parametrization to those
previously suggested by Horner and by Kemp-
thorne is indicated. The implications to these
results to the design of experiments for the
selection of genetically superior material in such
populations is emphasized.

9.17. A Theoretical Study of Population Changes
Under Stabilizing Selection. S. K. Jain and
R. W. Allard (Davis, U.S.A.).

The "intermediate optimum" and the "homeo-
static" models of stabilizing selection require

148

Section 9 — Population Genetics

fitness of an individual to be related to its metric
value and heterozygosis respectively and there-
fore have different consequences on the distri-
bution of gene frequencies and the level of
heterozygosity at equilibria. Data from a pre-
dominantly inbreeding population of barley,
however, did not allow a clear choice between
these alternative models. It would be particularly
significant to find clear evidence for the operation
of homeostatic model in this case in view of the
probable role of heterozygote advantage in in-
breeding populations postulated earlier.*1^ The
present study was taken up in order to charac-
terize changes in various statistics (mean, inter-
family and intra-family variances, fitness,
hybridity, linkages) of a population under diffe-
rent forms of stabilizing selection. The case of
complete selfing was analysed with the use of
generation matrices and of mixed mating with
the help of numerical work on a digital computer.
In the "variable" parameter runs, the selective
values and the proportion of selfing were made
random variables in turn and the initial values of
"fixed" parameters were varied among the paral-
lel runs. These results allow a distinction between
the consequences of different models and seem
to have interesting bearing on the mode of selec-
tive changes in inbreeding populations.

A mathematical study of natural selection in a
fluctuating environment was carried out by
analytic methods and computer simulation.
The average fitness of the population was deter-
mined for different genetic systems, and the optim-
al genetic system was found for different pattern
of environmental heterogeneity. Genetic variance
increases the average fitness provided the environ-
mental variation is large compared to the tole-
rance of individual genotypes, or when the corre-
lation between the environments of successive
generations exceeds a threshold that depends on
the model. If the first condition holds but not
the latter, the optimal system has no additive
variance but much epistatic and dominance
variance. If only the second condition holds, most
of the variance will be additive. In a fluctuating
environment the average frequency of a heterotic
lethal is below its optimal value so that mutation
to the lethal may increase fitness. Mutation also
reduces the variance of gene frequency and in-
creases the correlation between the state of the
population and its optimal state. The optimal
rate of recombination was also shown to depend
on the environmental variance and autocorre-
lation.

1. S. K. Jain and R. W. Allard, Proc. Nat.
Acad. Sci. 46, 1373-1378, 1960.

9.18. On the Theory of Genetic Loads. Howard
Levene (New York, U.S.A.).

Morton, Crow and Muller and Crow have
suggested a method of determining whether the
genetic load is mainly due to recurrent deleterious
mutations or to genes with heterozygous ad-
vantage. The method is based on the comparison
of individuals from random matings with inbred
individuals. A number of restrictive assumptions
are made in the derivation of the method, and
additional assumptions must be made to apply
it to actual data. The effect of relaxing these as-
sumptions, and the degree to which valid con-
clusions may be drawn from the types of data
so far employed will be discussed. The broader
implications of the concept of a "genetic load"
will also be considered.

9.20. Linkage in Selection Programmes. A. Robert-
son (Edinburgh, Great Britain).

Linkage is obviously a retarding factor in
artificial selection programmes. The extent of
this retardation is dependent on several factors
between which there may well be important
interactions. The population size in the selection
programme may well have an effect at intermedi-
ate values but not with very large or very small
populations. We may expect the effect to be
greater (i) when linkages are primarily in repul-
sion in the initial population, (ii) when we are
concerned to fix a large number of genes, and
(iii) when the initial frequencies of these genes
are small. Possible interactions between the
factors will be discussed theoretically and with
reference to results of computer programmes.
Finally a selection experiment with Drosopliila,
in which crossing-over was suppressed in one
series of lines, will be discussed.

9.19. Optimal Genetic Systems in a Fluctuating
Environment. Richard Levins (Rio Liedras,
Puerto Rico).

9.21. Heterosis and Genetic Balance. N. V. Turbin
(Minsk, U.S.S.R.).

At the present time there can be no doubt that
the two conflicting concepts of heterosis -the

149

Section 9 — Population Genetics

hypothesis of dominance and the hypothesis of
overdominance-are not mutually exclusive,
but the causes of heterosis with which they deal
may act, and as a rule do act, simultaneously.
The heterosis effect cannot be explained by a
single genetic cause, by one type of interaction
of hereditary factors, but is the result of the total
similar action of various genetic processes. Not
one of the proposed hypotheses determined for
any one type of interaction of hereditary factors
can be accepted as a general theory of heterosis,
although certain of them, and, in particular,
the two hypotheses mentioned, are in good agree-
ment with determined experimental data. They
contain the elements of exact knowledge and
can be considered as fragments of the general
theory of heterosis.

It is represented as extremely probable that
the concept capable of helping to unite these
fragments into a single whole and of supplying
the missing elements, taking into account the
types of interaction of hereditary factors (allelic
and non-allelic) and also the role of conditions
of the surrounding medium in the development
of characters in hybrids, is the theory of genetic
balance.

The value of any character in each parent
variety (line) is a result of a determined balance
(equilibrium) worked out in the course of selec-
tion, in the variously directed action on this
character of many hereditary factors and condi-
tions of the surrounding medium, in which the
development of the organism takes place. In
the cross-breeding of parent varieties (lines)
which differ in their heredity there takes place
in hybrid offspring a change in the genetic ba-
lance with relation to a larger or smaller propor-
tion of the characters which can cause a deviation
in the value of one or another character in the
direction of an increase or decrease by compari-
son with the parent forms (positive or negative
heterosis).

Departing from what has been said above,
an attempt is made to interpret the causes of
heterosis and in particular the role in this process
of various types of allelic or non-allelic inter-
action of hereditary factors on the basis of the
theory of genetic balance.

meiotic chromosome pairing. Up to the present,
the following 9 cytogenetic types have been
identified in natural populations. Plants of the
constitution AA (2/2 = 16), BB (2« = 18), and
AA BB (2// = 34) are propagated sexually by
seeds as well as vegetatively by bulb multiplica-
tion. Plants of the constitutions BBB(2« = 27),
BBBB (2w = 36), AAB(2« = 25), ABB (2// = 26),
ABBB (2n = 35), and AABBB (2« = 43) set some
seeds, instable sexually, but propagate by clon-
ing by bulb multiplication.

These various cytogenetic types are found
usually in juxtaposition in a population. An
index of homogeneity of the populations as to
these cytogenetic types ranged from 0.82 to
0.22, from population to population, where
1 .00 indicates a pure stand of a single cytogenetic
type. The indexes were computed as follows.
Let a be frequency of a type A, b that of another
type B, c that of another type C, and so forth.
Simple computations (a2 + b2 + c2 + . . .)/
(a + b + c + . . .)2 give the index values
ranging from 1 to certain values approaching 0.

9.22. Population Structure and Dynamics in a
Complex Scilla scilloides (Liliaceae). Tutomu
Haga and Shozo Noda (Fukuoka, Japan).

In a perennial plant Scilla scilloides Druce,
two basic genomes A (x = 8) and B (.v = 9) are
clearly distinguishable by karyotype and by

9.23. Progress From One Generation of Selection in
Nicotiana tabacum. Dixie Bright 244 X Coker

139. D. F. Matzinger, T. J. Mann, and C.
Clark Cockerham (Raleigh, U.S.A.).

Within the F2 generation of a cross of 2 na-
turally self-fertilizing flue-cured tobacco va-
rieties, Dixie Bright 244 x Coker 139, individual
plants were self pollinated and crossed to random
members of the population. Only the cross-
bred material was evaluated and estimates of
additive genetic and dominance variances were
obtained assuming no epistasis. Parental plants
high in per cent total alkaloids were identified
on the basis of mean cross-bred performance.
From remnant seed, selfs of these superior plants
were intercrossed to form a new population.
In this first selected cycle, both self and cross-
bred families were evaluated. Estimates of
additive genetic, dominance, and additive x
additive epistatic variances were obtained.

In both the initial and selected populations,
significant estimates of additive genetic variance
were obtained for cured leaf yield, per cent
total alkaloids, days to flower, number of
leaves, and plant height. Additive genetic
variance was not significant for leaf value in
either population. The only significant estimate
of dominance variance was for leaf value in the
original population. None of the estimates of
additive x additive variance were significant
in the selected population. Selection for increased

150

Section 9 — Population Genetics

per cent total alkaloids was effective; however,
the yield of cured leaf decreased. The response
to selection for total alkaloids and the accompa-
nying decrease in leaf yield was in good agree-
ment with the predictions.

9.24. The Evolution of Flower Colours in Populations
of Tulipa schrenkii Rgl. according to Statistical
Data. A. J. Kupzow (Moscow, U.S.S.R.).

The area of Tulipa schrenkii Rgl. extends over
the steppes of the European part of the U.S.S.R.
and of Northern Kazakhstan. Individuals, in the
wild populations of this tulip, have four main
colours of their flowers: red (anthocyanine +
anthoxanthine), yellow (anthoxanthine), rose
(anthocyanine), and white (no pigment). There
are also certain modifications of these: orange,
light rose, violet, and cases of uneven distribution
of pigments (striped). Red flowers are charac-
teristic of most of the tulipa species, in sect,
eiestemenes, and also of the bulk of individuals
of T. schrenkii in the virgin steppes. The in-
fluence of man leads to a decrease of the amount
of red flowers in the wild populations of T.
schrenkii and, thus, to an increase of other
colours, especially of yellow. The latter ultima-
tely, becomes exclusive in the environs of large
towns, such as Odessa. 31 populations of T.
schrenkii from different parts of its area have
been studied. The correlation between the
amount of red flowers and that of yellow, the
two main types in these populations, was found
to be - 0.9 + 5 ± 0.0150. The percentage of
red flowers can be taken as an independent va-
riable and that of flowers with other colours
as its function. Then, while the percentage of
red flowers decreases from 100 per cent to
51 per cent, all its functions rise, and so the
composition of the populations approaches the
ratio: 9 red ± 3 yellow ± rose + 1 white
(with 10-15 per cent of modified colours among
them, mainly orange and violet as variants of
red). In the interval from 51 per cent to 0 per
cent, of red flowers, the percentage curve of
yellow flowers continues to go up whereas the
proportions of rose flowers goes down, the
curve of white ones ascends up to 30 per cent
of the red ones and then also descends. The
percentage of the modified colours tends to
go down within this interval. The removal of
anthocyanine (in yellow) is observed more often
than that of anthoxanthine (in rose). Thus, in the
former interval many populations have 5-10
per cent of yellow flowers and 0-9 per cent of
rose ones. The aforesaid modifications are

observed only in anthocyanine: orange and
violet, as mutants of red being more frequent
(up to 10.7 and 6.0 per cent) than light rose, as a
mutant of rose (up to 1.5 per cent).

According to the available data, the following
concept is suggested:

1. The initial colour of the flowers of T.
schrenkii was red, and it subsequently produced
distinctive mutants deprived of anthocyanine or
anthoxanthine, and, also modifier genes (muta-
tions affecting an anthoxanthine).

2. Individuals with red flowers have, in
virgin steppes, gained success in the struggle
for existence. However, under man's influence,
various mutants, especially those with yellow
flowers, have become the predominant forms.

3. High percentage of individuals with red
flowers ensures a higher rate of mutation, but,
if the amount of red flowers diminishes, this
rate decreases in the population.

4. Any increase of individuals with yellow
flowers in a population is accompanied by a
decrease of the rate of mutation, and so all
individuals with differently coloured flowers
gradually become supplanted by these yellow
flowers.

9.25. Evolution in the Tropics. F. G. Brieger
(Est Sao Paulo, Brazil).

The basic mechanism of phylogenetic evolu-
tion is likely to be the same in tropical and in
north-temperate regions, where most studies
so far have been carried out. The course of evolu-
tion will have been different, since temperate
and tropical areas have had a different geological
history since the Tertiary and since ecological
conditions are also quite different, causing
changes in selective trends. The orchids offer
excellent material for such studies, since they are
not only one of the youngest families of higher
plants, but allow a detailed study of phylogene-
tic evolution owing to their dispersal, made pos-
sible by the smallness and large number of
their seeds. Long distance dispersal is most
easily studied between continents, and has
been followed, almost exclusively by phylo-
genetic diversification into new species or groups
of species or into new genera. In the case of the
formation of new genera, this has followed
generally the same lines as evolution in the
original area, but with strong predominance of
terrestrial orchids over epiphytes and with a
strong tendency to form saprophytic species in
the new area. In the cases of formation of spe-
cies after dispersal between continents, there

151

Section 9 — Population Genetics

exists a correlation between stronger evolution-
ary activity in the original area and the frequency
of dispersal. Intracontinental dispersal has been
studied in the American tropics. In addition to
long distance dispersal, followed by allopatric
evolution, slow penetration into new areas has
occurred followed generally by marginal dis-
ruptive evolution. Explosive or sympatric
disruptive evolution has also occurred after
slow penetration or dispersal into a new area.
In general, there seems to exist a correlation
between larger evolutionary steps (genus forma-
tion) with restricted diversity (fewer species)
and between smaller evolutionary steps and
high degree of diversification.

9.26. Deviations from Panmixia as a Consequence
of Sex-determination in the Marine Copepod,
Tisbe reticulata. Bruno Battaglia (Padua,
Italy).

Experiments have been carried out with the
purpose of testing if, between the various pheno-
types of the polymorphic Copepod Tisbe
reticulata matings occur at random. Observa-
tions concern the following forms: maculata
(f/m r/m)5 violacea (Vv Vv) and violacea-macu-
lata ( Vv Vm), controlled by two of the alleles of
the V series.

Results show that, at least under certain condi-
tions, the observed frequencies of some types of
crosses differ from the frequencies expected on
the assumption of panmixia. These deviations
are mainly due to an excess of backcrosses
whereas all crosses of other types are fewer
than expected. This situation seems to be condi-
tioned by the selective matings $ Vv Vm x £
ym f/m and (j) I/v I/m c ^ l/v i/v.

The hypothesis is made that the heterozygous
females are sexually more active than the homo-
zygous ones, and the homozygous males more
active than the heterozygous ones. This view is
supported by other observations on the different
fecundities of the three genotypes.

The results of other experiments seem to in-
dicate that the deviations from panmixia depend
to a certain extent upon the degree of relation-
ship between the animals which mate.

The problem of the possible bearing of the
above situation on the evolution of natural,
as well as laboratory populations is being in-
vestigated.

The data will be published in full, possibly in
Evolution.

9.27. Selection Experiments after Inbreeding in
Habrobracon. R. E. Scossiroli and R. C.
von Borstel (Pavia, Italy, and Oak Ridge,
U.S.A.).

In previous experiments, long-winged sub-
strains had been selected from the small wing,
white eye stock of the parasitic wasp Habro-
bracon after six generations of inbreeding
(expected homozygosity = 0.97(J). It was hypo-
thesized that selection for longer wings in the
inbred strains was made possible by either (1)
maintenance of genetic variance by persistence
of heterozygosity, or (2) a high spontaneous
mutation rate for quantitative characters.
Both long- and short-winged substrains were
selected from the same strain of Habrobracon,
using a family method and a selection pressure
of 1.47 s, that is, only those were selected that
were beyond 1.47 standard deviations from the
mean. Selection under identical conditions was
repeated after three generations of inbreeding by
back-crossing to the same haploid male of
daughters from subsequent generations. It was
again possible to select long- and short-winged
substrains (expected homozygosity = 0.94). On
the other hand, attempts to select these substrains
from three replicates after 12 generations were
unsuccessful (expected homozygosity = 0.99 +).
This shows that by prolonged inbreeding, homo-
zygosity can be obtained for characters affecting
wing length. Therefore, a high spontaneous
mutation rate does not contribute excessively
to the genetic heterogeneity for characters
affecting wing length.

1. Scossiroli and von Borstel, Atti Asso-
ciazione Genet ica Italiana, 7, 191, 1962.

9.28. Competition Research on Unisexual Poly-
morphism in Megaselia scalaris. Robert
Springer (Vienna, Austria).

The newly discovered type of alternative sex
determination in the Phoride Megaselia scalaris
brings about a unisexual polymorphism in the
natural population. It has to be assumed that
three differing types of males co-exist in nature.
In these three types always one of the three
non-homologous chromosomes bears the sex
realizator. Competition research on these three
sex-determining types of males were carried
out with artificial populations. Present results
showed that the third chromosome always has a
strong selective advance. In natural populations,
however, the first chromosome mostly seems to
be sex-determinating. Further results can be
expected in due course.

152

Section 9 — Population Genetics

9.29. Intensities of Selection in Natural Populations.

Leigh Van Valen (London, Great Britain).

With respect to any population parameter p,
the intensity of selection ip may be defined as
the minimum proportion of the unselected
population, per generation, that must die or not
reproduce in order to generate the value of p in
the selected population. Methods for evaluating
phenotypic selection intensities from estimates
of initial and final parameters have been develop-
ed and applied to new and old data in several
phyla of animals.

Selection by mortality on the mean is usually
less intense than but not negligible in comparison
with that on the variance, indicating possible
importance of temporal and spatial heteroge-
neity in the direction of selection. Intensities by
mortality so far observed range from very near
zero (blood groups in Englishmen) to 0.55 (tooth
proportions in some extinct cave bears) for the
mean, and to 0.3 (tooth width in an extinct
horse) for the variance. The central tendency for
randomly chosen characters is not yet evident.
Natural selection by fertility has been largely
neglected; the highest intensity known to me
is about 0.05 in a hydromedusoid.

Destabilizing selection with an average in-
tensity of about 0.2 is present on tooth width
in semi-commensal house mice in Great Britain.
Spatial heterogeneity exists for selection inten-
sity on the mean here; the average intensity
is about 0.08. The direction of selection on upper
tooth dimensions in an extinct horse population
is opposite to the direction of its evolution, also
suggesting heterogeneity in selection. All the
estimated juvenile mortality in this horse popula-
tion can be considered selective for tooth dimen-
sions.

9.30. Inherited Chai acteristics of Tribolium Popula-
tions. Daniel J. McDonald (Carlisle, U.S.A.).

vealed variations in the distribution of the life
cycle forms within the cage. Dead adults tended
to accumulate in corners and pupae preferred
regions where the medium had not been recently
renewed. On the basis of this information,
characteristics and interactions of the popula-
tions which may influence both intra- and inter-
specific competition were proposed. The pre-
sence of characteristics leading to density de-
pendent control of population size, was con-
strued as an adaptation to a stable, undisturbed
environment. Variations in these characteristics,
generated initially by genetic differences and
possibly amplified by population interactions,
evinced retention by the species of the adaptive
variability necessary for the maintenance of
population fitness.

9.31 . Polymorphisms in the Egg Albumen Proteins of
the Domestic Fowl. I. E. Lush (Edinburgh,
Great Britain).

The egg albumen of the domestic fowl is a
mixture of several different proteins. Starch gel
electrophoresis has been used to analyse the
albumen from individual hens at the Poultry
Research Centre, Edinburgh, and genetic
polymorphisms involving three of these proteins
can be demonstrated. Each polymorphic pro-
tein occurs in two forms which differ in electro-
phoretic mobility. The polymorphism of each
protein is determined by the segregation of two
alleles at a corresponding genetic locus. Data
from crosses will be presented. One of the poly-
morphic proteins, ovalbumin, is electrophoreti-
cally heterogeneous even in the albumen of a
hen which is homozygous at the corresponding
locus (named Ov.). Data on the analysis of this
heterogeneity by enzymic modification of oval-
bumin will be presented.

Several groups of Tribolium confusion popu-
lations were observed in population cages for
more than a year. Weekly samples taken directly
from the populations were used to determine the
number of live and dead adults, pupae and eggs
in the population. This revealed that the num-
bers of these forms were greater in some groups
than in others. In the larger populations, fatali-
ties were estimated to be higher and adult life
spans shorter, and, in separate experiments,
the population groups were also found to differ
in the interaction between fecundity and adult
density. The latter response was proposed as a
possible cause of various other population dif-
ferences. Total censuses of the populations re-

9.32. Heritability of Developmental Time and Via-
bility of Turkey Embryos in Three Environ-
ments. W. A. Becker and T. P. Bogyo (Pull-
man, U.S.A.).

The relationship between estimates of genetic
variation and environmental change was in-
vestigated by subjecting turkey embryos to
stress. The eggs from matings of 40 sires with 400
dams were stored for 1, 2 and 3 weeks before
incubation. For developmental time, 1 week
storage: x = 647.5 hr, hi = 0.31 ± 0.06,
h^ =■ 0.56 ± 0.08; 2 weeks storage: x = 652.2
hr, h| = 0.20 ± 0.07, h* = 0.53 ± 0.08: 3

153

Section 9 — Population Genetics

0.14 t 0.08, 9.34.

weeks storage: x = 656.5 hr, hf
hf, = 0.63 ±0.12.

The viability of embryos for 1 week of storage
was: x = 67.0 per cent, hf = 0.12 ± 0.03;

2 weeks: x = 50.9 per cent, hj = 0.08 ± 0.03;

3 weeks: x = 29.5 per cent, hf = 0.11 -_ 0.03.
Heritability was highest during the 0-7 day
incubation stage (hf = 0.08 to 0.16); the other
stage (8-29 day and pipping) had hf == 0.00 to
0.05.

Developmental time was increased by storage
while viability decreased. Heritability estimates
were not materially changed for these traits by
the environmental changes. Developmental
time was influenced by maternal effects. Most of
the genetic variation of viability of turkey em-
bryos occurred during the 0-7- day stage of
incubation.

9.33. The Effects of Temperature on Two Strains of
Mice of Different Body Weight and Tail
Length. F. Cockrem (Palmerston, New
Zealand).

Two strains of mice, one of high body weight
and short tail length (line A), and one of low
body weight and long tail length (line B), have
been developed by selection. As an initial study
of possible correlated changes associated with
tail length and adaptive ability, the two lines
were reared at temperatures of 7°C, 21°C
(control) and 32°C from 3 weeks to 6 weeks of
age. Tail lengths and body weights were mea-
sured at these two ages. Ten males and 10 fe-
males of each strain were used for each treat-
ment.

For both strains and sexes of mice, the cold
(but not the hot) treatment depressed body
weight growth. There were no interactions and
the effect of the treatments was proportional in
both strains of mice. For the variances of body
weight at 6 weeks, the only effect which was
consistent for both sexes was a reduction of
variance for the A line under the hot environ-
ment.

For both lines a higher temperature was as-
sociated with a longer tail length, but line B show-
ed a greater increase under the hot and control
environment. Under the cold environment, both
lines showed a similar increase.

Thus a genotypic difference in tail length was
associated with a difference in the degree of
response of this character to the three environ-
ments. Furthermore, a strain difference in tail
length and body weight was not associated with
a difference in the ability of the two strains to
adapt to the different temperatures.

An Investigation of Genotype-environment
Interaction in the Mouse. Peter Hull (Roches-
ter, U.S.A.).

It is to be expected that the genetic variance
between sires, estimated from the performance
of their progeny, will be small when the sires are
from the Fi of a cross between two highly inbred
lines, and larger when the sires are from the F2
of a cross of the same two inbred lines. From
previous work with poultry it was decided that
there was probably a relationship between the
magnitude of interaction of a group of genotypes
with environment, and the magnitude of the
additive genetic variance within the group of
genotypes tested. The results of the experiment
which is to be described are in agreement with
this hypothesis, since it was found that the inter-
action of sires with environment was greater
when the sires were from an F2 group than when
the sires were from an Fi group. This difference
was noted in the case of a metric character
(tail length), whose heritability was expected
to be comparatively high — in the case of a
second character (body weight), whose heritab-
ility might be expected to be lower, there was no
detectable difference in the size of the genotype-
environment interaction component of variance
between the two groups of sires.

9.35. Selection Progress Toward an Absolute Limit
for Amount of White Hair on Mice. W. H.

Kyle and H. D. Goodale (Lafayette and
Williamstown, U.S.A.).

Selection for amount of white hair was ini-
tiated in 1931 by one of us (H.D.G.) in a stock
derived from one male mouse with a few white
hairs on its forehead and four self-colored fe-
males. A sample (5 males and 6 females) of the
selected stock was transferred to Purdue late in
1959 from which eight generations have been
produced. All mice were scored at weaning for
percentage of white on the dorsal surface. After
the first generation, numbers of offspring ranged
from 74 to 210.

The unweighted mean per cent white of the
eight generations is 69.5 per cent for males and
64.7 per cent for females. Regression of sex
means on generations shows an average gain
per generation of 1.73 per cent for males and
0.55 per cent for females. Regressions of offspring
on parent are quite variable but indicate a low
degree of heritability. There is some indication
of a sex-linked effect. Standard deviations within
sex and generation are large.

154

Section 9 — Population Genetics

Beginning in the 5th generation, two or more
all-white dark-eyed males have been obtained
in each generation for a total of eleven. One
all-white female was obtained in the 8th genera-
tion from an all-white sire. Tests show the ab-
sence of albino and the absence of genes domi-
nant to wild type.

The tremendous changes in the mean and
variance in this closed population from an initial
level of essentially zero are of primary impor-
tance. Although the mean of this sample does
not approach the absolute limit of selection, a
dozen individuals have reached the phenotypic
limit.

9.36. Effects of Mating System on Ovulation,
Implantation and Litter Size in Mice. T. G.

Martin, F. E. Harrington and D. L. Hill
(Lafayette, U.S.A.).

Four inbred lines of mice were mated in a
diallel mating system producing sixteen genetical-
ly different groups of offspring. Samples of four
females for each trait in each genetic group were
drawn to estimate number of ova shed, number of
implantation sites and litter size. Embryonic
mortality was estimated by number of ova
shed minus litter size. Pureline versus crossline
comparisons made in the parental generation
yielded only one statistically significant differ-
ence with crossline and pureline matings having
9.17 and 8.73 implantations, respectively. Ave-
rages for the other traits were: litter size 8.33
and 8.38, ova shed 9.88 and 10.25, and embry-
onic mortality 25.9 and 26.9 per cent for cross-
line and pureline matings, respectively. In the
Fi generation, crossbred and pureline matings
were compared. Crossbreds performed at signi-
ficantly higher levels than pureline mice in all
traits except ovulation rate. Crossbred averages
were 11.24 ova, 11.39 implantations, 9.02 off-
spring and 19.8 per cent embryonic mortality.
Pureline averages were 10.29 ova, 8.63 implanta-
tions, 7.53 offspring and 29.0 per cent embryonic
mortality. Analysis of the Fi data indicated
strongly that specific combining ability was
a more important source of variation than gene-
ral combining ability thus indicating that a
large portion of genetic variation is due to
dominance and epistasis and a smaller portion
due to additive genetic effects. Mating system
influenced time of embryonic death with 36,
58 and 100 per cent of embryonic deaths occurr-
ing after implantation in pureline, crossline,
and crossbred matings, respectively.

9.37. Correlated Responses to Selection for Radia-
tion Resistance in Mice. Thomas H. Rode-
rick, (Bar Harbor, U.S.A.).

Selection for survival under daily doses of
X-rays was successful in two experiments: (1)
under lOOr/day, and (2) under 400r/day. Litter
size was a correlated response in both experi-
ments, and body weight only in the first experi-
ment. W

The correlated response in body weight was
only in the low direction, and that response was
more pronounced in the males than in the fe-
males. For the females, the means and standard
errors for body weight were: 39.3 ± 1.0 g for
the high line, 39.4 ± 1.1 g for the unselected
control line, and 35.6 ± 1.0 g for the low line.
For the males: 41.8 ±0.5 g for the high line,
44.6 ± 0.6 g for the unselected control line, and
29.6 :; 0.8 g for the low line.

The first selection experiment also produced a
correlated response in LDso:3o° LDso:3o means
and standard errors for the females were:
905 ± 18r for the high selected line, 833 ±
21r for the unselected control line, and 788
± 21r for the low selected line. Survival under
400r/day was also a correlated response in the
direction of selection.

This study suggests that, although the selected
trait appears to have responded symmetrically,
body weight (a highly correlated character)
has responded asymmetrically. These results in
turn suggest that the physiology of response
in the upward direction is very different from the
physiology of response in the lower direction.
It is possible that the loci involved in upward
selection of this trait are quite different from the
loci involved in downward selection.

1. Roderick, Genetics 48, in press.

9.38. Evidence that Selection for the Maintenance of
Developmental Homeostasis may Lead to the
Retention of Genetic Variability. H. T. Band

(Vancouver, Canada).

Changes in the lethal + semilethal frequencies
in the South Amherst D. melanogaster popula-
tion can be correlated with changes in environ-
mental variables (Band and Ives, 1961). Analysis
of homozygotes and random heterozygotes
containing second chromosomes from the 1960
population suggest that the gene pool is inte-
grated on two levels: (1) in the maintenance of
developmental homeostasis and (2) in the magni-

155

Section 9 — Population Genetics

tude and organization of the genetic load (Band
and Ives, 1963). Experiments by Band (1963)
indicate that if the gene pool remains coadapted,
then genetic changes which occur must be
accompanied by the maintenance of develop-
mental homeostasis among the random hetero-
zygotes.

Trends toward more adverse environmental
conditions have been observed during 1961 and
1962. The lethal allelism frequency of 1 per cent
in 1961 indicates reduction in population size;
tests for 1962 are in progress. Genetic variance
(a^) relationships between random heterozygotes
and nondrastic (nd) homozygotes in 1960, 1961
and 1962 reveal changes in genetic organization.
In 1960 erf het < a'l „,„„, in 1961 o'l het > os\ h0m
due to the genetic variance of the nd/nd hete-
rozygotes; in 1962 a\ het < ct| hom. Never-
theless, in both 1961 and 1962 heterozygotes
containing two, one or no "drastic" second
chromosomes possess comparable developmental
homeostasis, superior to that of the nd homo-
zygotes in each collection.

These findings provide evidence that the
genetic diversity carried in the gene pool enables
the population as a whole to shift from one of
Wright's adaptive peaks to another. They also
indicate that the attainment of new equilibrium
gene frequencies is influenced by a variety of
factors both external (environmental) and in-
ternal (the achievement and maintenance of
developmental homeostasis; Band, 1963). Thus,
selection for the maintenance of developmental
homeostasis may lead to the retention of geno-
typic variability in the population (Lerner,
1956).

Supported by grants from the National Science
Foundation and the National Research Council
of Canada.

9.39, The Estimation of Population Fitness. J. A.

Beardmore and W. van Delden (Gronin-
gen, The Netherlands).

In the simplest terms the fitness of a popula-
tion is the weighted average of the fitness of the
individual genotypes of which it is composed.
Two practical difficulties encountered in connec-
tion with this simple statement are: first that it
is almost impossible in outbreeders to specify all
the genotypes concerned (thus preference is
given to polymorphisms as objects for study)
and secondly even when the genotypes are
specified the estimation of the fitness of indivi-

dual genotypes may have to be made in isolation
and hence interactions of all types are ignored.
This leads to a situation where empirical obser-
vations of suitable populations may be valuable
in assessing ielative population fitness over both
short and long periods. We have attempted to
estimate the relative fitness of a number of labo-
ratory populations using as indices of fitness
biomass production, longevity, fertility and other
characters. We hope to report on the correlation
between genetic variance and fitness in a stan-
dard environment, correlation of fitness values
in different environments and the environment
as a component of fitness.

9.40. Synchronous Mutability Changes in Geogra-
phically Isolated Populations of Drosophila
melanogaster. R. L. Berg (Leningrad,
U.S.S.R.).

During 1937-41 high concentrations of yel-
low mutants and high mutation rates of the
yellow locus were detected in five geographi-
cally isolated populations of Drosophila me-
lanogaster. The studied populations were Uman,
Ukraine (1937), Nikita Botanical Gardens,
Crimea (1937-38), Dilijan and Erevan, Armenia
(1939), Kashira, near Moscow (1940-41).

During 1945-46 we studied the populations of
Kutaissi, Georgia (1945), Tiraspol, Moldavia
(1946) and Uman (1946). In Uman and Tiraspol
high concentrations of yellow mutants were
again observed. The rate of occurrence of the
yellow mutation proved to be lower than during
the previous period of investigations.

During 1957-62 low concentrations of yellow
mutants and low mutation rates were seen in
nine populations (Piatigorsk and Inosenitsevo,
North Caucasus 1957; Uman, 1958, 1962;
Nikita Botanical Gardens, 1960, 1962; Dilijan
1960-62; Kashira, 1960, 1962; Alma-Ata, 1961;
Tiraspol 1962).

The concentration of yellow mutants among
wild males was: in Uman in 1937 0.246 ± 0.49
per cent (25 yellow males among 10,159 wild
males), in 1946 0.301 ± 0.090 (11 yellow males
among 3652 males), in 1958 there were no yel-
low mutants among 13,131 males.

The mutability in the Uman population in
1937 was 0.149 ± 0.027 (30 yellow males among
20,144 males) in 1946 in a mutability test among
5394 males we found no yellow mutants and in
1958 among 84,831 males in the progeny of wild
flies, only three (0.035 ± 0.020 per cent), i.e.
one forty-third, of the percentage observed in
1937.

156

Section 9 — Population Genetics

Such a tremendous change in the occurrence
rate of visible mutations was not observed in
other loci of the sex-chromosome. A certain
decrease in the rate of occurrence of sex-linked
lethals was also observed in some populations.

Synchronous mutability changes in geogra-
phically isolated populations involving such an
extensive area ought probably be considered
as caused by some external geophysical or cos-
mical factor, influencing differently the mutabili-
ty of different loci. We can only guess at the
nature of the factor which may act either di-
rectly or through the medium of some bio-
ecological changes.

9.41. On the Evolutionary Signification of Heterozy-
gosity in Drosophila Populations. E. Bosiger
(Gif-sur-Yvette, France).

Natural populations of Drosophila melano-
gaster show a high degree of heterozygosity.

The vigour of males of stocks recently derived
from natural populations is higher than that of
inbred laboiatory stocks. Inbreeding lowers the
vigour. Hybridization of two stocks of low
vigour produces very vigorous males. This
hybrid vigour is maintained for at least sixty
generations in populations of at least 200 flies.

The advantage of heterozygous males in in-
traspecific sexual selection constitutes a mecha-
nism of genetic homeostasis which maintains the
high degree of heterozygosity of natural popu-
lations. Heterozygous flies produce more eggs
and spermatozoa and resist moulds and bacteria
better.

A high degree of heterozygosity seems to be
important for natural populations of Drosophila,
which are very reduced in winter. The mainte-
nance of a rich gene pool by heterozygosity avoids
rapid changes of the genetical constitution by
genetic drift, and enables the species eventually
to undergo rapid expansion or an adaptation to
changing conditions.

This work on intraspecific sexual selection
and on the comparison of the fecundity of homo-
zygous and heterozygous flies leads to a new
technique of augmentation of the vigour in
inbred lines without loss of the acquired selection
profit.

The enrichment of the morphological, physio-
logical and psychological possibilities of a species
by the creation of new loci seems to be the major
progress of evolution and could be more im-
portant than the substitution of alleles at existing
loci by mutation.

9.42. The Effect of Inbreeding on the "Fitness" and
on the Rate of Attainment of Acclimatization
to Temperature in Drosophila subobscura.
K. Bowler and M. J. Hollingsworth
(London, Great Britain).

A further aspect of the hypothesis that in-
breeding leads to a loss of "biochemical ver-
satility"d) has been tested. Males from the
B & K inbred lines of Drosophila subobscura and
from the recriprocal hybrids between these in-
bred lines were used.(-> The criterion of bio-
chemical versatility has been the rate at which
these males can adapt to changes in temperature.
Fitness has also been measured by comparing the
ability of the inbreds and hybrids to withstand a
high lethal temperature (34°C). All flies were
fully acclimatized at either 14.5°C or 25°C and
then placed at the reverse acclimatization tem-
perature for periods varying from 1 hr to 1 week.

The results have shown that there is no
significant difference between survival times at
34 C of the B/K and K/B hybrids. However,
both in the 14.5C and 25°C adapted groups
the hybrids survived longer than either of the
inbred lines, this being particularly noticable
in the 14.5°C adapted groups. Of the inbreds the
K males were slightly more "heat-tolerant"
than the B males. All flies gained adaptation at
25CC from 14.5°C much more rapidly than they
lost this adaptation when taken from 25°C and
put at 14.5C. The hybrids both lost and gained
acclimatization to 25 DC more quickly than either
of the inbred lines.

It is concluded from these results that in-
breeding leads to a loss of biochemical versatility
and also to a loss of fitness.

1. J. B. S. Haldane, (1948) Ric. sci. Supp. 54.

2. J. Maynard Smith, (1956) /. Genet. 54, 261 .

9.43. Life Cycle and Mating Activity as Criteria of
Heterosis in Heterokaryotypes in Drosophila
pavani. Danko Bracic and Susi Koref-
Santibanez (Santiago, Chile).

The study of chromosomal polymorphism due
to inversions, found in many species of the genus
Drosophila, has proven that these constitute an
adaptive character, and that frequently the hete-
rozygotes for different gene arrangements (hete-
rokaryotypes) are heterotic. Nevertheless, more
experimental data are needed in order to deter-
mine the exact mechanisms by which such
adaptive superiority is achieved, and why
balanced polymorphism is maintained in nature.

157

Section 9 — Population Genetics

The Chilean species, Drosophilu pavani, is
polymorphic for gene arrangements in its chro-
mosomes. Studies of the mechanisms which
contribute towards the maintenance of this poly-
morphism in the populations have shown that the
frequency of heterokaryotypes with respect to
some gene orders is significantly higher in
100-day-old flies than in larvae or in young 10-
day-old adults. This finding suggests that the
selective pressures which confer a higher fitness
to these heterokaryotypes may act both duiing
the preadult and the adult life stages.

When mating activity was taken as a criterium
for the superiority of heterokaryotypes, it was
found that, in a very heterogeneous population,
the group of males which courted or copulated
within the first 30 min of contact with the female,
contained a significantly higher proportion of
heterozygotes for inversions than the group of
males which copulated after this period. As
mating activity may be important in fitness, these
findings could in part explain the maintenance
of balanced polymorphism in the populations.

9.44. Sexual Isolation Between the "Sibling" Species
Drosophila pavani and Drosophila gaucha.

Susi Koref-Santibanez (Santiago, Chile).

Drosophila pavani and Drosophila gaucha are
two neotropical "sibling" species of the meso-
phragmatica group of the genus Drosophila. They
are mostly allopatric in their distribution, al-
though they have been found sympatrically in
certain regions. Hybrids have never been found
in nature, and in the laboratory, where they can
be produced abundantly, they are sterile. Chro-
mosomally the two species differ only in the gene
arrangement in their X chromosomes.

The different levels of sexual isolation were
studied in order to try to establish the mechanisms
by means of which gene exchange between D.
gaucha and D. pavani is prevented. Thus, among
others, courtship behaviour, mating ability,
sperm viability and early embryonic develop-
ment were analyzed. Previous work of Fernan-
dez in our laboratory had shown that spermato-
genesis in the hybrids was apparently normal.

Courtship behaviour in both species and in
their hybrids is very similar, although the hybrid
males are slightly more inactive. They copulate,
and hybrid females accept D. pavani, D. gaucha,
and hybrid males. Nevertheless, an embryonic
development is seen in the eggs of inseminated
females. In view of the above-mentioned data,
it seems that under laboratory conditions, the
main mechanism of hybrid sterility depends on
the lack of fusion of the male and female game-

tes. To support this hypothesis there exists
experimental evidence of the rapid inactivation
of hybrid sperm within the female vagina. The
factors involved in this inactivation are now being
analyzed.

9.45. On the Behavior of Lethals in Natural and in
Laboratory Populations. A. Brito da Cunha,
J. S. de Toledo, S. A. de Toledo, L. E. de
Magalhaes and C. Pavan (Sao Paulo, Brazil).

To study the behavior of lethals in nature,
7 wild and 7 radiation induced lethals were
introduced in two isolated island populations
in 2 experiments. The lethals, which before had
an allelic frequency of 6 per 10,000 chromosomes,
reached, after the introduction, a mean frequency
of 0.105 in one population and of 0.119 in the
other in the first experiment and of 0.184 and of
0.205 in the second. The introduced lethal fre-
quencies in the first experiments dropped to
0.012 in one population and to 0.001 in the other
after 8 generations, being practically eliminated
after 15 generations. In the second experiment
the mean frequencies dropped from 0.205 to
0.015 in one island and from 0.184 to 0.008 in
the other in 4 generations.

Parallel experiments with the same lethals were
carried out in the laboratory using population
cages. In these experiments every lethal had an
initial frequency of 0.250. After 15 generations the
lethals had frequencies between 0.1 74 and 0.030,
6 out 16 lethals having frequencies equal or
above 0.100.

There is a sharp contrast between the behavior
of the lethals in nature and in the laboratory.
In the laboratory the selection was very slow and
the lethals behaved as recessive. In nature the
elimination was not uniform. The lethals had
deleterious effects in the heterozygotes behaving
as incomplete recessives with effects which varied
with ecological factors. The period of drastic
selection was always correlated with the deterio-
ration of the environmental conditions. Radiation
induced and wild lethals behaved similarly.

9.46. Dynamics of Lethal Genes in Natural Popula-
tions of D. willistoni. L. E. de Magalhaes,
A. B. da Cunha, J. S. de Toledo and C.
Pavan (Sao Paulo, Brazil).

The frequencies of lethals released in the popu-
lations of the islands of S. Joao and Queimada
Pequena decreased in the 1st to the 4th genera-
tions according to the expectation due to eli-
mination through homozygosis and dropped

158

Section 9 — Population Genetics

very quickly in the succeeding generations to
almost zero in generation 15. The drastic
elimination of such lethals (14 in total, in two
different experiments) after so few generations
could be explained by changes, due mainly to
environmental factors, in the adaptative values
of the zygotic combinations carrying lethals.

If this hypothesis were correct, a change in the
frequencies of chromosomes bearing lethals in a
control island would be expected. We have four
samples analysed from a control island, Quei-
mada Grande, very close to the other two. The
samples were taken in June and July when the
frequencies of lethals should be high and in
October and January when the frequencies should
be lower. The frequencies found were: 0.41,
0.39, 0.39 and 0.34 per cent respectively. No
statistically significant difference was found. This
shows that there are no corresponding increases
of elimination of lethal chromosomes, in the
natural population for their own lethal, present
in very low frequencies.

This comparison suggest, that the relative fre-
quencies of the genetic components could be very
important in their own dynamics.

9.47. A New Type of Hybrid Sterility in Drosophila
paulistorum. L. Ehrman (New York City,
U.S.A.).

It has been shown that Drosophila paulistorum
is a group of at least six races or incipient species,
and that, at our time level, reproductive isolation
is in the process of formation between these
incipient species. Crosses between three (Centro-
American, Amazonian, Andean-South Brazilian)
of the six races result in the production of fertile
female and sterile male hybrids. The male sterility
has been found to depend on the genotype of the
male's mother. Any female which carries any
mixture of the chromosomes of different races
deposits eggs giving rise to sterile male zygotes
and to fertile female ones. The male sterility is in-
dependent of the genotype of the male parent and
the genotype of the sons themselves. What hap-
pens is evidently that the presence of a foreign
chromosome in the female so alters the structure
of her eggs, presumably by some modification
of the egg cytoplasm, that male individuals devel-
oping from these eggs are sterile, and this
regardless of the chromosomal complementwhich
they come to possess after fertilization. Further-
more, any one foreign chromosome (the species
has three pairs of chromosomes) suffices to in-
duce this male sterility.

More recent work involving all six of the D.
paulistorum races permits the categorizing of six

distinctly different manifestations of this new
type of hybrid sterility; these further emphasize
the unique dichotomy in the genetic causation of
the Fi and of the backcross hybrid male sterility
exhibited by this species complex.

9.48. Subspeciation in Drosophila melanogaster.

G. Eloff (Bloemfontein, South Africa).

Cultures were made of more than 30 popula-
tions of Drosophila melanogaster collected over
Southern Africa at localities, some of which are
almost 1000 miles distant and involving im-
portant ecological differences. These populations
were bred under similar conditions in the labo-
ratories of the Dept. of Genetics of the Orange
Free State (20°C; very dry conditions; banana,
agar and yeast moldex food recipe). Of each
population the average yield per 10 females was
determined. Great differences in number of off-
spring were observed. These different geographic
populations were intercrossed and the number of
Fi per 10 females of each cross counted. The
number of offspring ranges from nil to more
than the average for each parental population.

Furthermore, it was found that in some cases
a population would not cross with Oregon River
or Florida population of Drosophila melanogaster
but would cross with another geographic race,
which inks turn does cross with Oregon River.
Several of these transitional stages were recorded.

These three aspects of geographic populations,
viz. (1) differences in fitness under similar con-
ditions of breeding; (2) differences in average
number of Fi of the different interpopulation
crosses; and (3) degrees of succesful crossing
between the different geographic populations,
strongly suggest considerable genetic differen-
tiation and perhaps subspeciation in progress.

9.49. Isolation by Disruptive Selection. J. B. Gibson
and J. M. Thoday (Cambridge, Great Britain).

Thoday and Gibson*1) exposed a population
"Southacre" of Drosophila melanogaster to
disruptive selection for sternoplural chaeta
number using a breeding system that ensured
that all selected flies were together in a single
3 x 1 in. vessel for the limited period during which
all mating had to occur. Their population rapidly
split into two components between which little
if any effective mating occurred.

Selection was discarded in this line when ex-
treme fertility trouble occurred at generation 28.
However, isolated high and low chaeta number
lines were maintained. A new line was started

159

Section 9 — Population Genetic*

from the "Southacre" stock under the same
selection regime. Divergence was more rapid in
the second experiment than in the first and the
two halves of the population became completely
distinct by the seventh generation.

Mating preference tests in this new line using
chaeta number as a marker have indicated an
isolation index of +0.54 (where 0 would
represent random mating and +1 complete
positive assortative mating). There is therefore
evidence of strong mating preferences in the line.
It does not however seem sufficiently strong to
provide a complete explanation of the lack of
hybrids in the line itself. Further evidence sug-
gests that the hybrids that are formed may
compete poorly with the non-hybrids.

1. Nature 193, 1962.

9.50. Selection for Xanthine Dehydrogenase Activity
Levels in Drosophila melanogastei . Edward C.
Keller Jr. (Chapel Hill, U.S.A.).

In an attempt to obtain information about the
quantitative genetic control of individual pro-
teins, a series of selection and breeding experi-
ments were undertaken using xanthine dehy-
drogenase levels as the selected phenotype.
Various methods of selection have been effective
in isolating strains which have very high or very
low levels of this enzyme. One high activity line
(three or four times the "standard" Oregon-R
strain) is of a dominant or overdominant nature
and it has not as yet been possible to localize the
genetic cause or causes of this high activity. A
random sample of about 100 wildtype strains of
diverse geographic origin were also assayed for
their mean xanthine dehydrogenase activities and
many differences in activity levels were found.
Analyses of the data revealed that these differen-
ces were mainly due to genetic causes. Indeed,
several strains contained an autosomal recessive
gene which when homozygous limited the xan-
thine dehydrogenase activity levels to about 25
per cent of the „standard" strain. Subsequent
analyses have shown that this gene is located
near the center of the left arm of the third
chromosome. Therefore, this new mutant is not
an allele of the rosy or maroon-like eye color
mutants, which have no detectable amounts of
xanthine dehydrogenase.

9.51. Selection for Interpopulation Heterosis. Ken-

ichi Kojima (Raleigh, U.S.A.).

The base populations of this study were two
divergent long-term cage populations of D. pseu-
doobscura. The trait used was egg production.
Within each cage there was no significant
additive genetic variance, although there existed
substantial non-additive hereditary variances.
Their hybrid progenies did not show heterosis,
but crossbred half-sib covariance was significant.
Reciprocal recurrent selection, a scheme in
which two populations are selected for their
populational combining ability, was employed
in order to increase interpopulational heterosis.

Approximately 40 per cent gain in crossbred
progeny performance over the original hybrid
was obtained by the 11th cycle of selection, with
only slight increase in purebred performance of
the two populations. The increase in crossbred
performance was accompanied by a reduction
of additive genetic variance in crossbred proge-
nies. During the next 9 cycles the crossbred
performance more or less plateaued, while
significant improvement was observed in pure-
bred performance. Thus, in reference to the
average performance of the base populations,
the level of heterosis was increased from zero to
40 per cent. With reference to the performance of
purebred progenies at various stages of selection,
however, the degree of heterosis changed from
nil through the highest to a low value of 10 per
cent.

This research was supported in part by a
research grant (GM 08202-03) from the National
Institutes of Health.

9.52. A Chromosomal Analysis of Genetic Variation
in Drosophila melanogaster. J. F. Kidwell,
J. W. Gowen and Janice Stadler (Ames,
U.S.A.).

The marked inversion outcross technique
was used to form two lines of Drosophila that
were isogenic for all four chromosomes. The
81 possible combinations among females were
made. Egg production during the sixth, seventh
and eighth day and the number of chaeta on
the fourth and fifth abdominal segments were
measured. Theanalysis was that of a 34 factorial
experiment, which provided a complete partition
into additive, dominance and epistatic genotypic
effects. Additive and dominance effects, and some
two and three factor interactions, were important.
Single, double and triple heterozygotes showed
increasing heterosis. The quadruple heterozygote
exhibited less heterosis than the triple heterozygote
for chromosomes 1, 2 and 3.

160

Section 9 — Population Genetics

9.53. On the Influence of the Number of Inbieeding
Generations on Viability and Size in Droso-
phila melanogaster. F. A. Lints (Louvain,
Belgium).

As pointed out by Lerner (1954) attempts to
verify the general idea that heterozygotes are
better canalized than homozygotes are urgently
needed. A wild strain was submitted to in-
breeding immediately after capture, and in
order to obtain also reciprocal hybrids, each
generation was backcrossed to the wild strain
Swedish-B. Twelve generations were realized,
seven of which were measured for several
traits at five temperatures ranging from 30° to
18 C. This study concerns only viability and
size. A full report is actually in preparation and
will be submitted to Heredity.

Viability: In absolute values the viability of
hybrids does not vary during the successive
generations, but in relative terms, and with
relation to the control passes from a superior to
an inferior level. There is no relation between the
number of inbred generations and the percentage
heterosis for viability. The variance of inbreds
increases irregularly between the first and the
12th generation; the effects of inbreeding on
viability can be more adequately described by the
regression viability-temperature which increases
regularly with the number of inbred generations.

Size: The coefficient of variation of the size
for inbreds and hybrids, considered in the
complete array of environments used, is identical.
This unexpected fact is due, for the hybrids, to
a smaller variance in some of the environments
considered, combined to a significant regression
in function of temperature, for the inbreds, to a
large variance at each temperature and to a
quasi absence of temperature-size regression.

Conclusion: The incompatibility of these data
with the classical hypotheses of additivity and
of heterozygosity per se have to be stressed. In
view of the actual and earlier results inbreeding
degeneration has to be considered at two levels
one at which it has its maximal effect, on viability
namely, the other at which it is probably less
active, namely on various luxuriance and per-
haps on fitness traits of the hatched individuals.

9.54. Reproductive Isolation between Incipient Species
of Drosophila paulistorum in Brazil. Chana
Malogolowkin and A. Solima Simmons
(New York, U.S.A.).

Two races or incipient species of the D. paulis-
torum complex are known to occur in Brazil, the
Amazonian in the states of Amazonas and

Para and the Andean-Brazilian in the territory to
the south. At Belem, Para, both races occur
sympatrically. The strains of the two races are
difficult to cross, and when crossed produce
progenies consisting of fertile daughters and
sterile sons. A number of so-called "transitional"
strains were however found in the states of
Bahia and Ceara; they belong to the Andean-
Brazilian race, but they can be crossed and
produce fertile hybrids with some strains of the
Amazonian race. Sexual (ethological) isolation
has been studied, by means of the "male choice"
method, between various strains of the two
races. Females of the transitional strains show a
significantly weaker isolation from Belem
Amazonian strains than do females which
produce sterile hybrids with the latter. The
Amazonian females from Belem do not however
accept males of the transitional strains any more
easily than do males of the other strains of the
Andean-Brazilian race.

The results as a whole are consistent with the
assumption that sexual isolation is built up, or
at least strengthened, by natural selection in
territories where two races coexist sympatrically.
Territories inhabited by only one race have
populations which show relatively weaker
isolation when exposed to the challenge of
hybridization with other races.

9.55. The Races of Drosophila rubida. W. B. Mather
(Brisbane, Australia).

The species D. rubida Mather of the immigrans
species group occurs in northern Queensland,
New Guinea, New Britain and New Ireland and
is morphologically uniform. However, it has been
shown to be rich in inversion polymorphism by
mating males and despermed females from the
wild to an inversion free standard strain from
Northern Queensland and scoring seven larvae
per cross against a photographic giant chromo-
some map prepared from the standard strain.
By these methods eleven simple and three
complex inversions have been detected.

Sexual isolation tests between cultures from
Cairns (Northern Queensland), Port Moresby,
Madang, Lae and Samarai (Eastern New Guinea)
and Rabaul (New Britain) have been carried out
by confining ten males of one strain with ten
females from another and after a food change at
five days dissecting the female genital tract for
the presence of sperm after a total of ten days.
By these methods it has been shown that where-
ever Rabaul males are involved in the cross the
sexual isolation is high (usually less than 50 per
cent insemination) and that sometimes when

161

Section 9 — Population Genetics

Rabaul is involved the F2 does not come through.
However, backcrossing of the Fi female to males
from the original parents is successful in these
cases.

Cytologically the inversion complex ORE has
only been detected at Rabaul and is homozygous
there. Apart from IIRE only simple HID with a
frequency of 56 per cent has been detected.

Thus on the available evidence we may
distinguish two races of D. rubida between which
there is considerable sexual isolation and hybrid
sterility: Race A from northern Queensland and
Eastern New Guinea without inversion IIRE and
Race B from Rabaul with inversion IIRE.

9.56. The Relation between Fitness Components and
Equilibrium Frequencies in an Experimental
Population of Drosophila melanogaster. Ray

Moree (Pullman, U.S.A.).

In laboratory populations initiated with
equal frequencies of the alleles + and e", the
carriers of e" \e" fell rapidly to a mean stable
frequency of about 2.46 per cent. That similar
results obtain with several alleles of e and that
stabilization is probably a consequence of the
superiority of the carriers of +/e", is well known.
Several fitness components possibly contributing
to these effects were examined. Mass mating
tests (but not choice tests) indicated only slight
selection against e"/e" . Viability tests, with
crowding such that numbers of parents and
offspring were approximately equal., gave
coefficients of 92, 100 and 46 per cent for the
carriers of + / + , + /e" and e"/e", respectively
(Moree and King, 1961). Fecundity tests,
corrected for inviability, gave coefficients, in the
same order, of 88, 100 and 85 per cent (Moree,
1 962). Since such components measure sequential
events they may be multiplied to obtain partial
fitness coefficients of 81, 100 and 41 per cent
(same order). These coefficients indicate an
equilibrium frequency of q (e") = 24.28 per cent.
If panmixis is provisionally assumed, then
q2 (e"/e") = 5.9§ per cent. But this is an over-
estimate. If the above viability coefficients are
included the calculated equilibrium frequency of
e"/e" is reduced to 3.09 per cent, since, at
equilibrium, inviability changes genotype, but not
gene, frequencies. By t-test, 2.46 and 3.09 percent
differ (P<0.01, 7V$j 7000). Thus while heterosis
accounts for stabilization, and inviability and
infecundity for about 97 per cent of total se-
lection, a small fitness component, probably
related to selective mating, is yet to be accounted
for.

9.27. "Yellowish" in Drosophila bifasciata, con-
ceivable as an Incipient Species. Daigoro
Moriwaki and Osamu Kitagawa (Tokyo,
Japan).

In northern parts of Japan, e.g. Hokkaido,
Drosophila bifasciata which is known to inhabit
cold or mountainous districts in the rest of the
country can be collected at level ground even in
early summer. At three localities, so far confined
to Hokkaido, some mutant flies with yellowish
thorax color — designated by "yellowish" (yli)—
were collected once in a while. Interestingly, they
were found always together with wild type flies.

Besides the thorax color, "yellowish" has one
— not two — black stripe on the scutum, which
expands postlaterally forming a trident-like
pattern and it also shows some differences in
metric characters from the wild type.

"Yellowish" females can cross with wild type
males to some extent, while the reciprocal cross
is difficult. Isolation index obtained with the
aid of "male-choice" method, amounts to 0.8 on
the average. It seems that "yellowish" is consider-
ably isolated sexually from the wild type, and
can live sympatrically with it.

According to the test for fertility of the Fi
hybrid flies, the male hybrids are always sterile,
notwithstanding slight fertility of the female
ones.

On examining salivary gland chromosomes
of the hybrid between yh and +, no complete
paring has been seen in any pair of the homolo-
gous chromosomes. Although further careful
comparisons are being made, it is tempting to
speculate that "yellowish" may be an incipient
species.

9.58. A Study of the Genetic Structure of Natural
Populations by Means of Polygenic Viability
Mutation Rate in Drosophila melanogaster.

Terumi Mukai (Misima, Japan).

A single male Pm/ + from a cross of Cy/Pm x
W160 (Burdick's isogenic wild-type stock) was
sampled and multiplied to 104 by crossing to
Cy/Pm females, and each wild-type second
chromosome has been maintained through a
single male by the cross of Cy/Pm (5$$)xPm/

+ 7 Oc?) (*=1. ■ • • » 104) f°r tne Purpose of
accumulating spontaneous mutations affecting
viability.

In generations 10, 15, 20, and 25, the homo-
zygous viabilities of those 104 chromosomes were
estimated by Cv-method (Wallace, 1956) and
their increasing rate of genetic variance and

162

Section 9 — Population Genetics

decreasing rate of average viability in homo-
zygous condition were estimated.

Under the assumptions that the mutations in
question are distributed on the chromosome
according to Poisson distribution and that all
mutations are homozygously deleterious, the
average mutation rate was estimated to be at
least 0.1 per second chromosome pel generation
on the basis of counting about 1.8 million flies.
This mutation rate is of a surprisingly large
magnitude.

When the survival rate of eggs is calculated on
the basis of the classical hypothesis and this
estimated mutation rate, it becomes about 58 per
cent in random mating populations, and is
inconsistent with the actual situation. Thus, the
result is unfavorable to the classical hypothesis.

9.59. Let huls and the Parameters of a Southern
Population of Drosophila willistoni. M. Napp,
H. Winge, M. L. Reguly and A. R. Cordeiro
(Porto Alegre, Brazil).

Random crossing, mutation pressure and the
progressive betterment of gene coadaptation in
complex genie balance due to natural selection,
builds the genetic structure of natural popu-
lations.

Genetic analysis of a natural population
inhabiting an isolated, small wood in the grass-
land of Rio Grande do Sul State, disclosed sig-
nificantly different frequencies of II chromo-
somes bearing lethals or semi-lethals, averaging
44.7 ± 1.7 per cent for the period of March
1959 to April 1961 with 895 strains studied. The
III chromosome lethals + semilethals frequen-
cies showed no changes and averaged 29.5 ±
2.3 per cent in five samples from March 1958
to December 1959 (389 strains). The total
intrapopulation allelism frequency determined
is: 11=0.0102 111= 0.0029.

With the aid of S. Wright's formulae we
calculated that the rate of lethal incorporation
for II chrom. is 0.0046 and 0.0049 for the III.
The elimination due to random homozygosis:
11=0.0007 and 0.0001 for the III. This allows
increments of II = 0.0039; HI = 0.0048.
Nevertheless, taking the same mutation rate (*)
the number of lethals, if completely recessives,
would be in the II, 2.6 — 4.6 times, andinthelll,
5.6 — 10.3 times greater than the actually observed,
averaging 2.6 and 7.6 times, respectively.
Selection coefficients opposed to lethals in heter-
ozygous condition of s (II) = 0.018 and s(III) =
0.031 would be necessary to account for the
discrepancy. Contrary to others' results the s
values for semilethals in heterozygous state are

smaller than the ones for lethals. Local in-
breeding, F(II) = 0.01 65, might be another factor
of lethal or semilethal elimination. The estimated
effective population size: N= 4030 (ace. Wright's
formula) conforms with the values of six to ten
thousand individuals determined by release
experiments of marked flies in the same wood.

1. Determined by Dobzhansky et al.
Genetics 37, 650. 1952,
Work to be reported full in Evolution.

Work supported by the Rockefeller Foun-
dation, Conselho Nacional de Pesquisas and
the University of Rio Grande do Sul.

9.60. The Peisistence of Deleterious Genes in
Natuial Populations of Drosophila melano-
gaster. Chozo Oshima (Misima, Japan).

The distribution of viabilities manifested by
individual homozygous second chromosomes
isolated from the Japanese natural populations
of Drosophila melanogaster has been in the
course of several years repeatedly examined.
From the results of allelism tests between lethal
genes, some of them were proven to have been
retained for several years in the same population.
The pre-adult viability of heterozygous flies for
single lethal and double lethal chromosomes was
estimated and compared with that of normal
heterozygotes. A complementary effect between
lethal genes was recognized in the viability of
double lethal trans- or cis-heterozygotes and it is
assumed to be due to interaction between two
loci carrying lethal genes in the heterozygous
genetic background. The mean relative viability
of heterozygous flies for single semi-lethal and
double semi-lethal chromosomes was also
estimated. The deleterious effect of a single
semi-lethal chromosome on heterozygous con-
dition was almost none as compared with that of
a single lethal chromosome, but the detrimental
effect of double semi-lethal trans-heterozygotes
was emphasized. From the above mentioned
results, some mechanisms of persistence of
lethal genes in a natural population are consider-
ed and the difference in their frequency between
lethal and semi-lethal chromosomes will be
discussed.

9.61. A Comparative Survey of Genetic Variabilities
between Second and Third Chromosomes from
Korean Populations of Drosophila melano-
gaster. Yong Kyun Paik (Seoul, Korea).

138 second and 151 third chromosomes col-
lected in mid-July in a suburb of Seoul were

163

Section 9— Population Genetics

analyzed by Cy and TM3, Ser balancer chromo-
somes. The number of lethals and semi-lethals
are 13.0 per cent for second and 18.5 per cent
for third chromosomes. In a second collection
after three months, 160 second and 164 third
chromosomes were taken in mid-October and
17.5 per cent of the second and 16.6 percent of
the third chromosomes were found to be lethals
and semi-lethals. Another sample was taken in
a location at rural Kwangnung in mid-September
of the same year, and 16 per cent of 176 second
and 20 per cent of 221 third chromosomes
sampled were found to be lethals and semi-lethals.
Differences in lethal concentration between
second and third chromosomes as determined
by chi-square are not significant in time and
space. However, it is interesting to note that on
the average slightly more third chromosomes
than second carry lethals. Additional data
showed high value of homozygous viability of
both second and third chromosomes above
semi-lethals and of quasi-normals: on the aver-
age, mean viability of chromosomes above
semi-lethals appears to be 30 ± 0.3 per cent for
second and 31.0 ± 0.5 per cent for third chromo-
somes. Nearly complete tests showed compara-
tively high frequency of allelism among the
strictly lethals in both second and third chromo-
somes, showing no significant differences be-
tween collections in the same location or be-
tween locations (1 to 3 per cent for III and 1
per cent for II chromosomes). Total incidence of
visibles was low in each collection, but more
than half of the different visibles occurred more
than once within the respective populations. Many
of the wild males tested, which carried either bw
or p, were further proved to have more than one
lethal and/or other deleterious genes either on the
bw or p carrying chromosomes or on the other
part of the homologous chromosomes.

As far as we may judge by the present data
available, the frequencies and allelism of sec-
ond and third chromosomes of lethals and other
genetic variabilities agree well each other, and
a batch of the results falls within the expected
range showing smallness of population size.
It may be safe to conclude that Asiatic popula-
tions of Drosophila melanogaster are really
different from the other geographical popula-
tions so far reported, in respect of population
size and genetic structure.

9.62. (D.) Polymorphism in the Water Boatman, Tri-
chocorixella mexicana. W. Peters (Chicago
U.S.A.).

Dextral and sinistral forms displaying situs
inversus occur in a 1 : 1 ratio in the wild popu-

lation. Phenotypic characteristics are illustrated
and a probable mode of inheritance proposed.

9.63. Color Polymorphism in Cage Populations
derived from Hybrids between Drosophila I.
lebanonensis and Drosophila I. casteeli. S. B.
Pipkin (Panama, Panama).

Founders of four cage populations were Fi
hybrids between a homozygous pale strain of
D. I. lebanonensis, a species exhibiting a balanced
mesonotal color polymorphism in natural pop-
ulations of the Lebanon, and D. I. casteeli,
its dark subspecies, lacking a color polymorphism
in natural populations of Arizona, U.S.A. The
pale versus dark mesonotal color is dependent
upon a single pair of alleles, S,s, not associated
with a chromosomal inversion.

After twenty-four generations, two popula-
tions maintain a Ss superiority. An initial s/S
ratio of 1 declined to 0.9 in the fifth generation
and 0.45 in the fourteenth generation, after
which there was a rise to 0.7 in one population
and 0.65 in the other. Presumably coadaptation
of gene complexes resulted in improvement of the
selective value of the Ss heterozygotes in these
two populations from the fourteenth through
the twenty-fourth generations. In two other
cage populations, the SS homozygotes proved
superior to Ss heterozygotes, although the
selective value of the latter improved somewhat
after the fourteenth generation.

Mating propensity, known to be low in D. I.
casteeli, is high in cage populations derived from
hybrids between D. 1. lebanonensis and D. I.
casteeli, with no differences among the SS, Ss,
and ss genotypes. Culturing a high temperatures
of from 25 to 28°C of replicate half-gallon
bottle populations, derived from the two equilib-
rium cage populations at the thirteenth genera-
tion, resulted in a rapid selection strongly fa-
voring SS homozygotes. Length of egg to hatch-
ing life cycle in an equilibrium cage popula-
tion is approximately the same for Ss, SS, and
ss genotypes.

9.64. Selection in Experimental Populations of
Drosophila melanogaster with Different Ge-
netic Backgrounds. Sergey Polivanov (New
York, U.S.A.).

A study was made of the behavior of several
mutants of Drosophila melanogaster in experi-
mental populations, kept in laboratory popula-
tion cages. In all experiments the foundation
stocks contained mutant and wild type alleles

164

Section 9 — Population Genetics

of certain genes with equal frequencies, 0.5. As
expected, natural selection led in all cases to
decreases in the relative frequencies of the mut-
ant and to increases of those of the wild type
alleles. The selection rates proved, however, to
be in some cases very different depending on
whether the foundation stocks contained only
one or many kinds of wild type chromosomes.
The "single-strain" populations were derived
from crosses of mutant flies to flies from a highly
inbred wild-type stock. The "multiple strain"
population's non-mutant chromosomes were
derived from many (about a dozen) wild strains
of different origin. With the third-chromosome
mutants ebony, scarlet and stubble, and ap-
parently also with the second-chromosome
brown-75, the selection rates against the mutants
were initially much greater in multiple-strain
than in single-strain populations; in later
generations this difference tends to disappear.
With the sex-linked forked and the fourth-
chromosome polished, the single strain and the
multiple strain populations showed similar
behavior within the limits of the experimental
errors. The data are, on the whole, consistent
with the assumption that the selection rates
against certain mutant genes depend not only
on the effects of these genes themselves but also
on those of the polygenic complexes associated
with them in the same chromosome.

in two strains of Drosophila nielanogaster.
One strain, S, had been subjected to 40 genera-
tions of stabilizing selection on development
time. The other strain, D, had been subjected
to 40 generations of disruptive selection for the
same trait.

The primary effects of selection (previously
published) show a decrease in the within culture
variance of the S line and an increase in the
D line and further, that the increased variance
in the latter was probably due to a loss of buf-
fering.

The present report concerns further studies of
the comparative sensitivity (buffering) of the
two lines to (1) environmental variables which
operate among cultures, (2) temperature varia-
tion, and (3) variations in an utritional variable
known to effect development time.

The results of these studies indicate that the
two lines do not differ in their buffering against
these three factors.

It is concluded, therefore, that the genotypes
involved in the differentiation of these lines are
rather specific in limiting their effects to buffer-
ing only against the microenvironmental varia-
tion within cultures. If loci for "generalized
buffering" exist, apparently they were not segre-
gating in these lines. In general these findings
contrast somewhat with studies of the effects of
inbreeding on buffering.

9.65. Chromosomal Inversion Polymorphism and
Size in Drosophila subobscura. Antonio
Prevosti (Barcelona, Spain).

Data are presented showing that some chromo-
somal orders of Drosophila subobscura present a
clear frequency cline north — south. The results of
experiments of artificial selection for size car-
ried on in the laboratory are presented. It is
found that as a correlated response, a tendency
exists to fixation in homozygous condition of
complex orders, when selecting for small size.
However, selection for larger size increases the
frequency of the heterozygosity between the
standard and the complex inversion types of the
chromosomes. Experiments planned to correlate
the chromosomal structure of individuals and
its size are in preparation. The preliminary results
will be given.

9.66. The Effects of a History of Stabilizing Selec-
tion on Sensitivity to Foreign Environments.

T. Prout (Riverside, U.S.A.).

The sensitivity of the time of development to
certain environmental variables was investigated

9.67. Towards a Synthesis of Population and Physio-
logical Genetics. Forbes W. Robertson
(Edinburgh, Great Britain).

The genetic properties of components of fit-
ness, their inter-relations and importance in
determining the stability of other characters must
be related both to development and ecology.
Such information will enable us to specify the
conditions, genetic and environmental, which
favour particular kinds of adaptive change
and thereby bridge the gap between population
and physiological genetics. This approach has
been applied to the growth of Drosophila nie-
lanogaster by selecting for body size, develop-
ment time and egg production on different
controlled diets and also by analysing the
changes which have accompanied adaptation
by a number of populations to novel nutritional
conditions. Growth can be divided into an
exponential and a later, slower phase and changes
in either appear to be genetically independent,
within limits. Both contribute to the total variance
of adult size, but their genetic properties dif-

165

Section 9 — Population Genetics

fer, especially with respect to the distribution of
variation about the mean, the evidence for gene-
environment interaction and also whether or not
development time and body size are correlated.
Selection for either shorter larval period or high-
er egg production, under novel conditions,
led to substantially smaller or larger size, sug-
gesting that the normal stability of the latter is
maintained by opposing selection pressures.
Populations adapted to different diets have
shown consistent changes in body size and
components of fitness and thus provide material
for the experimental study of evolutionary
change. Most of this work is in press and
will appear in the Journal of Genet ical Research.

9.68. Disruptive and Stabilizing Selection on a
Mutant Character. W.Scharloo, (Leyden.The
Netherlands).

Both disruptive and stabilizing selection were
practised on the expression of the mutant cu-
bitus interruptus dominant of Gloor (ciD_G) in
Drosophila melanogaster. In this ciD allele the
relation between factors affecting expression
(i.e. the extent to which the fourth longitudinal
wing vein becomes deleted) and their phenotypic
effect is almost linear whereas the original ciD
shows a strongly nonlinear relation.

A base population (B) was founded by intro-
ducing the mutant into the Pacific wild stock.
In the stabilizing line (S) animals with expres-
sion values nearest the mean were selected as
parents of the next generation (4 33' 4$9, among
20 of each sex measured). In the disruptive line
(D) the two 3S and $$ with highest and the
two $9. and 3<$ with the lowest values were
chosen. Each generation consisted of four bottle
cultures. A rotational mating system was used.

The mean expression values did change in a
minor degree only, but selection was very
effective in both lines with respect to variances.
In D the phenotypic variance increased in 6 gen-
erations to about 2 times the value in B; in
S the phenotypic variance became reduced
to half this value. Estimated heritabilities were
0.50, 0.58 and 0.88, in S, B and D respectively.

The independent variance component (based
upon the differences between the two wings of
one fly) did not change in this period. The
common environmental variance component
(non-genetic differences between flies) is of mi-
nor importance in this character and in the se-
lection response. This is corroborated by the
finding that there was no difference between D
and S in the response of expression to temper-
ature change.

In generation 10 the phenotypic variance in
D showed a further increase to about 4 times
the original value, but in S there was no further
decrease.

This work was begun at the Institute of Ani-
mal Genetics, Edinburgh, during tenure of a
N.A.T.O. Science Fellowship awarded to the
author by the Netherlands Organization for the
Advancement of Pure Research.

9.69. Chromosomal Polymorphism and Position
Effect in Drosophila subobscura. D. Sperlich
(Vienna, Austria).

Chromosomal polymorphism is common in
natural populations of Drosophila subobscura.
The frequency and the distribution of various
structural types are different in northern and
southern populations. The degree of structural
heterozygosity and the inversion frequency
are higher in Italy than in Austria and again
higher in Austria than in Scandinavia. Both
values are lower in insular isolation than on
the neighbouring continent. The association of
linked inversions is not at random although the
map distances between the inversions are some-
times large. There are regions in the chromo-
somes which show accumulation of breaks for nat-
ural inversions. An X-ray induced inversion
was found to be heterotic with three other
natural inversions tested. The distal break of this
inversion is identical with that of some natural
inversions. Therefore one may assume that
position effect plays a great role for the origin
of chromosomal polymorphism in Drosophila
subobscura.

9.70. Mating Propensity of Gene Arrangement
Carriers in Drosophila persimilis. Eliot B.
Spiess and Bozena Langer (Pittsburgh,
U.S.A.).

Multiple choice matings in 24 hr tests of ho-
mokaryotype Whitney and Klamath arrange-
ments of chromosome III demonstrated a rela-
tive propensity of 2 : 1 for W/W 33 to K/K 33
(Spiess and Langer, 1961). Tests were repeated
with varying ratio of W^: K^ in the mating
chamber from 9 : 1 to 1 : 9; the 3 propensity
varied randomly from 1.2 to 2.2 W^ Per
K3 mating. W/W§9_ were a'so mated with more
frequently than K/K $$ except at the extremes
of the 3 frequency ratio when $9. mated about

166

Section 9 — Population Genetics

equally. To ascertain which sex was controlling
rate of mating, observations were made on ten
pairs from uniform cultures in small plexiglass
chambers for 1 hr. Pairs were introduced without
etherization; mating pairs were withdrawn and
time recorded. Cumulative rate curves for 60
to 100 matings give these results: (1) W/W X
W/W matings are most rapid, 85 percent within an
hour; (2) K/K x K/K are slowest, 15 per cent
within an hour. WT $$ x KL <$<$ were 20 per
cent less rapid than W x W, while KL $$ x
WToo were about that much faster than K x K.
Females control the rate of mating then, and
substitution of a WT $ of for a KL $ increases
mating speed. WT <$(§ court persistently and
indiscriminately while KL <$$ court very
little. KL $$ refuse to mate by extruding the
ovipositor or decamping while WT $$ accept
mates more frequently. F2 flies from homo-
karyotype sib matings maintain about the same
rates. Heterokaryotypes (W/K, K/W) mated
within cultures had intermediate rate (55 per cent
in 1 hr) but when mated to WT as either sex,
mating is increased to 80 per cent while to
KL it is lowered to 20 per cent. Maternal ef-
fect in heterokaryotypes was evidenced by su-
perior mating of both sexes with WT mothers
than those with KL mothers when mated to
W/W. Therefore these chromosomal rearrange-
ments contain genie complexes controlling
mating behavior, especially receptivity of $$ and
activity of <$<§.

9.71. Selection for Rate of Development and Gene
Arrangement Frequency in Drosophila persi-
milis. Luretta D. Spies and Eliot B. Spiess
(Pittsburgh, U.S.A.).

It was demonstrated by Spiess (1958) that the
third chromosome arrangement Whitney (W/W)
surpassed Klamath (K/K) by approximately
3 days in rate of development. To evaluate the
rate of development difference as a major com-
ponent of fitness in controlling frequencies
of these arrangements, 6 experimental popula-
tions were initiated in which food was changed
rapidly (3 "Fast" Cages) to favor fast developing
larvae (W/W) or changed slowly (3 "Slow") to
allow other karyotypes (W/K and K/K) to
eclose. KL decreased in fast and slow cages at
about the same rate to about 10 per cent KL
equilibrium. A second pair of populations (DF,
DS) were initiated in which (1) all emerging indi-
viduals were counted each generation and (2)
the first 500 (DF) or last 500 (DS) adults to
emerge were used to establish each discrete
generation. Selection curves were parallel ap-

proaching equilibrium at 10-15 per cent KL after
6-8 generations. That WT is superior in other res-
pects is evident from studies on mating behavior
(Spiess), but possibilities remained that (1) the
rate of development difference was reduced
under population cage conditions or (2) se-
lection was not sufficiently intense to effect a
change in frequencies. New discrete generation
populations were initiated with the same strains
(WT = KL = 50 per cent) but with selection
intensified 20-fold (DIF, DIS). These popula-
tions diverged immediately and KL increased
in slow (DIS) to 80 per cent but decreased in
fast (DIF) populations to 15 per cent. Selec-
tion was reversed after 4-6 generations in subpop-
ulations while continuing the original selection
scheme. Reversed selection was effective. Rate
of emergence increased under fast but was
unaltered in slow selection; progeny per $ parent
increased from 3 initially to 1 1 after ten genera-
tions in DF and DS populations, indicating
improved total fitness. Therefore K/K and W/W
differ in rate of development under population
cage conditions, but moderate selection does not
discriminate between these karyotypes.

9.72. Heritability of Wing Length in Natural Po-
pulations of Drosophila melanogaster and D.
similans. Abdel-Azim Osman Tantawy (Alex-
andria, Egypt).

The genetic variance of wing lenght in natural
populations of Drosophila melanogaster and D.
simulans has been studied. Flies were captured
twice a month from the same locality for six-
teen successive months, from October 1961 to
December 1962. Heritability of wing length in
both species was estimated by the half-sib anal-
ysis and all experimental work was carried out
at 250°C.

The results are summarized as follows:

1 . Wing length in both species measured on the
F2 generation of captured females remained
constant from October 1961 up to April 1962
(mild temperature) after which it declined grad-
ually from May 1962 to August 1962 (hottest
months of the year) after whichit showed a
steady rise again to the end of the experimental
period. Thus, the results are in agreement with
these reported on populations captured from
high altitude and from northern geographical
regions, as compared with low altitude and south-
ern regions, respectively.

2. Heritability estimates indicate that popu-
lations of Drosophila melanogaster possess
higher significant values for the h2 each month
than those found in D. simulans populations,
but the species behaved differently. Populations

167

Section 9 — Population Genet to

of Drosophila melanogaster showed a constant
value up till April 1961 after which it declined
significantly (from 41 per cent to 31 per cent)
up to August 1961. Heritability estimates rose
once again to about 42 per cent from Septem-
ber 1961 to December 1962, while populations
of D. simulans showed arise, though not signifi-
cantly so, in the heritability estimates (28 to
35 percent) from October 1961 up to December
1962.

Phenotypic, genetic and environmental monthly
correlations between wing and thorax length in
both species of Drosophila were analyzed and
will be discussed.

9.73. Evolution of the Mean Ovariole Number in
Experimental Populations of Drosophila me-
lanogaster. Georges Teissier (Paris, France).

The mean ovariole number of wild type Dro-
sophila melanogaster may vary by a factor of
two, according to the geographical origin of the
studied strains; it is conditioned by a polygenic
system, which can be studied by the usual tech-
niques. It seemed useful to observe the evolution
of populations maintained for several years in
cages of the type we have been using since
1932. Six of these populations have been meas-
ured since 1956, six others since 1958. More
than 500 counts of the mean ovariole number
m of samples of 50 females, hatched from eggs
placed on a superabundance of food, have been
made during the last four years.

In three populations, constitued by crossing
the French strain B U (m = 29,45) with the
Japanese strain O T (/;/ == 15.67), the values
of m, which were originally 21.12 for the Fi,
25.75 for Bi and 18.16 for B2, oscillated during
the last three years respectively around 20.93 i
0.07; 21.52 ± 0.13 and 21.36 ± 0.08. Similar
values have been found for the three other popu-
lations, constructed from several different
French strains, such as B U, and therefore or-
iginally more complex.

It appears from these facts, and others which
will be discussed, that there occurred in all our
populations over the years a selection which
adjusted the ovariole number to a value optimal
for the conditions of a strong competition for
food characteristic of the life of Drosophila in
population cages. Moreover this value is not
the same for all the types of experiments.

9.74. An Experimental Study of Environmental
Influences on Population Structure in Droso-
phila. J. A. Thomson (Melbourne, Australia).

Laboratory populations of Drosophila psetido-
obscura initially polymorphic for the Standard
(ST) and Chiricahua (CH) sequences of chro-
mosome III have been studied undei a variety
of experimental regimes involving differences in
temperature, humidity, illumination and food
composition. A variable amount of gene flow
between interconnected populations held under
different environmental conditions was permit-
ted. The degree of genetic differentiation achie-
ved during 30 generations by such island popula-
tions in relation to the imposed environmental
differences has been investigated by following
the frequency of CH, certain physiological
properties, sex-ratio and productivity, in addition
to comparison of the accumulated recessive le-
thals of chromosome III and the additive ge-
netic variance of the sub-populations. Evidence
has been obtained that increased resistance to
desiccation occasioned by larger body size is a
major factor contributing to the adaptive
significance of the ST/CH polymorphism in at
least certain of these populations.

9.75. Cycl ic Variation of Bristle Number with Parental
Age in Drosophila melanogaster. J. M. Wat-
tiaux and M. J. Heuts (Louvain, Belgium).

Emergent flies from successive daily egg
batches, laid by single pairs from pupal hatching
till death, showed, in 8 out of 40 cases a periodi-
cal variation in mean abdominal bristle number.
In all these cases Kendall's correlogram takes
the form of a sinusoidal function with a constant
period whose value lays between 6 and 13 days
and whose maxima and minima differ by 3 to 5
bristles, according to the sibships. In the most
favourable case 5 periods of 6 days each are
evident. Linear trends are not discernible.

Density influences can be ruled out as a cause
of the fluctuations on experimental evidence.
Separate treatment of males and females yields
highly significant correlations between daily
values. The heritability coefficients (h2) calculated
from parent males and from parent females are
± 0.67, viz. ± 0.35, which are considerably
higher than those found for sternopleural chae-
tae (Scossiroli, 1954).

Cyclical segregation of oligogenes in Dr.
melanogaster have been described (Heuts,
1956). Pertinent questions as to the nature of
the mechanisms involved and as to the apparent
restricted occurrence of cycles in quantitative or
qualitative characters of offspring with relation
to parental age will be discussed in forthcoming
publications (Genet ica).

168

SECTION 10

DEVELOPMENTAL GENETICS

10.1. Hereditary Ovarian Tumours of Drosophila.
R. C. King (Evanston, U.S.A.).

The hereditary ovarian tumours of D. melano-
gaster demonstrate that a complex genetic
system plays a decisive role in restricting the
multiplication of the cells destined to become
incorporated into an ovarian follicle. Three
recessive genes are known which cause in the
ovaries of adults uncontrolled cell proliferation
which results eventually in female sterility. The
genes in question are female-sterile (fes), narrow
{nw), and fused (fu). Females homozygous for/<?.s
or nw have ovaries which are completely tumor-
ous at the time of eclosion; whereas freshly
eclosedfu females show no tumours. Tumours be-
gin to appear on the second day, however, ande-
ventually the fu ovary becomes completely tumor-
ous. With respect to fu a study of eight alleles of
independent origin has demonstrated that in each
case tumors eventually form in the ovaries of
homozygotes. However, different fu alleles can be
distinguished on the basis of the speed with which
tumorous chambers appear in the ovary.
Dietrich Bodenstein and I have studied the
development of ovarian implants which were
made using as hosts or donors phenotypically
wild type adults or flies homozygous for fes or fu.
Implants from fes or fu females behave autonom-
ously when left in wild type female hosts for as
long as 6 days. Implants from + donors appear
cytologically normal after developing in fes or fu
abdomens for times as long as 9 and 12 days,
respectively. Thus there is no evidence for diffu-
sible tumorigenic agents as initiating factors in
the development of the ovarian tumours charac-
teristic of fes or fu homozygotes.

10.2. Studies on the Aberrant Function of the Mal-
pighian Tubes in the Drosophila Mutant rosy.

Ilse Schwinck (Storrs, U.S.A.).

Experimental studies of the ontogeny of the
pleiotropic pattern of rosy (ry) mutants and
maroon-like (ma-l) mutants indicate that the
Malpighian tubes perform a central function in

the aberrant pteridine and purine metabolism of
these mutants. W As compared to wild type, the
Malpighian tubes of the rosy mutant are shorter
and cells appear swollen. In all developmental
stages some globules and conglomerates of
globules, composed of fluorescent pteridines, are
found in the lumen of the tubes; the globules are
excreted by larvae and flies. The first occurrence
of the aberrant metabolites in the lumen can be
observed several hours before hatching from the
egg. During the major time of metamorphosis
no formation of new globules was found.
However, in old pupae about 10 hr before emer-
gence, small globules appear in a very regular
distribution in the lumen of the tubes, becoming
larger approaching emergence. The relation of
these findings to the other pleiotropic characters
will be discussed.

Hadorn and Schwinck, Z. Vererbungslehre
87, 1956; Schwinck, Proc. Xth Internat.
Congr. of Entomology, Montreal, 1956, Vol. 2,
1958; Schwinck, DIS 34, 1960; Ur-
sprung, Z. Vererbungslehre 92, 1961.

10.3.

Studies on the Female-sterile Mutant deep
orange of Drosophila melanogaster. John C.
Lucchesi (Berkeley, U.S.A.).

A high percentage of eggs laid by dor/dor
females, mated to dorjY or +/Y males, are
unfertilized: 33.7 per cent and 10.7 per cent,
respectively (these frequencies were correlated
to the fact that +/Y males inseminate females
more readily than dor/Y males). In addition,
about 6 per cent of the eggs from dor females are
beyond metaphase I when they are fertilized.
A disturbance in folic acid metabolism,
resulting in abnormal oogenesis (King and
Sang, 1959), is suggested as an explanation
of the high frequency of unfertilized and belatedly
fertilized eggs from inseminated dor females, and
as a possible cause for the nature of dor cyto-
plasm. Folic acid is structurally and functionally
related to the pterins (involved in eye pigment

169

Section 10 — Developmental Genetics

biosynthesis); therefore, in the hope of restoring
normal folic acid metabolism and fertility, dor
females were made homozygous for ry, ry2 and
bw; ry lacks isoxanthopterin (accumulated by dor
females: Counce, 1957); bw lacks all the pterins;
dor, bw females remained sterile; dor, ry or ry2
flies were lethal indicating that the type of inter-
action sought may have occurred, although in an
unexpected direction.

10.4. Genetic and Physiological Studies of Abnormal
Abdomen in Drosophila melanogaster. Ralph
Hillman, and Stephen D. Barbour (Philadel-
phia, U.S.A.).

Abnormal abdomen was first described by
T. H. Morgan in 1915 as one of the earliest cases
in which the environment controlled the pene-
trance and expressivity of a genetic factor. This
original mutation was subsequently lost. In 1953,
Hillman W reported a recurrence of this ab-
normality as a result of X-irradiation. The
mutation A53g exhibits environmental relation-
ships similar to the original abnormal abdomen
and is located in the same approximate position
as that reported by Morgan. Recent genetic in-
formation indicates that A$3g acts as a cross-over
suppressor of the left end of the X-chromosome.
Cytological studies have shown a rearrangement
of the banding pattern in the heterozygous A53gj
Canton-S salivary chromosomes involving two
to five bands in the 3C-3D region of the X-
chromosome.

Preliminary chromatographic studies of the
amino acid constituents of the mutant stock have
yielded a possible explanation of the morpho-
logical effects. Since only the adult abdominal
hypoderm and tergites are abnormal in A53g,
individual prepupae have been squashed on
Whatman-1 paper and developed in butanol —
acetic acid — water. Results with general and
specialized indicators show an apparent increase
in the glutamic acid concentration of A53g and a
decrease in staining of the spots corresponding to
glutamine and proline. Since proline is known to
be an important constituent of the hypodermal
protein and glutamine is on the metabolic path-
way from glutamic acid to glucosamine and
chitin, these results are suggestive of an upset in
glutamic acid metabolism as a cause of the
morphological abnormalities.

10.5. A Cytochemical Analysis of Deoxyribonucleic
Acid (DNA) and Protein in Salivary Gland
and Gut of the Lethal Mutant lgl of D. melano-
gaster. Robert M. Welch and Kathleen
Resch (Austin, U.S.A.).

Previous research, comparing mature lgl with
mature wild-type larva, has suggested a differen-
tial effect, notably on salivary gland in compari-
son with gut and on DNA of salivary gland in
comparison with protein. An alternative expla-
nation, however, is differential growth in wild-
type larva after onset of general retardation in lgl.
To decide between these two interpretations,
DNA and protein in sections of salivary gland,
proventriculus, and stomach of mature lgl have
been compared with corresponding compounds
of wild-type larva at a stage where lgl DNA is
approximately equal to wild-type DNA and
where lgl salivary gland is approximately equal
to wild-type salivary gland. Gland and gut areas
have also been determined. Also, wild-type
larvae, experimentally altered in DNA or protein
metabolism, have been compared with normal.
DNA has been determined by Feulgen cyto-
photometry, protein by naphthol yellow S
cytophotometry and photometric interferometry.
Results do not indicate increased synthesis of
protein over DNA in lgl in either salivary gland
or gut at stages compared, nor increased growth
of gut in comparison with salivary gland.
Therefore, they do not support the hypothesis
of a differential effect, either chemical or organ,
of the mutation in lgl, although results on ex-
perimentally altered larvae do not exclude a
differential effect on DNA, affecting protein
synthesis indirectly. In absence of contrary
evidence, however, results suggest that differen-
tial growth of wild-type larva after a stage of
general retardation in lgl has produced the
appearance of a differential effect in mature lgl
when compared with mature wild-type larva.

1. DIS 27, 56.

Supported (in part) by PHS Research Grant
GM-06492 from the National Institutes of
Health, USPHS, and by a grant from the Rocke-
feller Foundation.

10.6. Gene Action of esc (extra sex comb) in
Drosophila melanogaster. Chiyoko Tokunaga
(Berkeley, U.S.A.).

When homozygous, the esc gene (II L) initiates
the morphological change of the second and
third male legs into a first leg in terms of bristle
pattern which includes differentiation of an extra

170

Section 10 — Developmental Genetics

sex comb on each basitarsus. The differentiation
of extra sex combs could depend on (a) the
primary change of the second or third into a first
leg, or (b) the autonomous action of esc in the
sex-comb area. Which alternative is correct may
be decided by producing esc/esc mosaic patches
of tissue at the sex-comb sites on the second and
third legs in esc+/esc males.

In order to obtain such mosaics, males having
an X chromosome bearing the gene yellow (v),
and second chromosomes — one bearing the esc+
gene and an insertion of the y+ locus into the left
arm, and the other carrying the esc gene —
were irradiated during the larval period. Somatic
crossing over in a dividing cell yields two daughter
cells, one homozygous for the rf insertion and for
esc+, and the other homozygous for esc. The
former results in tissue which is not yellow and
not esc, and the latter in the absence of the y+
insertion, and the presence of the y allele on the
X chromosome produces yellow bristles which
are also homozygous for esc .

The differentiation of extra sex combs is
interpreted as having been caused by the auton-
omous action of esc.

Research performed under the auspices of the
U.S. Atomic Energy Commission.

10.7. Developmental Genetics of the Gene "Sex-
combless" in Drosophila melanogaster. Ard-
hendu Mukherjee (Berkeley, U.S.A.).

The mutant "sex-comb/ess" (sx, I) affects
differentiation of sexcomb teeth on the basitarsus
of the male first leg. Sx is associated with an
overlapping inversion, in the X-chromosome of
salivary glands, which involves the region
between HCand 15F. Studies on the morphology
and chaetotaxy of the basitarsus of sat male first
leg reveal that the mean number of sexcomb
teeth and transverse rows of bristles per basi-
tarsus is 1.7 and 6.9 respectively. Bristles, that
are intermediate between a typical tooth and a
macrochaeta, frequently appear in the sexcomb
area. Whenever present, the tooth is always
oriented at an angle less than that found in the
normal male, and may lie anywhere beyond the
fourth transverse row. Studies on gynandric
mosaics utilizing ring-X reveal that in a small
patch of sx male tissue in the sexcomb area (in an
otherwise sx/+ female basitarsus) sx inhibits the
formation of any tooth and thus behaves autono-
mously. When sx is combined with eyD, en
(both differentiate extra sexcomb on the basi-
tarsus of the first leg) or tra (tra/tra — transformed

female differentiates sexcomb on the first leg)
the mean number of teeth is greatly reduced in
every case. This shows that sx is epistatic over
the action of these genes with respect to the dif-
ferentiation of sexcomb teeth on the first leg.

10.8. A Gene which Transforms Males and Females
into Intersexes. Philip E. Hildreth and John
C. Lucchesi (Berkeley, U.S.A.).

A recessive gene on the third chromosome of
Drosophila melanogaster has been discovered
which when homozygous changes both chromo-
somal females and males into intersexes. The XX
and XY intersexes are extremely similar in
appearance, and both exhibit male and female
traits. The pigmentation of the tergites is similar
to that of the wild-type male. The seventh tergite
is present, and at its base is the seventh spiracle,
both female characteristics. The ventral part of
the eighth abdominal segment has a protuberance
similar to the female gonopod while the ninth
segment, as in males, bears claspers. The anal
plates are situated vertically as in males. Neither
the XX nor XY intersex has sex combs; however,
on the basitarsus of the forelegs of each, the
bristles of the last transverse row are enlarged
and rotated toward the area a sex comb would
occupy if present. Internally the XX intersex
usually has male and female reproductive parts
with varying degrees of completeness. Well-
developed ovaries are present in some. Frequent-
ly a single gonad may be attached to both the
male and female systems. The XY intersex usu-
ally has a predominantly male internal repro-
ductive system, but both a male and female
system are present in some. In all cases examined
the gonads are poorly developed.

Research performed under the auspices of the
U.S. Atomic Energy Commission.

10.9. Genetics of Gynandromorph Production in
Aedes aegypti. K. S. Rai and G. B. Craig Jr.
(Notre Dame, U.S.A.).

About 100 gynandromorphs have been isolated
from Aedes aegypti (L.) during three years of
genetical investigations. Although simple genetic
ratios are not apparent, frequency of their
occurrence in some strains and in particular
crosses indicates an hereditary basis. A line of
demarcation between male and female tissue

171

Section JO — Developmental Genetics

is always evident. Anteroposterior, oblique, and
lateral types occur with equal frequency.

Certain gynanders with female head and male
abdomen took a blood meal, then died when the
midgut broke open, presumably because the male
gut is not adapted to blood feeding. Conversely,
gynanders with male head and female body
attempted to copulate with females. One bilateral
gynander had components of external genitalia
from both sexes, one spermatheca (three in
females) and two ovaries, one of which contained
testicular tissue and mature sperm.

In anterior-posterior gynanders with female
head and male abdomen, normal spermatogene-
sis occurs. Several of these individuals were
crossed to normal females, using the forced
copulation technique. In crosses using genetic
markers on all three linkage groups, the markers
were recovered in expected frequencies in the
F1-F3 generations. Moreover, sex ratio was nor-
mal and no additional gynanders were recovered.
Fi larvae had normal chromosome complements.
Thus, it seems unlikely that these gynanders are
produced through abnormal chromosome segre-
gation.

Crosses with markers have demonstrated that
gynanders have different gene complements in
their male and female parts. It seems probable
that gynanders in A. aegypti are produced by
fertilization of a binucleate egg or an egg and a
polar body by two sperms.

Supported in part by Atomic Energy Com-
mission Research Contract AT (ll-l)-38 and
NIH Grant No. AI 02753-04.

1 0.10. The Influence of Temperature and X-rays on
the Sex of Carausius morosus Br. L. P. Pun-
acker (Groningen, The Netherlands).

The stick insect Carausius morosus Br., native
to India, reproduces parthenogenetically in
Europe. However, <$<$ may appear spontaneously
(0.5 per cent $$ at 16°-20° C) but apparently
have no sexual function. Sexually aberrant
individuals showing gynandromorphy and/or
intersexuality are also found at frequencies of up
to 2 per cent. Progeny, if produced, are pheno-
typically normal.

Eggs treated with higher temperatures gave
more aberrant individuals. Incubation at 25°-
27°C gave practically 100 per cent abnormals.
Temperature shock of 40° or 45 °C for two hours
also induced abnormals, the percentages depend-
ing upon the temperature and the stage treated.
Early stages (especially the stage at which the

embryonal layers and appendages are developed)
were most sensitive with maximum percentages
of abnormals being 9 and 32 per cent at 40° and
45° respectively.

Early embryonic stages are also sensitive to the
action of X-rays. Eggs receiving doses of lOOOr,
2000r, 4000r, 8000r, produced up to 60 per cent
abnormals. Only the first two series gave adults.
The progeny of these treated individuals showed
an increased number of $<$ dependent upon the
dose and the stage treated. $$ treated intheol-
est embryonic stages were most sensitive and
gave up to 26 per cent $$ in their progeny.

<$<$ (spontaneously arising in control cultures)
have one heterochromatic sex-chromosome
compared with the two similar but euchromatic
chromosomes possessed by all $$ (2n = 66?).
Spermatogenesis is abnormal in both meiotic
divisions (precocious segregation or division,
lagging, non-disjunction, bridges), though normal
bivalents are found. Karyological investigations
indicate that interspecific hybridization may
have taken place in this species and this may
account in part for the lability of the sexual
phenotype.

10.11. An Embryonic Lethal with Reversed Polarity
in Drosophila melanogaster. Alice Louise
Bull (Wellesley, U.S.A.).

An inherited disturbance in egg polarity has
been observed in three related inbred lines of
Drosophila melanogaster, the parental line and
two others separated 176 and 191 generations
before the abnormality was discovered. Normal
winged female flies from these stocks, heterozy-
gous for a double inversion second chromosome
with markers CyL4sp2 and an unmarked second
chromosome, produce embryos which develop
a second larval abdomen anteriorly and in
mirror-image symmetry to the normal abdomen.
The reversed posterior end consists of four
terminal abdominal segments with normal
posterior spiracles. The hindgut and Malpighian
tubules are present. The central internal region
of the embryo is characterized by an undiffer-
entiated sac connecting the two hindguts.

Although symmetrical bicaudal embryos are
rare in an egg collection, other embryos showing
partial reversal of symmetry are found. In these
one to three terminal segments connect to a
normally oriented posterior end of varying
length. Still other embryos have normal sym-
metry, but exhibit disturbances in the formation
of the cephalopharyngeal apparatus.

The occurrence of abnormal embryos (ap-
proximately 2-13 per cent) is always accompanied

172

Section 10 — Developmental Genetics

by a larger number of undeveloped eggs. The
variation in proportions of undeveloped, ab-
normal and normal embryos in different col-
lections of eggs from one female or among
females suggests a threshold response to an
undetermined physiological or environmental
factor.

The genetic factor or factors that produce the
bicaudal effect appear to be associated with
the unmarked second chromosome present in
all three CyL4sp-/+ + + inbred stocks. The
maternal, but not paternal, chromosomes in-
fluence the formation of these embryonic ab-
normalities.

10.12. Phenogenetics of the lozenge Loci in Droso-
phila melanogaster. Harvey A. Bendkk and
M. M. Green (Davis, U.S.A.).

The pleiotropic effects of the sex linked
lozenge (Iz) mutants include such seemingly
unrelated features as: modification of eye color,
structure, and size; absence of spermathecae and
parovaria; and reduced tarsal claws. Recent
studies (Bender and Green, 1 962) have, in
addition, demonstrated primary ovarian histo-
pathologies in the /~34k and lz34k; su(3) fe84k
mutants. Such investigations have been extended
to include lozenge mutants representative of each
of the four sub-loci demonstrated in the Iz
pseudoallele complex, and to flies compounded
in all possible permutations with such alleles.

Utilized were studies of fertility, fecundity, egg
hatchability and viability, as well as histological
and histochemical analyses of the primary and
secondary reproductive tissues. The ovarian
investigations were implemented with whole
mount as well as paraffin sectioned preparations
and included transplanted tissues. The histochem-
ical studies detailed nucleic acid, carbohydrate,
protein and lipid localizations.

Evidence was obtained linking the imaginal
corpus allatum with the primary cause of Iz
ovarian abnormalities.

This work was supported in part by National
Institutes of Health Grant No. GM 08697-02
and by Atomic Energy Commission Research
Contract AT (I l-l)-38.

D. gyrociliatus under constant environmental
conditions suggests a special mechanism of sex
determination. There is a non random distri-
bution of big female and small male eggs in the
egg capsules. The two types of eggs in their
production in the ovary depend on each other in
a way that resembles genetic segregation. But
this segregation must take place before a stage in
which meiosis was demonstrated by Nachtsheim
(1919) and Shen (1936).

10.14. Pigment and Pleiotropism: Combined Effects
of Ww/ww and ff/Ff Gene Substitutions.
Elizabeth S. Russell (Bar Harbor, U.S.A.).

Many different mutant genes in the mouse,
including W and f combine effects on distri-
bution of pigment in the hair-coat with patho-
logical effects in other tissues. A hyperadditive
interaction of Ww/ww and ff/Ff genie substitutions
in restriction of pigmentation has been demon-
strated through quantitative evaluation of
percent white-spotting in littermate animals of
four genotypes, Wwff (40 per cent white), WwFf
(II per cent white), wwff ( 14 per cent white) and
wwFf (solid black). All spotting variation was
attributable to these segregating genes, since the
animals were otherwise highly congenic with the
FL/Re inbred strain. The ///fY" substitution has a
significant effect on fetal and newborn blood-
picture, leading to a microcytic, siderocytic
anemia, more severe at 14-16 days gestation than
at birth, and completely cured by two weeks
postnatal. Although WW fetuses and newborn
mice are lelhally anemic, newborn WwFf and
Wwff individuals have higher erythrocyte counts
than do corresponding wwFj and wwff individuals,
and fetal anemia associated with Ww/ww is very
improbable. It is conceivable that severe fetal
anemia, resulting from //'gene-action, could have
resulted secondarily in ventral white-spotting
by inhibiting late stages of melanoblast mi-
gration. It is highly unlikely, however, that cither
the ventral while-spotting of WwFf mice or the
great increase in white area of Wwff over wwff
mice is in any way related to defective fetal
blood supply. The results may better be ex-
plained by tissue-localized effects of Ww/ww,
and probably also ff/Ff, gene substitution acting
independently in mclanoblasts and in hemato-
poietic cells.

10.13. A New Approach to the Problem of Sex

Determination in Dinophilus. Walther Traut 10.15. Interactions at the Agouti Locus. M. M. Dickie

(Saarbrucken, Germany). (Bar Harbour, U.S.A.).

An analysis of egg capsules deposited by

The agouti locus in the mouse now has nine

173

Section 10- — Developmental Genetics

identified alleles. They are: lethal yellow Ay,
viable yellow Av?, light bellied agouti Aw,
agouti (wild type) A, intermediate agouti A\
tanoid aia, black and tan a1, non-agouti a, an ex-
treme non-agouti, ae. Many remutations from non
agouti a to black and tan a1, and to light bellied
agouti Aw, have occurred. This report will sum-
marize the findings about these remutations; de-
scribe two of the newer alleles, viable yellow Avy
and intermediate agouti A1; report some of the
information obtained about the action of these
alleles on different backgrounds; the appearance
and identification of various allelic combinations,
and review briefly some of the biochemical
studies and attempts to produce phenocopies of
certain alleles.

Certain closely linked genes can modify the
expression of some members of this allelic series.
The results of crosses of members of the agouti
series with these modifying genes may contribute
to the understanding of the action of the agouti
locus and the interactions of the various members
of the agouti series.

The action of the alleles of this series may
affect pigmentation of the entire coat or only
portions of the coat. Are these true alleles or
pseudo-alleles at a complex locus? This review
is being presented to open new avenues of
discussion and research to try to answer this
question.

10.16. The Control of Bristle Pattern by Hairy- Wing
in Drosophila melanogaster. Frederick Jay
Gottlieb (Pittsburgh, U.S.A.).

An analysis of the control of developmental
patterns exercised by the mutant gene Hairy-wing
49c {Hwi9c) was undertaken. Inhomozygotesthe
thorax is disproportionately wider at the level of
the dorsocentrals, there are numerous extra
macrochaetae and an increase in the number of
achrostichal rows and chaetal density is noted.
The heterozygote has normal width, fewer
extra macrochaetae and extra achrostichal rows,
and no significant increase in chaetal density.

A developmental analysis of the differences
in differentiation of HwiSc/Hw^, Hw49c/Hw+,
and Hw+/Hw+ tissues was performed by means
of genetic mosaics, resulting from X-ray induced
somatic crossing-over in heterozygous larvae.
Measurements and counts in the dorsocentral
region were used.

X-irradiation causes heterozygotes to express
themselves in a homozygous-like fashion.

Interactions were observed between tissues of
the three genotypes in mosaics. Hwi9c/Hw49c
tissue acts semi-autonomously in mosaics.
Hw+/Hw+ tissue acts non-autonomously, oc-

casionally producing extra macrochaetae in the
presence of adjacent Hwi9c/Hw49c and, more
rarely Hw49c/Hw+ tissue.

The evidence suggests that Hw49c adds extra
width to the thorax on which new prepattern
properties can be exhibited. The rest of the
thoracic prepattern does not seem to be altered
by this mutant, but rather the competence of the
hypodermal tissue to respond to these areas of
the prepattern appears to be changed.

This work was supported by a Predoctoral
Traineeship in Genetics, National Institutes
of Health, and is part of a thesis submitted in
partial fulfilment of the requirements for the
degree of Doctor of Philosophy in Genetics at
the University of California, Berkeley.

10.17. The Developmental Control of Activity and
Expression of the Hairless Locus in Drosophila
melanogaster. David Nash (Cambridge,
Great Britain).

The dominant mutant Hairless (H) on chromo-
some III of Drosophila melanogaster commonly
affects the macrochaetae of the head. Absence
of macrochaetae is not accompanied by severe
derangement of neighbouring pnaffected bristles.
Each vacant site may or may not bear a vestigial
chitinized disc. Selection for changes in level of
mutant expression indicates that one main rule
governs the distribution of vestiges and is best
interpreted as follows;

(1) In normal development the chaetae at
different sites regularly develop at different
times.

(2) Within development of a chaeta, later and
earlier deficiency of a normal gene product
result in presence and absence of vestiges
respectively.

(3) By selection the form of the time-course of
deficiency of the normal gene product is changed.

(4) Production of the hypothetical substance
is under negative feed-back control, which can-
not correct for the deficiency caused by the
mutant, but is still sufficient to produce a rise of
concentration after a critical level of deficiency
has been reached.

Two specific modifiers of hairless exist. The
time-course of their effects can be deduced from
similar morphological considerations as above:
Suppressor- Hairless (Su-H) acts as if it sup-
presses Hairless more strongly as development
progresses. Enhancer Hairless (E-H) flies are
often nearly devoid of chaetae, the majority of
sites bearing vestiges. Enhancement of mutant

174

Section 10 — Developmental Genetics

effect becomes stronger as development progres-
ses.

E-H and Su-H are both closely linked to and
on the same side of the locus black on chromo-
some II. It is therefore quite possible that these
different modifiers are mutants at one and the
same locus within the system controlling the
level of production at the Hairless locus; they
act as if they were "constitutive" {Su-H) and
"super-repressed" (E-H) mutants.

10.18. On the Effects of rolled (rl) in Drosophila
melanogaster. S. T. Lakovaara (Helsinki,
Finland).

In homozygous rolled flies (chromosome 2,
locus 55.1), the following features are observed:
wing edges rolled downward, venation of the
wings defective, eyes of subnormal size, darker
than normal and roughened. In hemizygotes, the
eyes are of half the normal size, dark red and
rough-surfaced. The wing venation is rudimen-
tary. In hemizygotes, Df (2) rl gives a stronger
effect than Df(2)MS-10. Heterozygotes are
normal.

No change in hemizygotes is found after
addition of an extra Y or after loss of this
chromosome. However, factors decreasing wing
size increase the size of the eye in the combi-
nations tested ( W,BdG, vg) .

In both homo- and hemizygotes, the strongest
effects are found at 26°C, with a lessening at
temperatures above and below this value. In
histological sections, developmental defects are
observable in homo- and hemizygotes in the
ommatids as well as in the brain. Pigment is
apparently formed in normal amounts but its
location is changed.

In conclusion it may be supposed that : In their
effect on the production of a substance important
for the development of both the eye and the
wing, the genotypes may be arranged in the
order rl/- < rl/rl < H/ + . The gene rl governs a
switch mechanism between the metabolic path-
ways leading to these two organs. The amount
of substance generated in the wing pathway can
be reduced by adding factors which have a wing-
reducing effect. As a "side effect", larger eyes
are obtained, owing to increased flow of the
common substance towards the eye pathway.
The temperature-dependent changes are due to
thermodynamic properties of the enzyme con-
trolled by rl.

10.19. Transfer of Phosphorus Isotope from Parents
to Offspring in Drosophila melanogaster

Meig. I. Csukas-Szatloczky (Budapest,
Hungary).

The distribution of P32, incorporated by
Drosophila melanogaster, has been examined at
various stages of ontogeny, and also the con-
centration of phosphorus in the gametes of the
progeny has been studied. The method of
Schmidt and Tannhauser was employed for the
separation of fractions. The distribution of the
incorporated phosphorus was found to be
different in the examined fractions in different
phases of the ontogeny.

The amount of phosphorus transferred by the
female sex cell was 50 times that transferred by
the male cell measured at the end of the stage
of third instar larva.

A comparison of the distribution of phos-
phorus taken up from the nutrient medium with
that received by way of inheritance showed
sharp differences in the various fractions.

It is worthy to note that the percentage of
inorganic phosphorus received from the egg
was approximately the same as that taken up
from the medium, while inorganic phosphorus
originating from the sperm was only in -traces
present.

As regards turnover, its rate in respect of
phosphorus originating from the medium was
the double of that for transmitted phosphorus.
Within the latter category, the rate of turnover
appeared to be much higher in female than in
male imagoes.

10.20. Induction of Phenocopies in Cosmarium by
RNase and Actinomycin D. Robert W. Korn
(Kingston, U.S.A.).

The highly specific shape of the unicel-
lular Cosmarium is primarily controlled by a
series of chromosomal loci. Over 25 different
morphological mutants have been obtained to
serve as a basis for a study of phenotypic
induction. RNase and actinomycin D treated
cells produced daughter cells which manifested
both shape and symmetry alterations similar
to mutant types. These phenocopies differed
from mutant lines by persisting for only one
cell generation. Actinomycin D produced a
higher frequency and a wider range of types
than RNase. RNase frequently inhibited all
differentiation while Actinomycin D usually
affected only one distinct step. Preliminary
evidence indicates these changes are induced over
the final two hours before cell differentiation.
Considerations of time, distribution, and organ-
ization of morphogenetic RNA will be presented.

175

Section 10 — Developmental Genetics

10.21. Progressive Expression of Mating-type Genes
in Paramaecium. R. W. Siegel (Los Angeles,
U.S.A.).

Our data support the hypothesis initially
presented by Metz which states that the four
mating-type specificities in P. bursaria syngen 1
are brought about by the four unique combina-
tions of two independent pairs of complemen-
tary ciliary mating-type substances; substances
AB, aB, ab, and Ab characterize the cell sur-
faces of mating types I, II, III, and IV, respec-
tively. Conjugation depends upon the complemen-
tarity of either the alpha or the beta substances
or both. One pair of allelic genes controls the
alpha substances and an independently assorting
pair of alleles controls the beta substances.
Early in the life history of a clone mating does
not occur and neither alpha nor beta substances
are formed. Later, in adolescence, clones will
react with but two of the four standard mating
types and it has been shown that now some
clones form an alpha substance and others a
beta substance. With maturity, each clone forms
both an alpha and beta substance. These find-
ings mean that the two genetic loci concerned
with mating-type specificity come to expression
in a predictive temporal sequence; first neither
locus is expressed, then one locus alone is ex-
pressed and finally both loci are expressed.

The appearance of each successive life-cycle
stage in P. awelia is probably a consequence of
the orderly progressive differentiation of nuclei
(Siegel, 1961). In P. bursaria nuclear differentia-
tions may control the expressions of (or "switch-
on"?) the two loci concerned with the develop-
ment of sexual specificity.

10.22. Siime: a Plasmodioid Variant of Neurospora
crassa. Sterling Emerson (Pasadena, U.S.A.).

The heritable slime variant of Neurospora
grows on an agar surface by plasmodium-like
flows which are devoid of cell walls at the ad-
vancing margins. Spherical cells with thin cell
walls are produced in older parts of such colo-
nies. Growth in liquid culture consists of spheri-
cal cells and atypical hyphae, both having flexible
cell walls.

Heterokaryons between slime and wild type
have the wild-type hyphal morphology under
all growth conditions. In vegetative isolations
from such heterokaryons the slime phenotype is
invariably associated with nuclei of the slime
parent.

Three genetic determiners, segregating in-

dependently at meiosis, are essential to the pro-
duction of the slime phenotype, but are insuffi-
cient for its maintenance. Ascospore isolates
carrying these three determiners are often hyphal
from the start or, if germinating as slime, soon
become hyphal. By rigorous vegetative selection
from such isolates it has been possible to establi; h
sub-strains which retain the slime phenotype per-
manently.

The unique morphological features of slime
make it useful in a number of kinds of investiga-
tions. The absence of hyphal walls permits easier
isolation of cellular constituents than is possible
in hyphal forms. Slime grows in an extremely
thin sheet between an agar surface and a cover
glass, permitting observation of living material by
phase microscopy under conditions in which
cellular structures are well spread and do not
overlie one another, and in which light defraction
from cell walls is absent.

10.23. Transplantation of Nuclei in Neurospora
crassa. James F. Wilson (Salinas, U.S.A.).

Heterokaryons of N. crassa were produced
artificially by cell-to-cell transfer of protoplasm
of genetically marked strains. The injected hyphal
sections were excised and cultured separately.
The criteria of prototrophic growth and recovery
of conidia of the donor genotype were used to
prove that functional nuclei had been transplan-
ted. Reciprocal injections were made with 2
pairs of macroconidial and 3 pairs of micro-
conidial strains. Growth curves and conidial
ratios of these artificial heterokaryons were
compared to the corresponding natural ones.
In each pair of heterokaryons produced by re-
ciprocal injection, the growth curve of only one
was equivalent to the naturally formed hetero-
karyon of that pair. The growth curve of the
other artificial heterokaryon differed either in
rate or in the lag period before growth occurred.
There was no apparent relation between conidial
ratios of the natural heterokaryons and the
artificial heterokaryons with comparable growth
curves. Injection of normal nuclei into a morph-
ological temperature-sensitive mutant pro-
duced an apparent heterokaryon which grew
with normal morphology at 34°C. The apparent
stability of Neurospora nuclei may make in
vitro studies possible.

Published in extenso in Amer. Jour. Bot.
(in press).

176

Section 10 — Developmental Genetics

10.24. Alteration of Frog Zygote Nuclei by Macromo-
lecular Fractions obtained from Adult Liver
Cells. Heinrich Ursprung (Baltimore,
U.S.A.).

Experiments were carried out to test the hy-
pothesis that differential gene function and cyto-
differentiation are based upon complexes for-
med between chromosomes and specific mac-
romolecules. Macromolecular fractions were
prepared from nuclei and cytoplasm of adult
Rana pipiens liver. Aliquots containing approxi-
mately 0.2 mg of protein were injected into
fertilized frog eggs. Two of the fractions, the
globulin-albumin fraction and the histones, pro-
duced cessation of cell division and arrest of
further development at the late blastula stage.
When nuclei from such arrested blastulae were
transplanted into enucleated eggs, the recipient
eggs developed to the late blastula stage, but then
were arrested also, indicating a persistent nu-
clear change. Cytological examination of such
embryos showed no primary gross chromosomal
abnormalities.

The substance responsible for this alteration of
nuclear capacities is heat-labile and not dialyzable.
Specific activity, expressed as biological activity
per milligram of protein, is highest in the fraction
that precipitates between 57 and 65 per cent satura-
tion of ammonium sulfate. Removal of digestible
RNA by ribonuclease does not decrease the specif-
ic activity. DNA has not been detected. It is conclu-
ded that the active substance is or contains protein,
and it is speculated that its action consists in form-
ing complexes with the DNA portion of the chro-
mosomes in such a way as to impede those nuclear
activities that are required for continuation of cell
division and gastrulation.

Supported by NSF Grant GB-298.

10.25. "Brown Spots" (bsp), A Genetic Character
of Drosophila melanogaster which manifests
only after Copulation. Anna di Pasquale and
Laura Zambruni (Milan, Italy).

"Brown spots", a new character arisen spon-
taneously in a wild stock of Drosophila melano-
gaster in 1959, is due to the presence of one or
more recessive factors localized in the 2nd
chromosome. It is transmitted by the male as
well as by the female, but the phenotypical
manifestation, which consists in the formation of
browncoloured areas on the pleurae, is limited
to the female.

The manifestation shows a clear relation to
mating. Females kept apart from males and fe-
males isolated from them soon after courtship
phases but before copulation, never show spots.
A single copulation is sufficient to provoke
spotting.

bsp females mated with males of nine different
stocks show spots with high frequency; the
mating with y w males, after which the inci-
dence of spots is particularly low, is a peculiar
case.

Mating with sterile males (X/YLc and X/O:
non-motility of spermatozoa; X/X; trajtra:
females transformed into males = no sperma-
tozoa) also determine the appearance of spotting
in bsp females.

Frequency of spotting increases with the age of
the copulating females. No relation is found
between aging of the male and spotting.

From these data it is clear that the pheno-
typical manifestation of "brown spots" is strictly
dependent on copulation, irrespective of fer-
tilization and presence of spermatozoa in the
sperm fluid. The nature of the phenomenon
is not yet clear; recent experiments would
emphasize an activity of the sperm fluid:
mating with males lacking paragonial fluid owing
to repeated copulations, does not seem to cause
any spotting.

10.26. Gene-environment Interactions and the Pene-
trance of Melanotic Tumour Genes. James
H. Sang and B. Burnet (Edinburgh, Great
Britain).

The melanotic tumour phenotype of Droso-
phila melanogaster may be determined by any one
of a number of non-allelic, second chromosome,
recessive genes. Penetrance of some of these genes
depends on modifiers on other chromosomes,
usually the third, and also on the environment,
particularly the nutritional balance provided
to the larvae. The question examined in this
report is whether the same environmental mani-
pulations affect tumour penetrance in non-
allelic tu strains of different origin and, if they
do, what this implies with respect to gene action.

Two inbred strains were examined, each of
which showed a low incidence of tumours when
cultured on live yeast or on complete, chemically
defined media. One strain (tuk) carried a re-
cessive of very low penetrance on the second
chromosome (and probably an enhancer —
e-tu* — on chromosome three); the other also
carried a second chromosome tu gene but this
was hypostatic to a third chromosome recessive

177

Section 10 — Developmental Genetics

suppressor (su-tu). This second tu gene was
fully penetrant and not an allele of the first.
Despite these genetic differences, both strains
responded by an increase of tumour penetrance
to (a) a sub-optimal balance of dietary pentose
nucleotides, (b) cholesterol deficiency and (c)
excess 1-tryptophan. Their Fi responded simi-
larly to these abnormal conditions. Only the
homozygous tu; su-tu strain reacted with a
high tumour frequency to X-irradiation of
embryos, suggesting that X-rays affect only the
su-tu system.

10.27. Genes, Amino and Nucleic Acids, Progressive
Tissues and Cancer. Investigations on Poeciliid
Fish, Nicotiana, Drosophila and Vitis. F.

Anders, A. Anders, F. Drawert and E.
Steitz (Geilweilerhof/Pfalz, Germany).

Platypoecilus maculatus has a gene causing
black spots on its body-surface (Gordon,
Kosswig). These spots represent a "progressive
tissue" (Anders et al., Experientia 1963, in
press), because they enlarge in a positive allo-
metric manner, while the number of their con-
stituents, macromelanophores, increases. P.
maculatus has a low level of free amino acids. In
contrast, Xiphophorus helleri lacking this gene
has a high one. Certain hybrids of these species
show a high amino acid level, and, when car-
rying the spot-gene from P. maculatus, exhibit
an abnormal increase in the number of macro-
melanophores thereby producing melanomas.
Whether or not tumour formation takes place
seems to depend on presence or absence of
macromelanophores and on amino acid level.
Hybrids of 8 other poeciliid species and sub-
species have shown the same dependence.

In certain hybrids of Nicotiana producing ge-
neticaltumours, a similar correlation is found.
On the basis of this analogy, N. glauca for instance
may be compared to P. maculatus and N. langs-
dorffii to X. helleri. In Drosophila melanogaster a
correlation between amino acid level and tu-
mour formation exists too. In Vitis it was found
that injection of certain amino acids causes
neoplasms identical with galls of Phylloxera,
when meristematic ( = progressive) tissues are
present.

Further investigations have shown that high
concentrations of free amino acids may accelerate
biosynthesis of RNA and that autonomous growth
of tumour may be caused by a high level of
RNA. A simple genetic concept for cancer for-
mation will be discussed.

10.28. Induced Mutations in Barley causing Trans-
formation of Lodicules. O. P. Kamra (Halifax,
Canada).

During induced mutation experiments in
barley, using X-rays, Y-rays, neutrons and
chemicals, several single gene mutants with al-
tered sex organs were isolated. In these mutants
changes in the structure and function of the
lodicules were observed.

In the mutants 1 to 4 lodicules are produced.
The normal barley spikelet has two lodicules.
The extent of transformation varied greatly from
a slight modification in shape and structure to
complete alteration in form and function. The
two lodicules may fuse to form one highly vas-
cular lodicule or may be transformed into func-
tional pistils or stamens.

The lodicules are considered to have evolved
from the petals. From detailed histological and
cytogenetical investigation of these mutants a bet-
ter understanding of gene action in differentia-
tion of sex organs in higher plants, and of some
aspects of the evolution of these organs may be
expected.

10.29. Flowerstriping in Cyclamen persicum cv.
"Harlekin". Friedrich Bergann (Pots-
dam-Babelsberg, Germany).

Self-pollinated individuals of the striped-
flowering Cyclamen persicum cv. "'Harlekin" do
not breed true. Only a certain part of the off-
spring is striped-flowering, the rest self-coloured
red or pink. The numerical proportionsvary. If
a "Harlekin"-bulb is decapitated, the lower
part of it will survive and produce numerous
adventitious buds from the wound. In the follow-
ing year most decapitated plants are flowering
unicolored, generally with red petioles, more
seldom with pink petioles. Occasionally red,
pink and striped flowers are found in coexist-
ence on the same plant. It may be concluded,
therefore, that flowerstriping in "Harlekin"
is not due to a gene but to chimerical constitu-
tion.

If we suppose the shoot-apices of "Harlekin"
to have periclinal constitution (pink over red, or
red over pink), flowerstriping can be interpreted
as originating by tissue-rearrangements, namely
perforations in L 1, or partial replacements of
L 1 by L 2.

Considering the evidence on "Harlekin's"
chimerical nature, somatic segregations and tis-
sue-rearrangements must be concluded to have
taken place in the early ontogenesis and to have
consequently produced mosaicism within the

178

Section 10 — Developmental Genetics

floral tissues, not only in petioles but even in
the archespors of carpels and stamina. In a
haphazard manner self-pollination therefore
may render possible "intra-individual crossings"
and may produce de novo a varying number of
"ever-segregating" and "ever-rearranging" in-
dividuals, in the same way as cv. "Harlekin"
is reported to have originated by crossing pink x
red. The investigations are being continued.

10.30. Spontaneous Somatic Mutability in the
Tomato. W. Williams (Newcastle upon Tyne,
Great Britain).

A case of spontaneous somatic mutability
in the tomato causing leaf variegation will be
described. The condition occurs in a large num-
ber of genotypes but is confined to certain stages
of plant development. The histological basis of
variegation is a failure of normal development
in the palisade parenchyma. In addition there
is failure of differentation of the sporogenous
tissue in the immediate pre-meiotic stage which
results in complete male and female sterility.
By a process of elimination the condition may
be interpreted on the basis of a highly mutable
controller locus acting at a specific stage in
development.

10.31. Canalization of Flower Morphogenesis in
Wheat. O. H. Frankel and Anne Munday
(Canberra, Australia).

In common wheat (Triticum vulgare) normal
flower morphogenesis is an invariate character.
It is controlled by a factor associated with Q,
which, in turn, controls the "vulgare syndrome".
In speltoid mutants, where Q is deleted or in-
activated, we have obtained, as previously
reported, a range of stable genotypes from a
virtual absence of flowers to perfectly normal
flower morphogenesis. In certain compactoids
this extends in the supernormal direction, with
extra flowers in the empty glumes.

Flower morphogenesis in vulgare and in the
speltoids differs genetically and physiologically.
In vulgare flower morphogenesis is conditioned
by a single gene; in speltoids it is conditioned
by a complex multifactorial system. In vulgare
the flower formation is uniformly normal in
all experimental environments applied, includ-
ing drastic heat and radiation shocks; in the
speltoid series, frequencies of flower formation
are strongly affected by suitable combinations
of daylength and temperature applied during

the critical period of flower formation.

Thus, in vulgare flower morphogenesis is
buffered genetically against mutation and re-
combination among a multitude of polygenes,
and physiologically against environmental
shocks. There are striking analogies between
canalization systems uncovered in Drosophila
(Waddington, Rendel), mice (Fraser) and wheat.

It can be readily understood that there can be
no more than one canalization factor even in a
hexaploid; and it is not surprising that the
typical polyploid series have been found in
genes conditioning relatively superficial func-
tions. Canalization systems are opportunistic;
their transformation in polyploids may shed
light on the evolutionary changes in gene func-
tion itself.

10.32. Inheritance and Morphogenesis of Capsule
Spines in Ricinus communis. Hava Stein
(Rehovoth, Israel).

At the S (spiny capsule) locus, there are 4
alleles: S — spiny capsule, sr — very few spines
present, s, sf — entirely spineless, similar to each
other when homozygous hence considered isoal-
leles. S is partially dominant: 5 sr, S s, S sf
plant bear spines, but their number is much
lower than in S S. Number of spines decreases
from Ssf to Ssf: S 5> Ss?> S s> Ssf In Ss
and S-s+ plants, spine number varies in a predicta-
ble manner within each plant. Number of spines
on pods of the first raceme is always much
lower than in later racemes. Within each raceme,
spine number increases gradually from the
first capsule to later ones. This indicated an
inverse relation between spine number and
growth rate. Growth inhibition induced by
continuous removal of leaves resulted in a highly
significant increase in spine number, as expected.
The control of spine formation on the capsule
by a treatment applied to other plant organs
may well be mediated by mobile substances.

A spine is a multicellular cylinder crowned by
a single extremely elongated cell. Appearance of
this cell always precedes, and seems obligatory
to, the development of the multicellular organ.
Determination of spine formation is tentatively
assumed to consist of two steps: Excessive elon-
gation of random epidermal cells on the capsule
(perhaps controlled by the above-mentioned
substances) and stimulation of cell division in
the underlying tissues by the elongated cell
(through stress or similar forces). This model
affords an interpretation of the entire process
of spine morphogenesis through simple events
on the cellular level.

179

Section 10 — Development Genetics

10.33. The Developmental Genetics of Leaf Size in
Lolium. J. P. Cooper (Aberystwyth, Great
Britain).

The developmental genetics of leaf size in
Lolium has been studied in terms of the rates of
leaf initiation, leaf appearance, and expansion
of the leaf surface at the following levels: (i)
differences between populations, (ii) differences
between high and low selection lines, and (iii)
the effects of major mutants.

Population differences in leaf size in L.
multiflorum and L. perenne are based largely on
variation in cell number rather than in cell size,
but show marked interactions with temperature,
usually related to climatic origin. At 20°C
most populations of /.. perenne are similar in
leaf size, but at 5° the Mediterranean material,
with a winter growing season, shows considerably
greater rates of expansion of the leaf surface
than do the winter-dormant north European
populations. These differences are exhibited
mainly in the size of the individual leaves; there
is little variation in rate of leaf appearance.

Selection for high and low leaf size has been
highly effective within populations of L. multi-
florum and L. perenne, with realized heritabilities
over four generations of 30-40 per cent. A strong
negative genetic correlation with rate of leaf
appearance has appeared, although the rate of
initiation remains unchanged. Response to se-
lection has operated mainly through changes in
cell number rather than in cell size.

A number of major genes influencing leaf
development have been isolated, including
dwarf (d), branched culm (b), and shaving brush
(sh), which affect leaf size and rate of leaf ap-
pearance differentially. These single gene dif-
ferences are now being used to elucidate the
biochemical processes involved in leaf develop-
ment.

10.34. Hybrid Growth-Evidence of Differential
Physiological Development in Heterotic and
Non-heterotic Maize Seedlings. Igor V. Sar-
kissian (Morgantown, U.S.A.).

Earlier experiments^1) have led to an hypo-
thesis that stimulated photosynthetic carboxyla-
tion in young hybrid seedlings of barley and
maize is associated with heterosis in plants.
Additional evidence supporting this hypothesis is
presented from studies of photosynthetic C1402
fixation by heterotic and non-heterotic hybrid
maize seedlings. The patterns of distribution of
C14 to the intermediary compounds were studied
by paper chromatography and autoradiography.

Differential shifts in the distribution of C14 were
observed in the case of the hybrids, suggesting
systems undergoing altered patterns of physiolo-
gical development. Determinations and measure-
ments of the respiration patterns of young seed-
lings also supported this conclusion.

The significance of enhanced photosynthetic
CO2 fixation and differential physiological
development and their relationship to heterotic
growth will be discussed.

1. Sarkissian and Huffaker, Proc. Nat. Acad.
Sci (U.S.A.) 48, 735, 1962; and in press.

10.35. The Number of Chloroplasts in Different
Cells of Trisomic Sugar Beets. T. Butterfass

( Ladenburg, Germany).

It is shown that certain chromosomes (I, II,
VIII) of Beta vulgaris in the trisomic state in-
crease the number of chloroplasts in spongy
parenchyma cells by up to 60 per cent, compared
with eudiploids, but leave the numbers in guard
cells of stomata unchanged. Other chromosomes
(especially III, V, VI) increase the number in
the guard cells by up to 30 per cent, but not in
the spongy parenchyma cells. The augmentations
brought about by chromosomes II (50 per cent)
and VIII (60 per cent) turn out to be nearly
additive in double trisomies of certain combina-
tions, while in others they are not, thus showing
that some system of restriction exists which is
responsible for the reduced augmentation of
only about 45 per cent from diploid to triploid
level compared with 130 per cent by the summed
up effects of the nine chromosomes of the set.
The mean numbers of chloroplasts are indepen-
dent of cell size as well as of chloroplast size.
In adult epidermal and spongy parenchyma cells
the numbers mostly show a bimodal frequency
distribution. Endomitotic polyploidy, lacking in
guard cells, is considered one of the causes for
the existence of a second peak. The usefulness
of the plastid number as a character in differen-
tiation studies is emphasized.

10.36. Influence of the D-locus on Phenylalanine
Metabolism in Mice. Harold Rauch and
Martha T. Yost (Amherst, U.S.A.).

Using an inbred strain of mice segregating
alleles D (full pigmentation) and d1 (dilute-
lethal), we have followed the development from
birth through weaning of two liver enzymes

180

Section 10 — Developmental Genetics

involved in phenylalanine metabolism. In DD
and Ddl the activity of phenylalanine alpha-
ketoglutarate transaminase rises rapidly after
birth to a peak at 14 days and then falls off
about 50 per cent during the third week to the
adult level; the activity in dxdl increases similarly
only until 7 days at which low level it remains
until toward the end of the third week when
it falls slightly to the normal adult level. The
activity of phenylalanine hydroxylase increases
at the same rate in all three genotypes until
17 days; following this dhi1 remains the same
while DD and Dd1 continue to increase reaching
the high adult levels at about four weeks. The
low hydroxylase activity in dilute-lethals is due
to the presence of an inhibitor rather than to a
failure in enzyme formation. The inhibitor,
associated with the microsome fraction in brain,
kidney, heart, etc., as well as liver can be solu-
bilized with deoxycholate. Evidence suggests the
inhibitor may be an enzyme. Reduced transa-
minase and hydroxylase activities can account
for significantly higher serum phenylalanine
levels in dilute-lethals (e.g. dldl ten times DD
at 14 days). Excess circulating phenylalanine
may be associated with the myelin degeneration
observed in brains of dilute-lethals.

Supported by U.S. Public Health Service
Grant GM 05921.

10.37. Effect of Somatotropin^ the Weight Gain of
Yellow Dwarf (A* a dw dw) Mice. George L.
Wolff and Barbara Resnick (Philadelphia,
U.S.A.).

Possible involvement of somatotropin (STH)
in the increased fat deposition by yellow (/4>~)
mice was investigated with yellow dwarf (Ay a dw
dw) and non-yellow dwarf (aa dw dw) mice
known to be deficient in STH. Ay a dwdw and
aa dwdw mice of both sexes were injected with
0.5 USP unit bovine STH daily for 29 days be-
ginning at about 8 weeks of age. Weekly weights
were recorded for injected and control mice for
over six months. During the treatment period,
weight increase was about the same for both
genotypes. After injections were stopped, the
Ay a dwdw mice continued gaining weight while
the aa dwdw mice stopped gaining weight im-
mediately. By 9 months of age the A? a dwdw
males had reached a mean plateau weight of
about 50 g and females about 45 g. These are
similar to plateau weights of normal yellow
(Ay a Dw) mice. — Results from current ex-

periments with 0.05 USP unit Bovine STH
indicate that weight gain of yellow and non-
yellow dwarf mice is increased to the same
degree during the injection period and is propor-
tional to the STH dosage. Apparently the per-
centage increase in weight of yellow as compared
with non-yellow mice is independent of the
STH level in the body. Since excess weight gain
of normal yellow (Ay a Dw~) mice does not
usually begin until after about 8 weeks of age,
a metabolic process which depends on a cer-
tain maturation of the animal may be involved.
Administration of STH to Aya dwdw mice
may merely allow maturation to proceed to the
stage at which excess fat deposition begins.

Supported in part by USPHS Grants RG-6275
and GM 10112-01 and American Cancer So-
ciety Grant IN-49.

10.38. The Effect of Hormonally induced Changes
During Development and During Adult Life of
Drosophila. K. C. Sondhi (Baltimore, U.S.A.).

The present experiments describe the effect
of ring gland transplantations in Drosophila
melanogaster. The experiments were performed
on Samarkand inbred line, which was inbred for
over 200 generations and was structurally homo-
zygous for all the chromosomes. Three mature
ring glands were transplanted into the body
cavities of host larvae. Two synchronous control
populations were maintained, one consisting of
individuals which were allowed to develop nor-
mally and the other of individuals in which only
Ringer's solution was injected. The times of
emergence of flies were recorded. To minimize
the nutritional differences affecting body weight,
the flies were weighed on a torsion balance im-
mediately after eclosion.

The results of these experiments showed that
the transplantation of extra ring glands increases
the mean body weight of Drosophila over 35
per cent compared to controls. The response of
females to hormone secreting ring glands was
found to be much greater than in males. The
effect of altering the hormonal balance on re-
productive ability and on the adult life-span is
under investigation.

10.39. Growth Equations and Late Moiphogenesis of
Quantitative Characters. G. Fabian, J. Ern-
haft and M. Varga (Godollo, Hungary).

The basic growth equations were used pre-

181

Section 10 — Developmental Genetics

viously in the phaen analysis of some quantitative
characters by Brody and others. This method,
naturally with its limitations, still has many
really useful aspects and so the authors, analys-
ing during some years different species, races,
hybrid forms, back cross generations, are in-
clined to think that this effort had given some
results in the "kinetic" point of view of the
epigenesis of quantitative characters.

In any case one must take into consideration
that the so-called "allometrical ground plan"
in Needham's sense shows little if any change in
respect of the different quantitative varieties.
This fact precludes not the possibility of the
different manifestation of the adult quantitative
characters. In realizing the final state the quan-
titative characters are developing in many ways:
different startpoints, different velocity of growth
in the subsequent development phases, accelera-
tions and compensations. It is not difficult to
perceive sometimes the "transit-heterosis" state.
This has practical importance.

The differences of reciprocal hybrids during the
postembryonal development are also the results
of kinetic changes and describing them in this
sense is much more accessible than in the end state.

From the linear transformations of the growth
curves one can conclude about the degree of the
homoestasis by means of the deviations from
linear regressions for each separate genotype
and the so-called intraspecific allometry is

available in the phaen-analysis of quantitative
characters.

10.40. Contribution to the Teratogenic Effect of
Thalidomide in Golden Hamsters and Mice.

H. Stengel, K. H. Degenhardt, and G.
Badtke. (Frankfurt/Main, Germany).

Gross analysis of a control group of 124
newly-born golden hamsters has not shown any
malformations. However, after processing the
new-born animals for skeletal study (alizarin,
glycerin) the examination disclosed several
malformations: 1 x a slight reduction of the
maxilla, 2 / a bent radius and ulna on both
sides (right > left), and 19 < defects of the
centra of the vertebrae, especially in the cervical
region. For the treatment, thalidomide was
suspended in 1 per cent carboxymethylcellulose.
Daily doses of 10 mg or 100 mg thalidomide/
lOOg body weight were administered female
golden hamsters by oral intubation for a period
of eight months. Gross analysis of 1 19 offspring
in the 10 mg series showed no malformations.
The skeletal system, however, revealed various
malformations: 2x a slight reduction of the

maxilla and 15 x defects of the centra of the
vertebrae, especially in the cervical region. Fur-
thermore, in more than 50 per cent of the ani-
mals microphthalmia of various degrees occur-
red, and in some cases other eye abnormalities.
Of the group treated with 100 mg none of 127
new-borns was found to have external malfor-
mations. Again a number of skeletal malforma-
tions were found: 1 X a reduction of the
mandibula, 30 x defects of the centra of the
vertrebrae, and 4 x chondrodysplastic changes
in the long bones of the upper limbs (right >
left). The eyes are being examined histologically.
Analogous experiments have also been done
with mice of the inbred strain C57 BL/6 Han-
nover. In the control group of 149, 16 abnormal-
ities of the vertebrae have been found. Two ani-
mals had an extreme microphthalmia. Following
treatment with 10 mg, 117 new-borns could be
analysed. 21 showed abnormalities of the verte-
bral column, 5 defects of the jaws, 2 cleft palate,
3 extreme microphthalmia and 1 a kidney dys-
plasia on the right. Of the 118 animals from
25 litters treated with 100 mg thalidomide
100 g b.w. 109 skeletal systems have been analy-
sed, 10 x abnormalities of the vertebral column
occurred, 5 defects of the jaws, 7 x micro-
phthalmia, 2 x cleft palate and 1 x aplasia of
the abdominal organs. Histological investigations
are being carried out.

10.41. An Experimental Contribution to the Determi-
nation of Origins of Complex Abnormalities of
the Anterior Chamber of the Eye. K. H. Degen-
hardt and G. Badtke. (Frankfurt a/M,
Germany).

Complex abnormalities of the anterior part of
the eye known in human beings as "Peter's
malformation of the cornea, congenital leucoma
and staphyloma of the cornea", differ chiefly in
the various strengths of the single symptoms.
Features in common are opacity of the cornea
and adhesion between the opaque area of the
cornea and the iris or the persistent pupillary
membrane. Changes in the structure of the lens
consist of different degrees of cataract, leading
up to aphakia. Microphthalmia and coloboma
are also observed. The pathogenesis remains as
yet unclear. Peters thought that disturbance due
to the separation of the lens from the ectoderm
was the cause; but up to now it has not been
possible to prove this using embryological mater-
ials. The alternative theory that the cause is dis-
turbance by the development of the mesenchyma
has also not yet been proved.

Distinct disturbances due to the separation of

182

Section 10 — Developmental Genetics

the lens from the ectoderm have been observed
in the experimental investigations on the eyes
of new-born rabbits and mice by one of the
authors (K. H. Degenhardt) using the techniques
of oxygen deficiency and X-irradiation damage at
particular stages of embryonic development.

In these cases ruptures of the cornea, anterior
synechia, aplasia of the Decemet membrane
in the region of synechia, disintegration of the
fibres of the lens, aphakia, coloboma, microph-
thalmia and other known derangements were
observed. These malformations of the cornea,
iris and lens are to be considered as the beginning
of a teratological series of which the above
mentioned derangements of the human eye are
the most severe. If the animals had been allowed
to live longer secondary reparative changes
would have caused a scarred closure of the
rupture of the cornea and special changes of the
iris and the lens, as have been observed in
human eyes. This hypothesis is supported by the
condition of a rabbit eye, in which the rupture
of the cornea was already scarred. In human
beings the long duration of intrauterine develop-
ment prevents the observation of the early
stages of this special categoiy of eye defects.

We think disturbances of inductive relation-
ships between the optic vesicle and the ectoderm,
initiated experimentally by oxygen deficiency
or by X-irradiation, are responsible.

10.42. Response to a Teratogen of Mice Heterozygous
for Anophthalmia. Sidney L. Beck (Ann
Arbor, U.S.A.).

Normal-eyed female ZRDCT-N (>) and C57BL/
10 mice were mated with their own strain or
Anophthalmic (ZRDCT-An) males. Pregnant
mice were injected on the 7th, 8th and 9th days
post coituin with 0.25 cc. of 0.3 per cent trypan
blue (2), sacrificed during the 18th day of gestation
and examined for total implantation sites. Living
fetuses were examined for eye defects. Unilateral
and bilateral anophthalmia and various degrees
of microphthalmia on one or both sides were
found.

The ZRDCT-N x ZRDCT-An matings
produced many more defective progeny than the
ZRDCT-N x ZRDCT-N (P < 0.001); mortality
was higher among the pure line matings (P <
0.001). These findings suggest that the presence
of the anophthalmic mutant (ey) in the heter-
ozygous state brings an animal close to a
threshold for defective eye development in a
cross of two closely related lines.

Crosses of C57BL/10 x HL-ZRDCT-An
responded with a lower frequency of eye defects

(not significant) and mortality (P < 0.001) than
C57BL/10 x C57BL/10. Here the most obvious
phenomenon among the hybrids was heterosis,
suggesting that differences in polygenic modifiers
between the two lines were stronger than, or
could mask the deleterious effect of, the single
dose of ey.

In all cases the C57BL/10 crosses were affected
less than the corresponding ZRDCT-N crosses;
among all but C57BL/10 C57BL/10 there
were no spontaneously occurring eye defects.
In this latter group the teratogen doubled the
spontaneous frequency. In all crosses spon-
taneous fetal mortality was far lower (P < 0.001)
than mortality in stressed litters.

1. Beck, Am. Zool. 1, 436, 1961.

2. See Barber, Am. J. Ophtalmol. 44, 94, 1957

10.43. The Effect of Cortisone on the Penetrance of
an Eye Mutant in the House Mouse. Muriel
J. Watney and James R. Miller (Vancouver,
Canada).

The mutation elo which has recently occurred
in the C3H/MI mouse strain results in the
condition "eye-lids open at birth" (the mouse is
normally born with its eye-lids fused). This
mutant appears to be a simple autosomal
recessive with about 65 per cent penetrance in
homozygotes. In an attempt to increase this
penetrance, cortisone was administered to
pregnant females for several days prior to the
closure of the lids in utero which occurs during
the 17th day. Rather than increase the pene-
trance, the cortisone treatment resulted in a
significant decrease (to 4 per cent) in the frequen-
cy of the defect. On the basis of investigations
on the effect of cortisone on palate closure in the
mouse \ it is suggested that the cortisone
modifies the action of the elo gene through
mucopolysaccharides.

1. Walker and Fraser, J. Embryol. Exp.
Morph. 5, 201, 1957.

10.44. A New Gene Causing Cerebral Degeneration
in the Mouse. M. S. Deol and Gillian
M. Truslove (London, Great Britain).

A new recessive gene (symbol cb) causing cere-
bral degeneration in the mouse has been dis-

183

Section 10 — Developmental Genetics

covered. The degeneration produces an ex
vacuo hydrocephalus which is clearly visible in
the living animal. The time of onset of the
anomalies varies, but classification is usually
possible at birth. The most susceptible parts of
the brain are the cerebral hemispheres and the
olfactory lobes. In later stages the epithelium of
the nose and the trachea also degenerates. The
involvement of the skull is believed to be second-
ary. The homozygotes generally die before
reaching maturity, but if they live on they are
always sterile.

tically reduced. Those seen are small and pyk-
notic. In adjacent areas where myelin is intact
the oligodendrocytes are normal in size and
shape and are lined up in rows along the nerve
fibers. An astrocytic reaction is not apparent
although some cells with two or more nuclei were
seen. Microgliacytes are plentiful. Perivascular
cuffing by macrophages is not a prominent
feature, but is occasionally present.

Supported by U.S. Public Health Service
Grant GM 05921-05.

10.45. (D.) Hereditary Cerebral Degeneration in the
Mouse. Gillian M. Truslove and M. S. Deol
(London, Great Britain).

A demonstration showing the extent of the
fibre degeneration in the cerebral hemispheres
caused by a new recessive gene {cerebral degener-
ation, symbol cb) in the mouse. A series of
drawings based on sections through normal and
cb/cb brains with degeneration and hydrocepha-
lus. Photographs to show the distribution of the
cells in the nasal epithelium in different regions
of the nose. High power photographs showing
the normal and abnormal mucous membrane
and also differences in the bone structure of the
cranium.

10.46. Cellular Changes in the Central Nervous
System of Dilute-Lethal Mice. Diane E. Kel-
ton (Amherst, U.S.A.).

Previous study of the effect of the dilute-lethal
gene (d1) on the central nervous system showed
that the sequence and degree of myelinization
did not differ among DD, Ddl and dldl mice from
4 to 25 days of age. Death of dxdl mice occurs at
18 to 20 days of age. The Marchi method showed
increasing amounts of degenerating myelin in
the vestibulospinal, spinocerebellar and tecto-
spinal systems of dldl mice with increasing age.
The degenerating myelin appeared within a day
or two of the onset of myelinization. Current
studies indicate that although myelin disinte-
grates partially or completely in scattered foci
there is little or no destruction of axis cylinders,
nor is an axonal reaction of nerve cells observed.
Little cellular change is seen at 10 days of age.
By 20 days of age in the areas where the myelin
sheaths have disappeared the number of identi-
fiable interfascicular oligodendrocytes is dras-

10.47. A Developmental Analysis of Strong's Luxoid
Mutant. Paulinus F. Forsthoefel (Detroit,
U.S.A.).

Deficient growth of the facial processes and
anterior components of the chondrocranium
first detected at 12 days of gestation restricts the
forward growth of the brain resulting in excessive
upward expansion of the telencephalon and
mesencephalon. The pressure exerted by the
upward pushing brain inhibits hair follicle
development and ossification of the frontal and
parietal bones over the dorsal part of the brain.
Tension exerted on the tissues about the eyes by
the outward-pushing mesencephalon results in
defective formation of the eyelids. Excessive
growth of the preaxial margins of the footplates
can be detected at 11 \ days in the forelimb and
at 12 days in the hind limb. The pattern of
blastematal condensations in the limbs and
pelvis combines excesses with deficiencies,
resulting in Polydactyly, radial reduction and
duplication, reductions of the pubis and tibia.
Deficient skeletal elements lag in chondrification
and ossification and show weak alkaline phos-
phatase and ascorbic acid activity. The posterior
belly wall and the anterior half of the genital
phallus show deficient growth. Epidermis never
covers the belly between the phallus and the root
of the umbilical cord. Hair follicles (guard, awl,
auchene, zigzag) form at the normal times on
the dorsum, but in early stages appear to be
retarded. The failure of hair to persist on the
dorsum for several weeks after birth is probably
due to a weakness in their structure. The primary
effect of the Strong luxoid mutation is on
growth in localized body regions.

Research supported by U.S. Public Health
Service Grant C-3613.

184

Section 10— Developmental Genetics

10.48. Lethal Micromelia in the White Pekin Duck.

William J. Ash (Cornell, U.S.A.).

Routine inspection of unhatched pedigreed
eggs uncovered a lethal syndrome in the White
Pekin duck. Affected embryos appear smaller
than their normal sibs. Most conspicuous is the
marked shortening of the extremities. Shortening
of the upper mandible is observed in some but
not all birds. Feathering is greatly retarded, and
many embryos appear naked. Feathers which
develop often take on a "wormy" appearance.
Subcutaneous edema is conspicuous in the
cervical region.

Affected embryos survive to terminal stages of
incubation. Some pip their shells, but none has
ever hatched.

Histological examination of the long bones
reveals irregular cartilagineous architecture at the
epiphyses. The orderly transformation of young
discoidal chondrocytes into mature ovoid ones is
absent. Ossification does occur, but it is limited
presumably by the aberrant cartilagineous model
which precedes it.

Matings of phenotypically normal carriers indic-
ate that this syndrome is an autosomal recessive
obligate lethal mutation.

cribed by Eker in 1954 (x) and a preliminary
report on the genetic aspect has been published!2).

10.49. Dominant Renal Adenomas in the Rat.

Jeanne Mossige and Reidar Eker (Monte-
bello, Norway).

The renal adenomas which occurred in an
inbred strain of Wistar rats at this laboratory
have proved to be due to a dominant gene with
complete penetrance. No homozygote has been
found.

The earliest macroscopically detectible tumors
have been found at about 3 months, but they
may appear first at the age of one year or later.
Their development has been followed from
early stages in serial sections. They begin as
small, cystic tubules and develop into cystic,
cysto-papillary or solid tumors and are almost
always bilateral. Most of them are well limited
without cellular atypia, but many exhibit
atypical nuclei and invasive growth in the kidney.
Only a few, however, have metastasized, to the
liver and lungs, proving malignancy. In humans
they would be diagnosed as renal carcinoma.

They develop earlier in males than in females
and usually appear in the left kidney earlier than
in the right. Various experimental series are
being run to test factors which might influence
the development of the tumors.

The pathology of the tumors has been des-

1. Acta Path, et Microbiol. Scand. 34, 554.

2. Eker and Mossige, Nature 189, 858, 1961.

10.50. Periodical Hypotrichosis in Mice. N. Kobo-
zieff and N. A. Pomriaskinsky-Kobozieff
(Maisons Alfort, France)

Periodical hypotrichosis in mice is a recessive
mutation with incomplete penetrance. The
phenotypical manifestation takes place between
the 16th and 21st day after birth and may
reappear at more or less frequent intervals.

The genetical analysis of the normal descen-
dants of two abnormal parents (penetrance
30 per cent), shows that they behave genetically
as abnormal animals.

When crossed to abnormal, penetrance among
the offspring is lower (24 per cent), when crossed
inter se it is only 20 per cent, and in the F-2 it
drops to 10 per cent

The phenotype is variable, there are 6 classes :

— little loss of hairs, hair sparse on the whole
or part of the body (Type I),

— loss of hair in the scapular region (Type II),

— loss of hair on the whole thoracic region
(Type III),

- loss of hair as far as the sacral region, hairs
remain only on the head and the base of the
tail (Type V),

— loss of hair on the thighs and later than
normally (Type VI).

The gestation factor is important : the highest
frequency of abnormal is seen in the first litter.
It is lower in the second and inexistent in the
others litters.

The seasonal factor is also interfering: during
the whole year we observe litters comprising
normal and abnormal subjects but it has been
noticed that 54 per cent of the litters containing
abnormal mice appear in July- August; whereas
in the same period we have found 28 per cent of
the litters to consist exclusively of normals.
This shows clearly that the highest frequency of
litters with abnormal subjects is observed in
July-August.

10.51. Response of Polycythemic WWV Anemic Mice
to Erythropoietin. Margaret W. Thompson,
Elizabeth S. Russell, and Eleanor C. Mc-
Farland (Edmonton, Canada).

The hematopoietic defect in WWV anemic mice

185

Section 10 — Developmental Genetics

has been studied by comparison of the response
of WWV and normal ww mice to erythropoietic
stimulation, as measured by Fe59 incorporation
in erythrocytes. Adult WWV mice and normal
littermate controls were plethorized by trans-
fusion with C57BL/6 erythrocytes to hematocrit
levels of 60 per cent or higher, in order to
repress erythropoiesis and inhibit formation of
endogenous erythropoietin. Purified exogenous
erythropoietin, made available through the
kindness of Drs. G. Keighley and P. H. Lowy of
the California Institute of Technology, was then
administered to half the animals of each geno-
type. The response of animals of the two geno-
types to this stimulus was compared by measure-
ment of Fe59 incorporation in erythrocytes 24
hours and 72 hours after Fe59 injection. Pleth-
orized animals of both genotypes, without
erythropoietin, exhibited very low levels of Fe59
uptake. Erythropoietin increased the rate of
incorporation of Fe59 in mice of both genotypes,
but the increase in rate was much greater in the
normal than in the WWX mice. It is apparent that
WWV mice are not incapable of responding to
erythropoietic stimulation, as previous investi-
gations had suggested, but that they differ from
normal mice in degree of response. This new
finding implies that the ff-genes affect the rate
and end-point of the response to erythropoietin,
but do not inhibit its onset.

This investigation was supported by a Grant
(He-05748) from the National Heart Institute,
U.S. Public Health Service.

10.53. Effect of Folic Acid Inhibitor upon Morpho-
genesis of the Wings of vestigial Heter-
ozygotes in Drosophila. J. David (Lyon,
France).

When added to an axenic medium, aminop-
terin slows larval growth a little, but the adult
morphogenesis of the wild strains remains
normal. When vestigial (vg, 2rd chromosome
heterozygotes are reared with the above-
mentioned medium, the wings of the flies are not
normal and present some notches on the border.
This result appears to signify an imperfect
dominance of the wild type gene over its vestigial
allele and suggests that, in the heterozygous
condition, vg alters the folic acid metabolism in
the imaginal disc of the wing.

10.54. Variability of Sex Ratio and Sex-determining
Mechanism in Asellus. G. Montalenti and
G. Vitagliano Tadini (Rome, Italy).

10.52. Analysis of Gene Action and Characterization
of a New Hematological Abnormality, Hemoly-
tic Anemia. Seldon E. Bernstein (Bar
Harbor, U.S.A.).

This paper describes a new mutation in the
house mouse called hemolytic anemia (symbol
ha). Evidence is presented indicating that the
hemolytic condition results from the action of an
autosomal recessive gene at a locus as yet not
located in any of the 20 known linkage groups.
This blood dyscrasia is characterized as being a
severe neonatal hypochromic microcytic anemia,
resulting neither from a maternal-fetal incom-
patibility nor from the presence of an abnormal
hemoglobin. Erythroblastosis, jaundice, sple-
nomegaly, and cardiac hypertrophy are promi-
nent features. Homozygotes of hemolytic anemia
(liaha) resemble in nearly every detail jaundiced
homozygotes (jajd). Genetic evidence will be
presented, however, which will show that the
two conditions are caused by unrelated genes.
An analysis of the action of hemolytic anemia
genes in terms of a pedigree of causes will follow.

In Asellus aquaticus sex ratio shows a great
statistically significant variability between the
progeny of different pairs, the total ratio of the
population is approximately 1: 1. In the popu-
lation of the river Sarno (Naples) the percentage
of males in different sibships varies from 0 per
cent (absolute thelygeny) to 95 per cent (high
androgeny) with higher frequencies toward 50 per
cent ("normal" s.r.). The distribution is of the
gaussian type. Sex is genetically determined and
so is this variability; intersexuality or hermaphro-
ditism do not occur with appreciable frequency;
parthenogenesis does not occur. In previous
papers the authors have put forward, as a first
approximation, the hypothesis that sex in
Asellus is under control of a series of polygenes,
with the assumption of a clear-cut threshold
effect between maleness and femaleness. Further
experiments however failed to bring evidence in
favour of the loose recombination of a number of
genes. A modification of the former hypothesis
is proposed, which requires a limited number of
genes and assumes that they are more or less
closely linked. Three pairs of genes (or gene
blocks) would be involved, two (A, B) closely
linked, a third (Z) more loosely linked with the
former two. Two female genotypes would

186

Section 10 — Developmental Genetics

exist, with none or one dominants; males would
belong to three genotypes, with two, three or
four dominants. The variability of sex iatio and
the results of different crosses are thus explained
by a relatively simple oligogenic hypothesis,
without introduction of modifying genes,
cytoplasmic factors, etc., as has been implied by
other authors in other species. The selective
value of this situation versus a clear cut 1 : 1
mechanism is investigated and discussed.

10.55. (D) Genetic Analysis of the True Slime Mould,
Physarum polycephalum. Jennifer Dee (Lon-
don, Great Britain).

The true slime mould (Myxomycete) Physarum
polycephalum undergoes simple morphogenetic
changes during its life-cycle and passes through

a uninucleate phase and an acellular multinu-
cleate phase, either of which can be indefinitely
prolonged in culture. It should be a highly
suitable organism in which to combine genetical
and biochemical approaches to the problems of
morphogenesis. Biochemical investigations are
becoming advanced, but very little genetic
analysis has been done. The demonstration
illustrates an attempt to develop genetic analysis
of P. polycephalum.

Simple techniques are shown for culturing the
organism through its life-cycle in two-membered
culture. Stages of the life-cycle are illustrated by
living cultures or photographs. The uninucleate
phase ("amoebae") can be handled by the usual
techniques of microbial genetics including
plating, which is demonstrated. The mating-type
system, methods of isolation of genetic markers
and the results of a cross showing recombination
between genetic markers are also described.

187

SECTION 11

IMMUNOGENETICS

11.1. Am Immunochemical Study of the NADP linked
Glutamic Dehydrogenase and Mutant Forms of
the Protein in Neurospora crassa. D. B.

Roberts (Cambridge, Great Britain).

Sera containing antibodies against the normal
NADP-linked glutamic dehydrogenase of Neu-
rospora crassa, have been prepared in rabbits.
These sera have been used to study the immuno-
logical relationships between the normal and
mutant forms of the enzyme. The survey has
been carried out on double diffusion plates and
on immunoelectrophoretic plates. The line of the
enzyme-antibody complex in the gel has been
identified by a stain which acts specifically on
this complex.

A detailed comparison has been made be-
tween the wild type enzyme and the protein
from one of the mutants, am-3. The am-3 protein
shows activity if incubated with the substrate
glutamate. At a concentration of 33 mM of
substrate the am-3 activity is about one-third of
wild type activity and at a concentration of
133 mM is slightly greater than wild type activity.
At a concentration of 33 mM of substrate small
quantities of serum appear to enhance the activ-
ity of the am-3 protein, larger quantities inhibit
the activity slightly. At 133 mM concentration
of substrate the am-3 activity is greatly inhibited
if incubated with the serum before incubation
with the substrate. If the am-3 protein is incu-
bated with the substrate before the serum or
with both together, the serum has less of an
inhibitory effect. At both substrate concentra-
tions the serum inhibits wild type activity and
there is no substrate protection.

These results suggest a different relationship
between antibody-enzyme in wild type to that
in am-3, and also a difference in the enzyme
activity-antibody relationship of the am-3
protein at different substrate concentrations.

11.2. The Role of Cytoplasm and Nucleus in the
Determination of Serotype in Paramecium.

John R. Preer, Jr., Mary Bray, and Sa-
daaki Koizumi (Philadelphia, U.S.A.).

Sixteen years ago T. M. Sonneborn demon-

strated that in Paramecium the cytoplasm at
nuclear reorganization is important in deter-
mining the expression of genes for serotype.
Although it has been assumed that a system of
cytoplasmic inheritance is involved, an alter-
native possibility is that different cytoplasms
induce stable macronuclear states in the develop-
ing macronuclei. Dryl recently'1' showed that
serotypes change more readily at nuclear reor-
ganization than at vegetative reproduction. On
the hypothesis of macronuclear determination,
conditions inducing change in macronuclear
anlagen should not cause developing macronu-
clear fragments produced at macronuclear re-
generation to change so readily.

We have studied the frequency of change from
serotype B to A in macronuclear regeneration
induced in exconjugants of stock 51, Paramecium
aurelia. Five hours at 380 C followed by 31°
induced both macronuclear regeneration and
serotype change. Four lines were obtained
from each of several hundred exconjugants.
24 exconjugants produced both changing and
stable lines, as well as macronuclear regenerating
and normally reorganizing lines. In 22 of the
24 groups of four the macronuclear regenerates
were stable B, while 75 per cent (35\) of the nor-
mally reorganizing lines changed to A. These
results support the notion of nuclear control.
Possibly, however, secondary factors associated
with macronuclear regeneration favor serotype B
more than A, and the results, while suggesting
nuclear control, do not really prove it. Attempts
are being made to extend the study to the A to B
change which is induced by low temperature.

1. S. Dryl, /. Protozool. 6 suppl.), 25, 1959.

11.3. Blood Group Investigations of Two Marine
Animals. J. E. Cushing, D. Vann, D. Bo-
raker (Santa Barbara, U.S.A.).

Current investigations involving the blood
group antigens of two marine forms are reported.
The first concerns the use of Dolichos bifiorus
lectin in detecting an antigen in the California

189

Section 11 — Immunugenetics

bonito with a specificity related to human Ai.
The second concerns the exchange of blood
samples between individual sipunculids (Den-
drostomum zostericolurri) as a techniuue for the
investigation of potential immunological in-
compatibilities involving hemerythrocytes and
other hemocytes of this species.

11.4. Immunogenetic Relationships of Trout. James

E. Wright, Robert Sklenarik, and Suzanne
M. James (University Park, Pennsylvania,
U.S.A.).

Antisera were prepared in rabbits and chickens
against erythrocytes of individuals of four trout
species— brook (Salvelinus fontinalis), lake (Sal-
velinus namaycush), brown {Salmo trutta), and
rainbow (Salmo gairdneri). Agglutination tests
show cross reactions of any of these sera with
erythrocytes of any of the trout species. Compa-
rative studies of dilutions of each serum provided
evidence of extensive antigenic homology be-
tween the two Salvelinus species. Also, marked
similarities in the cellular antigens of rainbows
and the two Salvelinus species were found. In
contrast, brown trout apparently lacked practi-
cally all antigens common to any of the other
three species.

Absorptions of each serum by erythrocytes
from individuals of each species provided anti-
sera specific to each species; cross-match aggluti-
nation tests confirmed the antigenic relatedness
of brook, lake, and rainbow trout and the lack
of homology between brown trout and any of the
other three species, even rainbow trout, the other
Salmo species.

Although genetic blood group systems pre-
viously identified in brown and in rainbow trout
are species-specific, these results suggest that
blood groups may be identified that are common
to rainbow, brook and lake. Absorptions of
anti-brook trout sera with cells of individual
rainbows produced reagents that identify several
blood types in rainbows, but these reagents
failed to differentiate among brook trout. Ab-
sorptions of anti-lake or anti-brook sera have
produced reagents that identify a major blood
group system in lake trout; these reagents also
agglutinate erythrocytes of some individuals
among brook trout.

11.5. Sex-linked "Histocompatibility Reactions"
in the Salamander Triturus (diemictylus)
viridescens. Alexander Wolsky, C. Edward
Quinn, Jose Squadroni S.J. and Maria de
Issekutzi Wolsky (New York, U.S.A.).

In these salamanders female skin grafts are

better tolerated than male ones (Pizzarello and
Wolsky, Squadroni and Wolsky). This was
interpreted by assuming the existence of histo-
compatibility genes on the sex chromosomes
(although no sex chromosome dimorphism is
known in the species). Recent work, however,
indicates that the phenomenon may be due,
at least partly, to histological and histochemical
rather than immunological differences between
the sexes.

Male and female skin grafts behave differently
even on the same host (Squadroni and Wolsky).
Female grafts, when alone on a host, remain
histologically normal, whereas male grafts
quickly lose their structure; especially the break-
down of the epidermis and the skin glands is
characteristic (Pizzarello). A double graft
elicits typical lymphocyte accumulation around
both grafts but the female skin structures are not
attacked immediately and appear normal at a
time when the male graft has been resorbed
(Squadroni). Studies with the Ouchterlony tech-
nique, involving rabbit sera immunized against
Triturus skin homogenates, indicate no difference
in the antigenicity of the male and female
skin (Quinn).

The situation in the female Triturus skin seems
thus comparable to that found in the cheek
pouch skin of the golden hamster by Billingham
and co-workers, except that in the case of Tritu-
rus one must assume a double barrier in the
path of histocompatibility reactions (both to
and from the site of the antigenic stimulus)
whereas in the hamster only the latter seems to
exist.

11.6. Different Degree of Compatibility between
Several Stocks of Drosophila melanogaster,
tested with Ovary Grafting. Carla Halfer
(Milan, Italy).

Since it is generally known that grafting of
organs in Drosophila is successful, I have tried
to analyse the degree of compatibility between
genotypically different stocks of Drosophila
melanogaster, using the method of ovary grafting.
The compatibility between grafted ovary and
recipient has been measured as percentage of
flies where the grafted ovary was able to deliver
viable eggs, flies which allowed the ovary to
develop without becoming functional, and flies
which rejected the graft. As recipients, two dif-
ferent wild tumorous stocks have been used
(tu A2 and tu B3), one tumourless (Varese);
as donors: Chieti— vermilion, vermilion and
yellow white. The first data show a different

190

Section II — Immunogenetics

behaviour between recipients and implanted
ovaries in the different combinations. The more
significant differences have been obtained in
the survival frequencies; the highest percentage
found is 55.1 per cent in the grafts of Chieti —
v in tu A2, while the lowest percentage was of
1.0 per cent grafting yellow white in tu B3. As
regards both development and functionality,
the differences are less significant, even if there
is significance when the recipient stock is tu B3.
The grafts generally are more successful in
stock tu A-2 than in tu B3 and in Varese. Such a
different behaviour leads to consider the dif-
ferences between the two stocks as a result of
behaviour of lymph gland and the number of
cells swimming free in the haemolymph. In fact,
in tu A2 the number of free cells is high, very
low in tu B3 and in Varese.

to hemolytic disease — this problem is under
investigation.

11.7. Blood Groups of the Domestic Mink. Jan

Rapacz and Richard M. Shackelford
(Krakow, Poland, and Madison, U.S.A.).

Immunogenetic investigations in the domestic
mink (Mustela visori) started in 1961 by Rapacz
and Shackelford at the Fur Animal Research
Laboratory, University of Wisconsin, have
been reported in three papers. (^ At least seven
blood factors have been recognized by using
reagents from hetero- and iso-immune sera. All
reagents functioned as agglutinins and saline
agglutination tests were employed for dis-
covery of all antigenic factors except the anti-
globulin test for D specificity. Thirteen sera of
the 790 tested showed normal agglutinins;
three anti-A, six anti-B2, one unidentified and
three nonspecific auto-agglutinins (these three
mink, mother and sons, died).

A total of 1599 individuals were tested against
anti-A, B, B2 and 428 of them with anti-C
also; each mink reacted with one or more of
these reagents. Pedigree analysis of 668 off-
spring from 171 litters resulting from planned
matings indicate that these four specificities are
determined by triple alleles. Using the reagents
anti-A, B and C, six phenotypes corresponding
to the six possible genotypes were identified in
this system. Blood factors A, B and C corres-
pond with genes A*, A0 and Ac, respectively.
The B2 specificity is produced by gene Ab as
well as by gene Ac, and consistently accompanies
factors B and C in the mink; a single specificity
similar to B has been found in the Stone marten
(Martes foina). Reagents in the A system,
especially anti-B2, show clear dosage effects.
There is slight evidence that the mink is subject

1. Nature, 196, 4861; Immunogenetics Letter 3
(No. 1), 55-61 ; Nature (in press).

11.8. Full-thickness Skin Grafting in Pigs. L. N.

Baker and E. Andresen (Ames, U.S.A.).

Results of full-thickness skin grafting in pigs
have demonstrated the anticipated existence of a
histocompatibility mechanism. This grafting
technique has been introduced in connection
with blood-typing to detect animals which
are either monozygotic twins or erythrocyte
mozaics (natural chimeras). The dorsal surface
of the ear provides a suitable graft bed while
satisfactory grafts can be taken from the dorsal
or ventral side of the ear. A circular cutting tool
and a scalpel were used to prepare the graft bed
and to excise the graft. An adhesive patch held
the graft in place 4 to 6 days. Fifty-six pure-
bred pigs from outbred Duroc and Hampshire
stock were used. Forty of these were paired as
littermates of the same sex and same blood type
as detected with twenty blood-typing reagents.
Each paired pig received three grafts: an auto-
graft; a homograft from its pair-mate; and a
homograft from another littermate of the same
sex but slightly different blood type (not avail-
able to 4 pairs). All autografts were accepted.
Four of the homografts exchanged between lit-
termates of the same blood type were lost by ac-
cident; the remaining 36 were sloughed after
37.0 ± 2.5 days. Nine homografts taken from
littermates of a different blood type were lost
by accident; the remaining 23 were sloughed
after 32.7 ± 3.8 days. Although the difference
in rejection time is not statistically significant, it
suggests an association of blood type with rejec-
tion mechanism. Thus, the combined tech-
niques have proved satisfactory for routine
examinations.

This work has received assistance from Con-
tract AT(1 l-l)-707 from the U.S. Atomic
Energy Commission.

11.9. Gene Interaction and the A-O Blood-group
System in Pigs. Benjamin A. Rasmusen
(Urbana, U.S.A.).

Anti-pig-A antibodies are present in normal
sera of certain cattle, sheep, and pigs, and anti-

191

Seel ion 11 — Imnuuwgenetics

pig-O antibodies are present in normal sera of
certain cattle, goats, and pigs. Reagents pre-
pared from these sources were used in hemolytic
tests to type the red cells of 961 Duroc, Yorkshire,
and Duroc-Yorkshire crossbred pigs and their
sires and dams. Three phenotypes, A, O, and -
("dash", i.e. A-negative, O-negative), were
observed. Blood-typing results were in agree-
ment with the hypothesis that the allele for A is
dominant to that for O, and that the expression
of A and O phenotypes is prevented by a homo-
zygous recessive genotype at another locus,
resulting in — . Five matingsof — — gave 22 —
offspring; 33 matings of O > O gave 213 O and
3—; 23 matings of O -gave 18 A, 70 O and
56 — ; 27 matings of A . -gave 66 A, 29 O and
66—; 36 matings of A : O gave 147 A, 98 O
and 30 — ; and 23 matingsof A A gave 96 A,
21 O and 26 — . Red cells of some pigs reacted
only weakly with available A or O reagents, and
the normal serum of an occasional pig appeared
to react most often with red cells of — pigs,
suggesting additional complexities of A-O
genotypes in pigs.

This work was supported in part by a research
grant, GM-08752, from the National Institutes
of Health, Public Health Service.

11.10. Studies of Equine Blood Groups. Clyde
Stormont, Yoshiko Suzuki, and E. A. Rhode
(Davis, U.S.A.).

Studies on equine blood groups have been
underway in our laboratory for the past several
years. Although there have been no published
reports of the data, some of the progress may
be mentioned here. Sixteen specifically different
equine blood-typing reagents have been produ-
ced, and the blood factors corresponding to
these reagents have been named Ai, A2, B,
C, D, E, F, G, H, J, K, L, M, N, O and X.
Our main interest is that of determining the
genetic systems in which these reagents act. It
is found that reagents for blood factors Ai,
A2, B and C react in a system named A in which
five heritable combinations (phenogroups) Ai,
A2C, B, A2BC and — vthe negative alternative)
are established. A second system, named D,
involves blood factors D, M and X, and the
phenogroups DX, MX and — . Blood factors F
and G appear to be independent of each other
and of all the other blood factors. Consequently,
they have been assigned to respective systems
F and G. Likewise, H appears to be indepen-

dent of all the others with the possible exception
of E. Although the remaining blood factors J,
K, L, N and O are excluded as members of the
systems A, D, F, G and H, their possible rela-
tionships with respect to one another have not
been fully ascertained. Thus, it would appear
that genes at a minimum of six loci are involved
in the control of equine blood groups.

11.11. Genetic Polymorphism in the Transferrings of
the Horse. Mikael Braend (Oslo, Norway).

Plasma samples from 220 horses belonging to
the Norwegian Dola breed have been subjected
to starch gel electrophoresis. Nine different
transferrin patterns were found. They could be
explained by a theory of five different alleles
at a single locus. These alleles were named TfD,
TfF, TfH, Tf° and TF. The TfR and TF alleles
were most common, the TfD gene was very rare.
Results from studies of sire families, dam-
offspring pairs and gene frequency analyses were
in agreement with the genetic theory.

In the Fjording breed 44 horses were investi-
gated. Three transferrin alleles only were
recognized in this breed, but two genotypes were
found which had not been seen in the Dola horse.
The gene frequencies in the Fjording material
were markedly different from those found in the
Dola breed. Most common was the TF allele,
but the TfD and TF alleles were seen quite
frequently too.

In horses of foreign origin additional trans-
ferrin patterns have been detected. Most im-
portant is the occurrence of a sixth transferrin
allele, TfM. In total, 14 different transferrin
genotypes have been found in this study.

11.12. The Inheritance of Bloodgroups in the B and
C Blood Group Systems in Cattle. J. Bouw
(Wageningen, The Netherlands).

Investigations on blood groups in cattle have
demonstrated that these bloodgroups can be
classified into at least 1 1 genetic systems.

Within the systems the blood groups are
composed of one or more serologically deter-
minable antigenic factors.

In the so-called B- and C blood group systems
numbers of at least 240 and 50 blood groups,
composed of various antigenic factors, controlled
by these systems, have been established.

Concerning the inheritance of these blood
groups mainly 2 explanations have been ad-
vanced:

192

Section 11 — Immunogenetics

1. The possibility of closely linked genes each
controlling one single antigenic factor.

2. The assumption of series of multiple alleles
of which each member produces one anti-
genic complex.

The data on blood groups in cattle as collected
in the U.S. and in Europe have contributed
largely to the second assumption as the blood
groups were usually found to be composed of
serologically closely related antigenic factors.

The blood grouping work in cattle, as per-
formed by now in more than 20 laboratories all
over the world, has as a result of this been based
upon this assumption of multiple alleles of which
each is controlling one antigenic complex.

During the last decade several authors have
drawn attention to irregularities by taking this
assumption into consideration.

Recent studies in Holland including over
8000 parentage cases are indicating that the
assumption of non-crossover units each control-
ling a part of the blood groups on the loci
controlling the B- and C-groups are presenting
a more satisfactory explanation for the inherit-
ance of the blood groups in these systems.

11.13. Possible Somatic Recombination in Twin
Cattle with Erythrocyte Mosaicism. W. H.

Stone, Janis Friedman, Audrey Fregin and
Joan Caulton (Madison, U.S.A.).

About 90 per cent of dizygotic cattle twins
contain a mixture of two antigenically distinct
kinds of blood cells derived from genetically
different tissues. This condition is known as
erythrocyte mosaicism (chimerism) and presuma-
bly results from an anastomosis of embryonic
membranes in utero followed by a reciprocal
exchange of primordial erythropoietic tissues.
Chimeric twins are immunologically tolerant to
erythrocyte and to transplantation antigens of
their co-twins. Recent observations indicate
that the proportion of the two kinds of cells in
co-twins is not constant, suggesting that a twin
may lose tolerance for the antigens of the co-
twin. In 6 sets of twins tested over a period of
years there was evidence of a shift in the propor-
tion of the two kinds of cells to a predominant
type. In addition, one surviving twin of a set
showed a shift toward a "recombinant" cell
type containing blood group factors that were
previously unique to each of the two original
types. This recombinant type represents over
95 per cent of the erythrocytes and may have been
associated with an abrogation of tolerance. We
have sub-lethally irradiated one member of
each of 4 pairs of chimeric twins, the other

serving as a control, to determine if there are
changes in the proportion of the two cell
types and if "recombinant" types appear. At 10
weeks post-irradiation, only 3 of the irradiated
twins have survived, but there were no definite
changes in their bloods.

11.14. Production and Effects of Antileukemic
Globulins from Animals pretreated with Normal
Human Blood. B. Sekla (Prague, Czechoslo-
vakia).

Immunogenetic conception of the origin of
malignant cells through somatic mutation and
thus acquisition of distinctive antigenic proper-
ties of these cells led to trials at production of
specific immune sera by biological means; the
principle of acquired immunological tolerance
(or lowered reactivity) towards antigens applied
during early ontogenesis and in large amounts,
has been exploited for this aim.

Pretreatment of new-born sheep was made
with large doses of whole human blood mixed
from many normal donors. Later, the pretreated
and control sheep were immunized with human
leukemic cells, every patient having his own
experimental and control group of animals.

In vitro tests have shown that immune sera
from pretreated animals do not agglutinate
erythrocytes of normal persons as well as those
of the patient in question in any considerable
measure. Furthermore, these sera have in vitro
a relatively specific cytotoxic effect against the
leukemic cells in question.

First trials in vivo have shown that relatively
large doses of immune globulins, produced by
ammonium sulfate fractionation of immune sera,
did not alter the red blood cell count of the
patient. Indications of specific effect upon the
white blood cell count which have been obtained,
must be considered cautiously in connection
with previous other therapy.

11.15. Independence of Genetic Variants of Egg White
Proteins and Blood Group Alleles of the A and
B Systems of the Chicken. G. R. J. Law

(Johnston, U.S.A.).

Egg whites obtained from hens of six inbred
lines of chickens were subjected to starch-gel
electrophoresis. Variations were observed in
three regions corresponding to those reported
by Lush d) and by Baker and Manwell (2>. No
one line of chickens exhibited segregation of
each of the genes controlling the variants at all

193

Section 11 — Immiinogenetics

three loci. The genetic potential of sires with
regard to egg white protein types was inferred
by pedigree analyses. Blood types of all birds
were determined by saline agglutination tests
employing specific blood-typing reagents made
by iso-immunization within each inbred line.
Linkage analyses of egg white protein types and
blood types were made on data from two
generations and part of a third generation. No
evidence was found for close linkage between the
A and B blood groups and the three loci con-
trolling the egg white protein variants.

1. Nature 189, 981. 1961.

2. Brit. Poultry Sci. 3, 161.

1962.

11.16. Effects of B Locus Genotype on Immunological
Tolerance in Fowl. L. W. Schifrman and
A. W. Nordskog (Ames, U.S.A.).

Similarity of the B blood group locus in
chickens with the H-2 locus in mice has previous-
ly been established. Techniques employed to
study immunological tolerance were similar to
those of workers using birds of unknown histo-
compatibility relationships except that B geno-
type was determined by blood typing. Of 70 B
compatible skin transplants exchanged between
closely related chicks at 17 days of age only
three were rejected prior to 34 days post-grafting.
All of 47 transplants between B incompatible
birds were rejected early; median survival time
(MST) being 6.6 1 0.4 days. Survival time of 47
transplants between B incompatible birds
injected at hatching with erythrocytes of the
same B incompatibility did not differ signifi-
cantly (MST 6.8 i 0.5 days) although the bird's
ability to produce B hemagglutinins at 3 months
of age was markedly reduced. Neonatal injection
of donor-type leukocytes was relatively effective
in inducing tolerance to later B incompatible
transplants. In a subsequent experiment injection
of erythrocytes at hatching and leukocytes at
17 days induced a greater degree of tolerance to
B incompatible transplants, exchanged on the
27th day, than did injection of leukocytes at
17 days only. Of interest was the fact that
heterozygous B incompatible transplants re-
mained intact significantly longer than homo-
zygous B incompatible transplants; presumably
an effect of differences in concentration of
foreign B antigens at the cell surface. Partial
tolerance existed in some cases such that a
homozygous incompatible (e.g. fl'/fi1) transplant
was rejected early while a heterozygous incom-
patible (e.g. Bl'B2) transplant on the same

individual (eg. B2/B2) showed no apparent
rejection during the bird's lifetime.

11.17. Genetics of Rabbit 7S y-globulins. S. Dray,
J. E. Colberg, G. O. Young, L. Gerald
( Bethesda, U.S.A.), and A. Nisonoff (Urbana,
U.S.A.).

Isoantibodies identify several rabbit y-globulin
allotypic specificities which are heritable. Analy-
sis of progeny confirm the hypothesis that the
Al, A2 and A3 allotypic specificities are deter-
mined by three autosomal allelic genes at the
a locus; the A4 and A5 specificities, by two
autosomal allelic genes at the b locus. Progeny
tests show that the a and b loci are not closely
linked. An allotypic specificity P is determined at
a third locus. Using I181-labeled y-globulins,
molecules with Aal, Ab4 and Ab5 specificities
were estimated quantitatively by repeated
precipitations with antibody. In the Ab4 or
Ab5 homozygotes or the Ab4-Ab5 heterozygote,
80-90 per cent of the y-globulin-I131 molecules
have Ab4 or Ab5; 10-20 per cent of the y-
globulin molecules have neither Ab4 nor Ab5.
In the Ab4-Ab5 heterozygote, the quantities of
y-globulin molecules precipitable by anti-
Ab4 (64 percent) or anti-Ab5 (27 per cent) are
independent of the order of precipitation indi-
cating that allelic specificities Ab4 and Ab5 are
not found on the same y-globulin molecules.
However, cellular studies of lymph nodes, using
anti-A4 and anti-A5 conjugated with fluorescein
isothiocyanate or lissamine rhodamine B, show
that allelic specificities Ab4 and Ab5 are found
within the same cell. Aal, Ab5 and Aa3, Ab4
double homozygous rabbits were mated and
y-globulin from a doubly heterozygous offspring
tested: 68 percent of the molecules with Aal also
had Ab4; 16 per cent of the molecules with Aal
also had Ab5 specificity. If, as is probable, non-
allelic specificities are present on different
polypeptide chains, the data are consistent with
random assortment of non-allelic chains in
molecules of the offspring.

11.18. Immunogenetics of Rabbit y-globulin.

A. S. Kelus and P. G. H. Gell (Birmingham,
Great Britain).

There are known six (Al, A2, A3, A4, A5
and A6) allotypes of rabbit y-globulin dis-
tinguishable serologically.

The inheritance of all six allotypes has been
studied in nearly 100 matings with several

194

Section 11 — Immunogenetics

hundreds of offspring and, in some cases,
through several generations. The allotypes
appear to be determined by six genes equally
divided between two loci (a and b) giving a
maximum of nine possible and not closely
linked "gene pairs" on chromosomes.

The phenotypic distribution of the allotypic
determinants on the y-globulin molecule and its
component parts will also be reported.

11.19. Non H-2 Antigens of Mice. D. Bernard Amos
(Durham, U.S.A.).

Mice of one H-2 genotype hyperimmunized
with tumor from another H-2 genotype produce
antibodies detectable as hemagglutinins and
cytotoxins. Most of the antibodies are directed
at H-2 antigens, but in certain combinations anti-
bodies reacting with other antigens are present.
An antibody produced in C57BL against
CsH/St tumor 6C3HED reacts with cells
carrying H-2 antigens C, K, H, A, but also with
cells from 129 mice which carry none of these.
The antigen on 129 was tentatively called alpha.
Since the antigen is a single factor controlled by
a gene H-5a present in C;3H/St and 129 but not in
CsH/He or C57BL, the designation was changed
to H-5A.

Another antigen, H-6A (first designated delta),
is present in a different C3H subline, C3H/HC
but is not in C3H/SL Both antigens are hemag-
glutinogens. H-5A is present in quantity on
kidney, testes and lung; H-6A in testes, spleen,
lung, brain and gut. A strongly cross reactive
antigen is present in feces independently of the
H-6 genotype of the host.

Both antisera react with other antigens not
related to the hemagglutinogens. One cytotoxin
has been found in antibody to CsH/St and a
mixture of several cytotoxins in antibody
against C^H/He. These antigens appear to
segregate independently of H-2, H-5 and H-6
and are being studied in reciprocal crosses
between C3H/He and C3H/SL Strains co-isogenic
for the various factors are being established for
a study of the relationship between graft rejection
and a variety of cytotoxic or hemagglutinating
antibodies.

1 1 .20. Developmental Genetics of H-2 Antigens of the
Mouse. G. Hoecker, O. Pizarro and P. Ru-
binstein (Santiago, Chile).

H-2 antigens of the mouse offer to be good
materials for the study of gene action at the

cellular level. The H-2 locus determines a
complex system of tissue and red cell antigens
which are inherited en bloc. The H-2 locus
exhibits pseudoallelism, some antigens crossing-
over in about I per cent of the gametes.

H-2 antigens seem to be present in very small
amounts — basal — in embryos and in newborn
mice. After birth, they increase and reach adult
concentration in about 5 to 6 days. During this
period different antigens from this system
increase their concentrations at the same rate and
reach maximum concentrations at the same time.
The phenotypic expression of H-2 genes is
strongly modified by the cellular environment
during development, both in quantity and
quality. As a result, different tissues vary in their
antigenic contents and some lack them alto-
gether. Besides these main variations in pheno-
typic expression, subtler changes were found:

(1) H-2 antigens from tissues having the same
amounts of antigens vary greatly in their ability
to induce and sustain antibody production and in
the types of antibodies induced preferentially.

(2) Differences in solubility of H-2 antigens in
saline were also found in some strains of mice
but not in others.

11.21. An H-2-associated Serum Protein Variant in
the Mouse. D. C. Shreefler (Ann Arbor,
U.S.A.).

A 20- to 25-fold difference in the concentration
of a specific seium a-globulin has been detected
among inbred mouse strains by immunodiffusion
with rabbit antisera. Studies of the inheritance
of this system and evidence suggesting associa-
tion with the histocompatibility-2 locus have
been reported. A pair of additively acting
alleles at an autosomal locus determine, in
homozygous condition, the low concentration
serum serological phenotype (Ss-L), or the high
type (Ss-H); the heterozygote has an inter-
mediate phenotype (Ss-HL). The Ss components
of Ss-H and Ss-L sera have the same apparent
electrophoretic mobility, precipitation behavior,
and pH and heat stability. No differences in
antigenic specificity have been detected, even
with specific rabbit anti-Ss serum. The Ss protein
is a euglobulin, probably of high molecular
weight, and has little or no lipid or carbohydrate
content. Tests for esterase, phosphatase, and
other enzymatic activities have been negative.
Present evidence strongly suggests control of the
Ss trait by the H-2 locus. All seven Ss-L lines
tested have the H-2k allele; eleven Ss-H lines
surveyed have various H-2 alleles, but none has
H-2k. Offspring from backcrosses of (C57BL/10

195

Section 11 — Immunogenetics

C3H)Fi ', C3H and (DBA/2 C3H)Fi x
C3H have been classified for serum type by
immunodiffusion and H-2 type by hemagglutina-
tion. No recombination has been observed
among 363 individuals typed, indicating at the
95 per cent confidence level a recombination
frequency not greater than 1 per cent. Absorp-
tion, inhibition and blocking tests reveal no
antigenic relationship between Ss protein and
H-2 antigens.

1. Genetics 46, 898, 1961 ; Genetics 48 (in press),
1963.

11.22. Gamma-globulin Isoantigens (Allotypes) in
the House Mouse. Leonard A. Herzenberg,
Robert I. Mishell and Leonore A. Her-
zenberg (Bethesda, U.S.A.).

Although genetic differences in gamma-
globulins have been recognized for some time
in the human and the rabbit, it was not until
1961 that a genetically determined isoantigen
in this class of serum proteins was found in the
house moused1) We have reported a second such
antigen, designated it Gg-2 and shown it to be
determined by a single segregating genetic
factor. (2> We have also shown that this antigen
is on antibody molecules of diverse specificities.

Data to be presented will show that Gg-2
and an antigen (very likely identical to the one
reported by Kelus and Moor-Jankowski)
which we label Gg-1 are controlled by a pair of
allelic genes, designated Gg1 and Gg2. Appro-
priate crosses with linkage testing stocks have
been set up to establish the linkage relations of
the Gg locus to known markers in the mouse.

Whether these antigens are found only on
separate molecules of gamma-globulin in hetero-
zygotes and the submolecular location of the
antigenic determinants are questions under
study.

1. A. Kelus and J. K. Moor-Jankowski,
Nature 191, 1405, 1961.

2. J.WuNDERLiCHandL.A. Herzenberg, Records
of the Gen. Soc. of America, 31, 126, 1962.

11.23. A Genetic Approach to the Mechanism of
Partial Immunological Tolerance. A. Len-
gerova, V. Matousek, and M. Vojtiskova
(Prague, Czechoslovakia).

The clonal selection hypothesis of immunity

presumes the existence of multiple clones of
globulin-producing cells, each genetically pre-
determined to form one (or a small number of)
specific antibody. The cells of a particular clone
can also develop specific non-reactivity (immuno-
logical tolerance) if their contact with the res-
pective antigen (or antigenic determinant) takes
place within a critical period of their matura-
tion. Tolerance of an antigenic complex is not
an all-or-nothing phenomenon occurring in
various degrees between complete reactivity
and non-reactivity. The hypothesis was being
tested that complete tolerance is the issue of a
series of independent events; the likelihood of
their occurring simultaneously thus greatly
depends upon the total number of clones involv-
ed in the formation of antibodies against the
given complex.

Assuming the existence of n antigenic differ-
ences between two inbred mouse strains (A and
CBA), then the F2 individuals represent a dis-
tribution of antigenic differences from 0 to n
with respect to each parental strain. Under the
hypothesis of n independently segregating histo-
compatibility loci and further hypothesis con-
cerning the probability of induction of tolerance
of k antigens from the inducing complex (Fi
hybrid spleen cells), the distribution of survival
times of both parental skin grafts has been cal-
culated and compared with that obtained in the
experiment. The consequences and limitations
of the model are discussed.

11.24. Transplantation of Mammary Glands from
Two Strains of Mice to Fi Hybrids of these
Strains. James S. Thompson and Flavia
Richardson (Edmonton, Canada, and Bar
Harbor, U.S.A.).

A technique has been developed for the trans-
plantation of mammary glands from C3H/HeJ
and C57BL/6 female mice to dorsal and nipple
line sites in female Fi hybrids of these two strains.
The effect of site of transplant and hetero-
chronicity upon the grafts has been investigated.
The site of transplant apparently had little
effect, except that glands in the nipple line were
sometimes suckled, whereas those in the dorsal
area were not suckled even though they lactated.
The percentage of successful grafts varied with
the age of both host and donor. With young
mice, four to six weeks of age, as both donors
and hosts, 80 to 100 per cent of the grafts per-
sisted in most series. As age of either host or
donor increased, the percentage of grafts accepted
declined, but there was considerable variation in
this respect from series to series.

196

Section II — lmmunogeneiics

The effect of pregnancy and of estrogen stimu-
lation upon the functioning and the histological
appearance of the glands has been investigated.
Some of the transplants reacted normally to
pregnancy, with lactation and. on occasion,
suckling being observed. Histologically these
glands had the typical appearance of lactating
glands. The effect of estrogen stimulation will
be discussed.

11.25. A Specific Alteration of Histocompatibility
Expression in the Progeny of a Homograft
Tolerant Male. Ronald D. Gutmann (Bet-
hesda U.S.A.) and J. Bradley Aust (Min-
neapolis, U.S.A.).

The following results are from experiments
designed to study the effects of continual expo-
sure of maturating male germplasm in the mouse
to living cells of a foreign genotype. An adult
C3H male was rendered tolerant to A strain
tissue by the neonatal intravenous injection of
A splenic cells. Breeding this CsHtoierantA
male with (A ■ C3H)Fi hybrid females produced
a backcross population of mice which accepted
an (A X CsH)Fi hybrid mammary adenocar-
cinoma with an incidence of 91.3 per cent and
an 18th transplant generation A strain mammary
adenocarcinoma in 30.4 per cent of the cases
compared with 7.7 and 0 per cent respectively
in the backcross control group. Other control
groups for the tumors were composed of A,
C3H, (A C3H)Fi, and (A C3H)F2 hybrid
litters which accepted the tissue in accordance
with the expectations of the genetical theory of
transplantation based on susceptibility control-
led by multiple dominant histocompatibility
genes. Subsequent litters of backcross mice
sired by the C3Htoieranti^ male were tested
with normal A, (A C3H)Fi, and (C57BL

C.3H)Fi hybrid skin and accepted the A and
(A CsH)Fi hybrid skin in increased incidence
when compared with controls, but rejected the
(C57BL C3H)Fi hybrid tissue showing that
this change in histocompatibility relationship
has a donor-host specificity and is demonstrable
using normal as well as neoplastic tissue.

11.26. Mutations of Histocompatibility Genes in the
Irradiated Mouse. Donald W. Bailey and
Henry I. Kohn (San Francisco, U.S.A.).

Tail-skin grafts were exchanged among
(BALB/c rS C57BL/6$) Fi or the reciprocal
type hybrid mice, referred to here as CBFi

and BCFi, respectively. Their fathers received
spermatogonia! X-irradiation (522 rads) or were
not irradiated. Of 2641 Fi mice skin-graft
tested, 30 were mutants. Of these, 26 showed a
gain in graft specificity (dominant), 3 showed a
loss in specificity (recessive), and only 1 showed
a loss and a gain in specificity (co-dominant).
The higher frequency of gains might be explained
thus: if the great majority of histocompatibility
genes or subgenes in the two parent strains were
identical, then by the laws of chance the gain
would be the most likely type of mutation de-
tected by our methods.

Irradiation did not significantly affect the
mutation rate. The relative frequency of mutants
was: treated groups, 0.009 (BCFi) and 0.012
(CBFi); untreated group. 0.014 (CBFi). It was
noted, however, that many mutants occurred
as clusters within families, and thus presumably
arose in earlier generations. The high spon-
taneous mutation rate may therefore have mask-
ed the effects of irradiation. Nevertheless most
mutants had different specificities and thus must
have arisen independently.

All observed histo-incompatibilities that were
adequately tested were shown to be inherited.

11.27. Heritability of the Natural Immunity. Gyorgy
Szemere (Szeged, Hungary).

Searching the genetic circumstances of the
natural immunity, the author has pointed out
that rats belonging to different races (brown,
laboratory white and Wistar) differ from each
other regarding their immunobiological titres —
namely the complement titre and bactericidal
properties — of their sera. Sera of brown rats
had the highest and that of laboratory white
rats the lowest complement titre and bacte-
ricidal activity. If Wistar and brown rats were
crossed, the complement titre and bactericidal
power of the hybrids' sera showed intermediate
values when compared with the titres of the
parents.

It has been also pointed out that similarly to
the complement titre and bactericidal activity,
the sera of brown rats contained the greatest
amount of properdin, while sera of laboratory
white rats the smallest one. It was supposed,
therefore, that the properdin level, and other
factors which play a role in the formation of the
natural immunity, are different not only be-
tween species, but between races too.

Crossing of brown and laboratory white
rats showed a surprising result. While the aver-
age properdin content of brown rats' sera
was 36.6 U./ml and that of laboratory white

197

Section 1 1 — Immunogenetics

ones 18.3 U./ml, the sera of the hybrid animals
contained 48.3 U./ml of properdin. These re-
sults suggest that immunobiological titres are
determined by genetic factors, and heterosis
effects may occur in them, though it seems to be
evident by the results of the author's other in-
vestigations that environmental factors (such as
exposure to cold) can change the degree of
immunity within a comparatively short period.

11.28. Histocompatibility and Linkage Relationships
of Loci Determining Isoantigens of the Rat.

Joy Palm (Philadelphia, U.S.A.).

Histocompatibility and linkage relationships
of genes determining cellular antigens in three
inbred rat strains (Lewis, N.B. and W.I.F.) are
being investigated. Allelic genes determine two
red cell antigens, 1 of Lewis and 3 of B.N.11'
The histocompatibility function of the locus was
indicated by the rapid (under 12 days) rejection
of Lewis or B.N. grafts on Fo hybrids which
always occurred when donor and host were
incompatible for these antigens. W.I.F. strain
rats lacked antigens 1 and 3 but did possess
an antigen with the B specificity. The segrega-
tion ratios in backcross tests indicated that the
genes determining antigens 1 and 3 were allelic
with the gene for the B antigen, but were trans-
mitted independently of sex and the non-anti-
genic loci for albinism and hooding. Tests for
linkage with other mutant genes are in progress.
The C-D antigenic locus, defined by Owen
(loc. cit.), was not associated with histocompati-
bility reactions even with the exchange of tissue
grafts between inbred rats differing only for
these antigens. The locus was not obviously
sex-linked or linked to loci controlling albinism,
chocolate coat color and hooding, or to the
locus determining antigens 1 and 3. A search
is being made for evidence (either by linkage
relationships or serological cross-reactivity of
specific antisera) indicating an homology of the
antigenic loci with those of the mouse.

1. Palm, N. Y. Acad. 97, 1962

2. Bogden and Aptekman, loc. cit .

11.29. (D.) Genetics of the Anaphylactoid Reaction in
Rats. H. Kalmus, J. M. Harris and G. B.
West (London, Great Britain).

A single injection of dextran or egg white
produces hyperemia, pruritis and oedema in the

face, ears and paws of most laboratory rats.
This reaction resembles an anaphylactic reac-
tion, but no prior sensitization is necessary.
Rats which do not react in the described way
even after repeated injections were found in some
Wistar albino colonies and pure strains of
reactors and non reactors were bred. The dif-
ference appears to be caused by a recessive auto-
somal gene dx which in the homozygous state
prevents the reaction; Dx dx and Dx Dx animals
are reactors. The gene was outbred into other
strains and segregates independently of the fur
colour genes c, b and a. In 14 colonies of dif-
ferent origin non-reactivity occurred in 3 Wistar
strains at frequencies of 17.5, 23.4 and 100 per
cent. Two of these colonies were maintained by
brother sister matings and the third as a closed
population.

J. M. Harris, H. Kalmus and G. B. West,
Genetical control of the anaphylactoid reaction
in rats. Genetical Research 1963 (in press).

11.30. New Crossreacting Hetero- and Iso-antigenic
Systems in Serum of Mammals including
Man. J. Moor-Jankowski and K. S. Brown
(Bethesda, U.S.A.).

On the basis of our previous results on gene-
tically controlled variations in serum isoantigens,
a working hypothesis was established for experi-
mental determination of antigenic markers in
animal sera. Experiments were performed in
(1) closely related species, e.g. monkeys and
man, and in (2) inbred strains of mice.

(1) Numerous selective antisera produced
recognized polymorphisms of several serum
components in isologous, and in closely related
species. A system of notation was developed to
define the antigen antibody relationships between
the antigen donor, the antibody producer, and
the reacting propositus: I, isogenic iso-antibodies
(ii). II, isogenic heteroantibodies (ih); III,
heterogenic isoantibodies (hi) (still hypothetical);
IV, heterogenic isospecific heteroantibodies
(hih); and V, heterogenic heteroantibodies
(hh). The abbreviations are used to classify the
following experimental results.

Individual human isoprecipitating antisera
recognized polymorphisms in serum lipopro-
teins and gamma-globulins of (ii) man and of
(ih) Papio cynocephalus, and yet undefined
serum polymorphism in (ih) Macacus rhesus.

Individual isoprecipitating antisera of Papio
cynocephalus recognized polymorphisms in se-
rum lipoproteins and gamma-globulins of

198

Section 11 — Immunogenetics

(ii) Papio cynocephalus, and yet undefined serum
polymorphism in (in) man.

Individual heteroprecipitating Papio cynoce-
phalus anti-human sera recognized polymorphism
in serum gamma-globulins and other fractions
of (hih) man, and yet undefined serum poly-
morphism in (hh) Macacus rhesus.

(2) Isogenic strain isoantibodies (isi) in mice
recognized the antigen gamma-BA present in
BALB/c, C3H/He and "Champagne-Glaxo" but
not in C57B1, and the antigen gamma-CA
present in C57B1 but not in BALB/c.

Genetic transmission of the above systems and
their postulated role in maternal-foetal incompa-
tibilities in primates including man, and in
mice, are being investigated.

11.31. Immunological Studies of Genetical Markers
in Tissue Cell Populations in Cultures. J.

Moor-Jankowski (Bethesda, U.S.A.).

Investigation of the genetics of tissue cells in
cultures depends on demonstration of hereditary
characters. Immunological tools for this pur-
pose are: (I) Mixed agglutination (Coombs),
based on specific agglutination, by the same
antibody, of tissue cells and indicator cells
sharing the same antigen; and (II) Double dif-
fusion in agar gel (Ouchterlony), consisting of the
diffusion of antibody and of antigen (here, cell
homogenate) in a concentration gradient to-
wards the reaction area, where they meet in
proportions optimal for precipitation.

In present tests, antisera for (I) were made
against erythrocytes, and antisera for (II)
against tissue cells.

The investigated characters of tissue cells were
species specificity and blood group specificity.
Diachronic studies were performed on conse-
cutive cell generations.

The cells tested were derived from (1) primary
animal and human explants, (2) human diploid
cell strains, and (3) long established aneuploid
human and animal cell lines.

Species specificity was ascertained, and no
diachronic changes were observed by (I) and
(II) in (1), (2), and (3); selective advantage of
some species of (3) was demonstrated. Blood
group antigens A, B, or H, and M and Tja were
found by (I) in human (1) and (2); no diachronic
changes occurred in diploid cells observed from
a few, up to 20 passages. Findings on blood
group antigens in (3) were not consistent. Partial
or complete loss of blood group phenotype in
aneuploid cells is evident.

The well studied human diploid strain WI-38
is being used to investigate antigenic markers

and their changes after viral-induced transforma-
tion to aneuploidy.

11.32. Experimental Studies on the Prevention of
Rh Haemolytic Disease. C. A. Clarke and
R. Finn (Liverpool, Great Britain).

The results are described of experiments in-
volving the injection of Rh positive blood into
96 Rh negative men and designed to find out
whether or not the production of immune anti-D
can be prevented.

Giving 10-20 ml of anti-D sera containing
high titres of complete antibody half an hour af-
ter the Rh positive blood it was found that only
about 50 per cent of the injected cells had been
cleared within 48 hr and immune anti-D pro-
duction was enhanced as compared with controls
who received only the Rh positive blood.

Using 35-50 ml of plasma containing piedomi-
nantly incomplete antibodies we found that only
3 of 21 "treated" men developed immune
antibodies after three or four stimuli as compared
to 11 of 21 control men, the difference being
statistically significant (P = 0.02).

Examination of these results and those of
other experiments which are described suggests
that about 95 per cent of the injected cells have
to be cleared from the circulation within 24 hr
if immune antibody production is to be prevent-
ed. The anti-D antibody most likely to be effec-
tive in this should have no saline activity and as
high an incomplete titre as possible.

Preliminary work with anti-D gamma-glo-
bulin given intramuscularly has shown that in
appropriate dose it is even more effective in
rapidly clearing Rh positive cells than the most
powerful plasma used.

The next step proposed is to give Rh positive
foetal cells to Rh negative infertile women, half
of whom will act as controls and half will be
given gamma-globulin. It is hoped that the
results of this experiment will be available.

11.33. Anti-RH Inhibition by RNA Derivatives and
Amino Acids. Emanuel Hackel (East Lans-
ing, U.S.A.).

Earlier studies from this laboratory have
shown that several ribonucleic acid derivatives
and some amino acids are capable of inhibiting
specifically the antibodies of the Rh series. This
inhibition is presumed to be the result of partial
neutralization of the antibody by the inhibitor

199

Section 11 — Immunogenetics

and, in turn, leads to the tentative conclusion
that the inhibitor, at least in some way, resembles
the antigen.

Recent experiments have shown that mixtures
of some RNA derivatives and amino acids have
a greater inhibitory effect on the Rh antibodies
than equivalent amounts of either substance
alone. Adenosine monophosphate, uridine mo-
nophosphate, and cytidine monophosphate
were the nucleotides used in this investigation.
The amino acids used were histidine, arginine,

lysine, phenylalanine, and serine. A number of
quantitative relationships between inhibitors
and antibodies have been determined. These
further support the idea of the complexity of the
Rh antigens and therefore the complexity of
the genetic material governing Rh. Furthermore,
while it is not possible to postulate a definitive
chemical composition for the Rh antigens on
the basis of these studies, they do offer an im-
portant insight into the nature of Rh and into
the ultimate unravelling of this problem.

200

SECTION 12

PLASMATIC INHERITANCE

12.1. Chemical Characterization of Metagons in
Paramecium. I. Gibson (Edinburgh, Great
Britain).

Metagons are gene-determined factors in
mate-killer paramecia. They control the mainte-
nance and multiplication of visible ia-particles
in the cytoplasm of these paramecia and are
thought to consist of RNA. Metagons have now
been extracted from cells and re-infected into
other cells. Results will be described with phenol
extracts and other techniques for the purification
and characterization of the metagons.

The possible role of the wild type mt xm-
XIV and N alleles will be discussed in terms of a
mechanism which assumes that the mt locus
controls an essential reaction in the synthesis of
the XIV mating type substance, that this locus is
repressible and that it does not function in
cells expressing type XIII.

This research was suppor ted by grants from the
U.S. Public Health Service and the National
Science Foundation.

12.2. Mutations affecting Mating Type Differentiation
in Paramecium. Stephan R. Taub (Cambridge,
U.S.A.).

The mating type of an individual Paramecium
aurelia is controlled by its macronucleus. Two
paramecia of identical genotype, however, can
differ in mating type. The nuclear differences
responsible for mating type differences become
established soon after the origin of the new
macronucleus. In certain strains (including those
of syngen 7), cytoplasm transmitted by the
previous sexual generation directs the differ-
entiation of the new macronucleus to control
permanently one of two mating type potentiali-
ties. The mating type-determining state of the
cytoplasm is, in turn, controlled by the differ-
entiated macronucleus and its specificity usually
is such as to maintain the same mating type
from one sexual generation to the next (Sonne-
born, 1954).

In syngen 7, mutations affecting these pro-
cesses have occurred at two unlinked loci
(n and mt). Paramecia carrying the N and the
mt xm, xiv genes can express either mating type
XIII or XIV, the type actually expressed depend-
ing on the state of the cytoplasm received from
the previous generation. Cells homozygous for
either the /?, the mt xm or the mtz gene express
type XIII regardless of the state of the inherited
cytoplasm. In addition, cells either heterozygous
or homozygous for mt xm and cells homozygous
for mtz carry XlV-determining cytoplasm
regardless of their mating type.

12.3. Transmission and Segregation of a Cytoplasmic
Factor for Streptomycin Resistance in Diploid
Chlamydomonas reinhardi. Nicholas W. Gill-
ham (New Haven, U.S.A.).

Resistance to 500 \xg per ml of streptomycin is
controlled by a nonchromosomal factor (sr-2)
which exhibits a uniparental pattern of inheri-
tance via the mating type plus {mt+) parent.
When resistant mt+ cells are crossed to sensitive
cells (ss) of the minus mating type (mt~), the
resulting tetrads segregate four sr-2: zero ss
progeny. The remaining 10 per cent or so of the
tetrads are exceptional and in these tetrads the
sr-2 factor is transmitted to the meiotic progeny
by the mt~ parent. A study of these exceptions
has shown that the meiotic products segregate
pure clones of resistant or sensitive cells or
mixed clones containing both types of cells.
Therefore, segregation of resistance and sensitivi-
ty may occur during either the meiotic or post-
meiotic mitotic divisions.

Recently, a method for obtaining diploids in
Chlamydomonas reinhardi has been devised by
Ebersold (unpublished). Cells are allowed to
mate as in an ordinary cross, but through the
use of appropriate selective markers it is possible
to isolate clones of cells that appear to behave
like diploids. In diploids made between resist-
ant and sensitive cells the uniparental pattern of
inheritance of the sr-2 factor appears to break
down and the factor is transmitted efficiently by
either parent. In the resulting diploid clones
segregation of resistance and sensitivity seems
to occur within the first few mitotic divisions.

201

Section 12 — Plasmatic Inheritance

12.4. Interactions between a Cytoplasmic Factor and
Nuclear Genes in Neurospora crassa. Selma
Silagi (New York, U.S.A.).

Mitchell and Mitchell showed that the slow-
growing aspect of the phenotype of poky, a
maternally inherited condition, could be sup-
pressed by a nuclear gene, f.

As a result of crosses between poky and several
other strains of Neurospora it has been found
that the f locus is closely linked to inositol on
linkage group V, with a centromere distance of
approximately 35 units. It has also been observed
that the f gene manifests itself in the poky strain
not only by increasing the growth rate but also by
shortening the lag period associated with ascos-
pore germination.

Additional attempts have been made to see if
other genes might modify the growth rate of
poky strains, including crosses with pem fl.
Poky strains containing fluffy, and therefore
aconidial, were found to have a relatively short
lag phase, resembling in this respect the behavior
of hyphal fragments of "cured" growing
mycelium. From this and other evidence which
will be presented it appears that the formation
and/or germination of conidia or ascospores are
a prerequisite for the full expression of the poky
phenotype.

12.5. A Gene-induced Cytoplasmic Mutation in Yeast.

M. Grenson (Brussels, Belgium).

Following the application of X-rays, a mutant
of Saccharomyces cerevisiae was isolated (D77)
which simultaneously requires glutamic acid
and is respiratory deficient.

When crossed with normal strains, D77 give
a mixture of normal and respiratory deficient
diploid clones, all glutamic-acid-sufficient. It
behaves thus as a suppressive petite. Tetrad
analysis of normal diploids show a 2 : 2 segre-
gation of both deficiencies, which are linked with-
out exception.

However, the respiratory deficiency behaves,
at the same time, as an extrachromosomal
character, since it does not complement a
neutral cytoplasmic petite.

Two nuclear petites, N2 and N4, were shown
to be complemented by D77.

A hundred glutamic-acid-sufficient revertants
isolated from D77 remained respiratory deficient
without exception.

The above may be understood if we assume
that the glutamic deficiency block is responsible
for the induction of a cytoplasmic petite mu-
tation.

This is further supported by the observation
that the cytoplasmic petite mutation can be
avoided in the ascospores carrying the glutamic
acid deficiency when germination takes place on
glycerol, lactate or acetate, instead of on
glucose, as the carbon source. Under these con-
ditions, the clones arising from the two glutamic-
acid-less spores exhibit normal respiration as do
the two glutamic-acid-sufficient clones.

12.6. The Inheritance of a Killer Character in Yeast
(Saccharomyces cereviseae). M. Makower
and E. A. Bevan (Oxford, Great Britain).

In certain strains of yeast three phenotypes
have been observed: "killer", "sensitive", and
"neutral". When killer and sensitive cells are
grown together in the same broth or agar medium
a high proportion of the latter are killed. No
killing takes place when neutral are grown with
either killer or sensitive cells.

Crosses between different killer and neutral
strains followed by Ascus analyses of over 800
tetrads reveal that the difference between killer
and neutral cells is controlled by two loci N/n and
K/k, showing 36 per cent recombination. The
presence of N determines the neutral phenotype;
its recessive n, the killer phenotype. K is epistatic
to N. Thus, all neutral strains are Nk, but either
of three genotypes, NK, nK or nk, may determine
the killer phenotype.

Sensitive cultures are never recovered follow-
ing crosses of sensitive by killer or neutral. But
the results of such crosses show that sensitive
cells may possess any of the above four
possible nuclear genotypes all lacking a cyto-
plasmic component which is present in both
killer and neutral cells.

Mutant sensitive strains have been recovered
at a frequency of 0.26 per cent following treat-
ment of killers with ethyl-methane-sulphonate.
These mutants retain the nuclear genotype of the
original strain. No mutation of sensitive to either
killer or neutral has yet been observed. These
observations provide evidence for the conclusion
that sensitives differ from killers and neutrals in
lacking a cytoplasmic component.

12.7. The Physiological Basis of the Killer Character
in Yeast. E. A. Bevan and M. Makower
(Oxford, Great Britain).

A high percentage of sensitive cells are killed
when incubated in cell-free filtrates of buffered

202

Section 12 — Plasmatic Inheritance

medium in which killer cells have been grown.
The percentage killing depends on the period of
incubation of both (1) the killer cells prior to
filtration, and (2) the sensitive cells following
filtration. So far, up to 39 per cent of the sensitive
cells have been killed when incubated for 3 ru-
in a filtrate derived from a 48 hr growth of killer
cells. A filtrate obtained from homogenized
killer cells resuspended in fresh medium also
shows killer activity.

The particular nature of the killing factor is
suggested by three types of observation: (1) the
retention of activity after dyalasis of the cell-free
filtrate for 24 hr in distilled water, (2) the con-
centration of activity following its ultracentrifu-
gation at 40,000 rev/min for 1 hr, and (3) the
appearance of particles approx. 350A in size
when a concentrated filtrate is examined under
the electron microscope.

Killer cells have been recovered from an
18 hr growth of sensitive cells (nk genotype) in
cell-free killer medium. They are presumed to
arise by infection since, by appropriate crosses,
they have been shown to possess the same nuclear
genotype (nk) as the original sensitives. Killer
or neutral cells have not yet been recovered
following incubation of sensitive cells in cell-free
neutral medium.

12.8. Male Sterility in Castanea. Richard A. Jaynes
(New Haven, U.S.A.).

Male sterility was observed in Castanea sativa
Mill., C. mollissima Bl., and several Castanea
hybrids. In most cases the event(s) leading to
sterility occurred before meiosis. The arrange-
ment and early growth of individual flowers on
catkins was normal; however, staminate pri-
mordia failed to develop anthers. The number and
fertility of pistillate flowers of the male sterile
trees compared to male fertile trees was not
diminished. Sterility observed in these trees may
be genie, chromosomal, or the result of a gene-
cytoplasm interaction. Somatic chromosome
counts of one male sterile tree and progeny
(germinating nuts) of six other male sterile trees
revealed normal complements, 2«=24, in all but
one tree. Neither abnormal chromosome number
nor meiotic irregularities appear to be the
predominant cause of male sterility among the
trees examined. Data obtained indicate that
male sterility may be cytoplasmically controlled
in a few trees. Final proof will depend on
completing enough successive backcrosses to
accomplish genome substitution in male steriles
by their male fertile parent.

12.9. Genic-Cytoplasmic Interactions in Peanuts
(Arachis hypogaea L.). A. Ashri and E. Gol-
din (Rehovot, Israel).

There are two geotropically distinct growth
habits in peanuts — runner (spreading) and
bunch (erect). Hitherto their control was
attributed to duplicate genes. Reciprocal crosses
were made between Virginia Beit-Dagan 4(V.4)
and Georgia 2, N.C.2, Dixie Giant, Line 123
and Red Virginia, all bunch. In all crosses, when
V.4 was female parent, the Fi hybrids were
runners. Their F2 progenies segregated (pooled
data) 3178 runner: 2511 bunch (fit to 9:7 ratio
P=0.70-0.50). When the other varieties were
female parents and V.4 male, the Fi plants were
bunch. Their F2 progenies segregated (pooled
data) 433 runners: 672 bunch (fit to 6 : 10 ratio
F = 0.30-0.20). The backcrosses of [(runner
Fi hybrids) • (their male parent)] gave 10
runners: 15 bunch. Reciprocal crosses between
the last mentioned five varieties and also with
Virginia Improved (bunch) produced only bunch
Fi and F2 plants. Conclusions are: I. There are
two types of cytoplasm — one found only in
V.4 and another found in all the other tested
varieties; II. There are two genes, to be termed
Hbi and Hb2, which interact differently with each
other in each cytoplasm and with each cytoplasm.
It appears thatV.4 isHbiHbihb2hb2while all others
are hbihbiHb2Hb2. In V.4 cytoplasm Hbi-Hb2
produce runners, other genotypes give bunch. In
the cytoplasm of the others Hbi-Hb2andhbihib-
Hb2Hb2 produce bunch, other genotypes give
runners. Implications of this genic-cytoplasmic
interaction and evolutionary aspects will be
discussed.

12.10. Cytoplasmic Effects in Nicotiana. D. R. Came-
ron (Berkeley, U.S.A.).

The observation that there were differences in
male-sterility in reciprocal species hybrids and
failure to obtain certain hybrids in one direction
among species of section Tomentosae, led to a
study of various associations of genomes with
plasmones of related and unrelated species.
Within the Tomentosae, morphological effects
ranged from no detectable deviations from nor-
mal to those in which flowers had no visible
corolla and stamens were absent or vestigial.
Some results indicated that failure to obtain
certain hybrids was not attributable to the
cytoplasm of the ovule parent. When TV. tabacum
genomes were incorporated in cytoplasms of
several species, the flower morphology was
altered depending on the species involved.

203

Section 12 — Plasmatic Inheritance

TV. tabacum in sylvestris cytoplasm and syivestris
in tabacum gave typical tabacum or sylvestris
plants, indicating that the sylvestris parent had
provided the cytoplasm in the original hybrid
from which N. tabacum was derived by am-
phiploidy. Association of tabacum with cyto-
plasm of certain Australian species and bigelovii
resulted in the development of split corollas and
complete pollen sterility. When plumbagimfolia
cytoplasm was involved, corollas were reduced
in size and there were only scanty amounts of
pollen formed. In the combination, tabacum
chromosomes with rustica cytoplasm, plants were
normal tabacum in appearance and had abun-
dant germinable pollen. They were fully female
fertile but, when selfed, set only an occasional
small capsule with a greatly reduced number of
seeds in each. When pollen of this type was
applied to stigmas of tabacum, virtually all
pollinated flowers abscissed.

12.11. Nucleo-cytoplasmic Interactions. Luis B.
Mazoti and Ruderico S. Velasquez (Llaval-
lol, Argentina).

A pure line of Zea mays (Selfing20) was used
as male recurrent parent during 14 back-crosses
X Euchlaena mexicana. Among both lines of the
same homozygous genotype, which were differ-
ent only in their cytoplasm, were found the
following differential characters:

(a) Significative of mean differences in per cent
of pollen sterility. In cytoplasm of Euchlaena,
x = 52.1 per cent, in cytoplasm of Zea, x = 8.8
per cent; «i + m = 480 anthers, P<i\ percent.
The environment affects the percentages, but the
signification is permanent.

(b) Correlation in the percentages of pollen
sterility between neighbor anthers. In cytoplasm
of Zea, r — 0.575; in cytoplasm of Euchlaena,
r = 0.948; m + n% =-- 480 anthers. This results
are indicating that the initial factor responsible
of pollen sterility is independent from meiosis.

(c) Significative of variance differences in the
nucleolus diameter. In cytoplasm of Euchlaena
V -■ 2.653 n, in cytoplasm of Zea V == 0.751 u,
ni + n2 = 86 and P<T per cent. This results
would indicate that the variance in nucleolus
diameter could be an index of the nucleo-cyto-
plasmatic harmony.

(d) Significative of mean differences of the
Knobs volume. The Knobs volume in Zea cyto-
plasm is x = 17.41 |i3, in cytoplasm of Euch-
laena x = 26.62 m3, ni + «2 = 960, P<1
per cent. This results could indicate that the
cytoplasm had modificated the chromosomic
structure.

When, after 14 back-crosses of (Euchlaena x
Zea) ', Zea14, we obtain autofecundations, the
pollen sterility decreased in a significant measure:
x == 20.8 per cent in the back-cross to x = 16
per cent in selfing, m -f «a = 960 (anthers),
jP<T per cent. When this genome of Zea, which
was modified by the cytoplasm of Euchlaena,
was transported again to Zea's cytoplasm, an
increase of the per cent of pollen sterility is
produced from x = 8.92 per cent with the ori-
ginal genome) to x = 13.75 per cent (with the
recuperated genome), n\ + W2 — 1161, P<C\
per cent. It wasn't changed in one year selfing.
This results would indicate that the variation
produced by Euchlaena' s cytoplasm on Zea's
genotype, are inheritable and favorable to
Euchlaena's cytoplasm and unfavorable (and
irreversible in S1) to Zea's cytoplasm.

The percentage of pollen sterility was esti-
mated in every "item" in base of 100 counted
pollen grains of one anther.

12.12. Morphology, Cytology, and Biochemistry of
Male-sterile Lines of Maize. Patricia Sar-
vella and C. O. Grogan (Mississippi,
U.S.A.).

Normal, cytoplasmic male-sterile, and restored
versions of five lines of maize were studied mor-
phologically, cytologically, and biochemically.
Measurements were taken from meiosis to
anthesis (about 15 days after meiosis). At an-
thesis only slight variations occurred in ear loca-
tion, ear height, tassel length , and number of
internodes to the ear. Some internodes and
sheaths above the ear, and tassel culms
and sheaths were shortened in the male-
sterile plants. Internode-sheath ratios show-
ed large differences between the versions. Stalk
lengths above the ear were correspondingly af-
fected which sometimes was reflected in the total
stalk length. Restored plants usually were shor-
ter than normal which could be attributed in
some cases to fewer internodes above the ear
rather than shorter internodes. Variations of the
different versions in different environments
showed a cytoplasmic-environmental interaction.
Pre-anthesis plants showed that shortening start-
ed in the male-sterile versions between 10 and 14
days after meiosis. At ten days after meiosis it
could even be the longest of the three versions.
The exact time of the shortening depended on
the line, on the internode location in relation to
the ear, and when elongation started. Pollen
degeneration in all lines occurred about 5 days

204

Section 12 — Plasmatic Inheritance

after meiosis, which was before the shortening
effect was evident. Amino acid content of the
leaves and tassels at various stages were analyzed
by means of paper chromatography. Certain
amino acids varied quantitatively and qualita-
tively, depending upon the stage of develop-
ment of the plants.

The very fact of the control on and the mode
of the transmission through grafts indicated that
the cytoplasmic hereditary elements have at-
tributes of both plasmids and viruses. This, and
the induction in one case of an apparent chromo-
somal integration of the trait, after its induction
in the progeny of the fertile graft symbionts,
resemble an episome-like behaviour.

12.13. A New Interpretation of Cytoplasmic Inheri-
tance in Streptocarpus (Gesneriaceae). Hans

Marquardt (Freiburg, Germany).

In Escherichia coli and yeasts regulator sys-
tems (i.e. the interaction of structural genes,
operator-regions and regulator genes, producing
a repressor-protein) have been found. Oehlkers
analysed in numerous papers the cytoplasmic
inheritance in the formation of the androeceum
and gynaeceum in the flowers of Streptocarpus
hybrids (Sympetalae, Gesneriaceae). His results
can be interpreted on the assumption, that in
the genome there are androeceum-determining
and gynaeceum-determining genes with their
corresponding operator regions. Instead of a
regulator gene, regulator units may be present
in the cytoplasm. The repressor-protein produced
by the regulator-units of the plasmone of various
Streptocarpus species either reacts with the
operator-region of one of the two structural
genes, thereby blocking the normal development
of the androeceum and the gynaeceum; or else
inactivating substances in the cell metabolism
are formed, which affect (inactivate) the repres-
sor-protein. On the basis of these assumptions
it is possible to interpret the observed variable
anomalies in the development of the androe-
ceum, especially in the Str. wendlandii hybrids.
The hypothesis is supported by a temperature
experiment conducted by Oehlkers (1956) and
by the observed switching of sex-expression in
the androeceum of suitable hybrids during
flowering time. Further experimental tests of the
hypothesis are envisaged.

12.14. Alternative Cytoplasmic and Nuclear Heredi-
tary Elements in Petunia. Rafael Frankel
(Bet Dagan, Israel).

Asexual transmission of cytoplasmic male
sterility in Petunia hybrida through heteroplastic
grafts induced two types of male sterility in
the progeny of fertile graft components: One of
these was maternally inherited, as was the origi-
nal trait in the male sterile graft symbiont, whe-
reas the other one exhibited mendelian segrega-
tion. The progeny of the donor plants for the
graft components served as controls.

12.15. Plasmon Analysis of Species of Solanum.

Paul Grun, and Mary Aubertin (Univer-
sity Park, Pennsylvania, U.S.A.).

Plasmon factor /«s is sensitive to dominant
genes of the In series, the expression of the gene-
plasmon interaction being a failure of pollen
dehiscence. To date five different In genes have
been located any one of which can lead to inde-
hiscence when it is combined with plasmon factor
/«s. Separate accessions of Solanum chacoense,
which have the /«s plasmon factor, are different
in the number of alternative In genes to which
they are sensitive. The geographical distribution
of the individual //; genes appears to be rather
wide, for plants sharing the same In genes have
been located in populations from Ecuador,
Colombia, and Peru. No single distinctive ab-
normality in cytological development has yet
been identified as the cause of indehiscence,
for indehiscent plants even within single proge-
nies vary in their anther contents. Some have
anthers containing normal pollen, some shrun-
ken pollen, some a mixture of normal and shrun-
ken pollen, and some degenerate bubble-
surfaced sporocytes. It seems, therefore, that
the gene-plasmon interaction may operate
through failure of normal anther development
rather than failure of pollen development. Ab-
normality of anther development can, as a
result of specific epistatic interactions, lead to
other pollen or sporocyte irregularities.

This research was supported by a grant from
the National Science Foundation.

12.16. Extranuclear Inheritance of Induced Plastid
Mutations in Arabidopsis thaliana. G. Rob-
belen (Gottingen, Germany).

Plastid development towards an active, nor-
mal green chloroplast is controlled by intra-
as well as extranuclear factors. The mutations of
both block these processes at many different
points.

205

Section 12 — Plasmatic Inheritance

Towards the various types of "chlorophyll
mutants" the contribution of gene mutations
is well recognized in general. In Arabidopsis
thaliana we made a detailed analysis of many of
these Mendelian factors considering mutation
rate, phenotypical manifestation in different
genetic backgrounds, and effects on plastid de-
velopment.

Objections are constantly raised by other
authors as to the second genetic component of
plastid development, i.e. the "plastom" as being
the sum of the autonomous heritable elements
within the plastid (Renner). In the course of
the mutation experiments reported above,
however, we got some strong evidences in favour
of the existence of this plastom.

Just as has been found for other plants,
plastom mutations in Arabidopsis are to be
induced by a homozygous recessive nuclear gene.
Plastids so mutated remain in that state indepen-
dent of following changes in the nuclear gene
contents. Moreover, plastom mutations have
been produced for the first time by X-raying
of egg cells or zygotes as well as by treatment of
seeds with some alkylating agents (ethyl-
methane-sulfonate, di-ethyl-sulfate, etc.) The
extranuclear constitution of the resulting so-
matic variegation in all three cases above is
supported by the following evidences:

1. In light- and electron microscope true
"mixed cells" (Correns) with both normal and
mutated plastids have been found. The appear-
ance of the mutated plastids observed does not
seem to be principally different from that one
following after an equivalent gene mutation.

2. The pattern of variegation within a leaf is
correlated to the margin of a cell descendence.
In mixed cells of our standard variety of Arabi-
dopsis the rate of sorting out of both plastid
types is rather high; this effect, however, varies
with different residual genotypes. Using gene
dependent "chlorophyll mutants" for induction
of plastom mutations we found the resulting
variegated regions to be larger, pointing to a
smaller competitive superiority of the initial
plastids in these mutants.

3. Wherever in plant development the mutated
plastids reach the egg cell, a clear proof can be
made on their continuity by means of cytologi-
cal investigations as well as by demonstration
of non-Mendelian inheritance.

and colour of the roots, have been effectuated,
obtaining a total of 12 hybrids.

The study of reciprocal hybrids effectuated
by us during the 1959-1961 period showed the
following:

1. The number of plants of maternal type was
considerably higher than those of paternal type.
This phenomenon was noticed in Fi and F2,
manifesting itself as regards the habit in general,
as well as the observed characters in particular
of form and colour of roots. For instance out
of the 135 plants from the four reciprocal hy-
brids of Fi between various sorts of the form, a
total of 97 plants (71.8 per cent) were of
maternal type, 29 plants (21.5 per cent) were of
intermediate type and only 9 plants (6.7 per
cent) were of paternal type. In F2 out of a total
of 639 hybrid plants, 435 plants (61.1 per cent)
were of maternal type, 189 plants (29.5 per cent)
were of paternal type and 15 plants (2.4 per
cent) were of intermediate type.

2. The variation of hybrids was generally
very high, appearing after the segregation in a
large scale of colours and intermediate forms,
between parents. At the same time new characters
appeared, such as: lilac and violet colours of
the roots, or a longish-global form in the higher
part of the roots. These new characters appeared
in a great number of individuals (from the total
of 12 hybrids, 10 hybrids).

3. The appearance of the hetorosis pheno-
menon is in great measure influenced by the
way in which the crossing is effectuated. The
biometrical measure of plants showed that
sometimes great differences exist as regards the
vitality between direct hybrids and the reciprocal
ones. In some cases the direct hybrids mani-
fested heterosis such as: Eiszapfen $ Red with
white end <§, Tara Birsei $ : Red with white end
^, andSaxa$ Round white $', while the reci-
procal hybrids presented a biological depression.

4. The effectuated study by us showed the
outstanding importance of the way of crossing
on the segregation of hybrids and their vitality.

The predominant maternal type of the reci-
procal hybrids and the great difference as re-
gards their vitality could be explained by taking
in consideration the cytoplasmic heredity and the
metabolic influence of the maternal organism
on the hybrid seeds development.

12.17. Reciprocal Crosses by Raphanus sativus L.

P. Raicu and I. Popovici ( Bucharest, Rumania).

In our researches reciprocal crosses between
seven sorts of radishes of various forms, size

12.18. Hereditary Infections and Plasmatic Inheri-
tance in Drosophila. D. F. Poulson (New
Haven, U.S.A.) and B. Sakaguchi (Misima,
Japan).

The demonstration that several striking in-

206

Section 12 — Plasmatic Inheritance

stances of presumed cytoplasmic inheritance in
Drosophila are the consequences of hereditary
infection raises the question of the nature of
all non-nuclear inheritance in that genus.

The maternally transmitted condition referred
to as "sex-ratio", SR, in D. willistoni and D.
nebulosa was shown to be the result of the pre-
sence in the particular strains of hereditarily
transmitted spirochetes closely resembling those
of the genus Treponema. This was subsequently
demonstrated to be true in the cases of SR in
other related species, equinoxialis (Malogolow-
kin) and paulistorum (Malogolowkin and Poul-
son), of the neotropical willistoni group. On the
other hand, no direct evidences of spirochetes
could be found in the SR strain of the palearctic
species bifasciata. Success in transfer of the SR
condition from D. willistoni into D. melanogaster
and D. pseudoobscura provided unequivocal
proof that the spirochetes are the etiological
agents of SR. Expression and stability of the
transferred SR have been shown to be depen-
dent on a number of factors of which host geno-
type, spirochete strain, level of infection, and
environmental conditions, such as temperature,
are important.

Introduction of SR spirochetes from D. wil-
listoni into D. bifasciata has been carried out
with the result that a "sex-ratio" condition has
been artificially established showing many of
the features associated with naturally occurring
"sex-ratio" in D. bifasciata. The data suggest that
the SR condition in that species, although of
such long standing as to have lost its infective
character, had its origin in a similar way from
an infection which has now become wholly
stabilized. Other evidence indicates that here-
ditary infections are widespread in nature and
are the probable sources of most cases of plas-
matic inheritance. Our data will appear in a
series of papers being publishedin Geneti cs.

The relationship between both mechanisms
has been studied by means of ovary transplanta-
tions and of injection of cell-free extracts. Es-
pecially the last technique proved to be useful,
consisting in injecting extract of tumourous
flies tu A2 (recessive abdominal and thoracic
tumours) into tumourless ones. The results point
to a transmission of a factor which — originally
located in the cytoplasm and capable to migrate
from cell to cell — becomes eventually integrated
in the germ-plasm, and is transmitted with equal
efficiency by the egg and by the sperm.

Using the dominant mutant Freckled, lethal
in double dose, and apparently located in the
2nd chromosome, it proved possible to obtain
again a transmission to the progeny of non
Freckled individuals, injected with Freckled
extract. Transmission through the sperm has
been also demonstrated.

Moreover, Freckled can be transmitted also in
a way which recalls paramutation in maize,
since from heterozygotes Freckled, one recovers
a proportion of cychromosomes "infected" with
Freckled.

The results obtained so far show a mode of
transmisson which can be understandable in the
best way interpreting the involved factor as a
unit similar to an episome, the integration of
which within the nucleus and the cytoplasm is
demonstrated, while the integration within the
chromosome still requires further work to be
elucidated.

12.20. Action of FUDR on the Multiplication of
Virus of Drosophila. N. Plus (Gif sur Yvette,
France).

12.19. A Case of Episome-like Transmission in Dro-
sophila melanogaster (Melanotic Tumouis).
C. Barigozzi, A. M. Kravina and M. Sari
Gorla (Milan, Italy).

Melanotic tumours in Drosophila prove to be
a chromosomally inherited trait, when tested
with mendelian crosses; nonetheless, there are
different reasons to believe that an additional
extrachromosomal mechanism is at work, which
collaborates with the former one. The chromo-
somal mechanisms seems to be the most efficient,
while the extrachromosomal one seems to be
rather weak.

FUDR delays the multiplication of o virus,
when injected in the flies together with the virus.
This inhibition is not reversed by thymidine.
This nucleoside, by itself, acts as an inhibitor
of cr multiplication and its action is synergic
of FUDR action. The inhibition of virus produc-
tion by either FUDR or by thymidine is reversed
by addition of an equal quantity of deoxycytidi-
ne. FUDR seems, with respect to the virus multi-
plication, to act as an analogue of thymidine,
blocking deoxycytidine formation. These facts
seem to indicate that a virus contains DNA. This
conclusion is enhanced by the finding that both
FU and RNAase are ineffective on a virus multi-
plication. It has been so far impossible to ana-
lyse the virus nucleic acid directly.

207

Section 12 — Plasmatic Inheritance

12.21. Genetic Recombination with Drosophila <j

Virus. A. Ohanessian (Gif-Sur-Yvette, France).

The hereditary virus of Drosophila (<J virus)
shows numerous genetic variations. Several
genetic factors which are described, are con-
troling aspects of the relationships between the ct
virus and its host. It has been possible to estimate
the mutation frequency of two of them.

Genetic recombination has been demonstrated
with ct virus. The recombination takes place

when Drosophila eggs are mixedly infected with
two genetically different viral particles. One
resulted from the infection of an oocyte in a
non stabilized female and the other is brought
by a spermatozoon of a stabilized male.

Genetic recombination probably occurs at the
beginning of Drosophila ontogenesis. The re-
combinants are detected in the resulting adult fly.
In the course of these experiments some par-
tial heterozygous particles are found. They are
heterozygous for one of the viral genes only. This
phenomenon has been observed several times.

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SECTION 13

PLANT GENETICS AND BREEDING

13.1. Studies on the Interspecific Hybrid Lupinus
rothmaleri Klink. x lupinus luteus L.

T. Kazimierski (Poznari, Poland).

Lupinus rothmaleri possesses several qualities,
e.g. small-seedness, delicacy and others which it
would be very advisable to combine with the
properties of L. luteus. For this purpose attempts
were made to cross these two species. Hybrid
seed was obtained when L. rothmaleri was the
seed parent, and L. luteus the pollen parent.
Out of 174 L. rothmaleri flowers pollinated by
L. luteus 5 pods yielding 14 seeds were set. On
the other hand, out of 505 L. luteus flowers
pollinated by L. rothmaleri 134 pods were set but
they contained no well-developed seeds.

Hybrids of the first generation possessed inter-
mediate morphological features and physiologi-
cal properties. They were in the blooming period
since April 20 to October 20, but set only 4,
i.e. 0.4 per cent one-seeded pods.

Backcrossed hybrid flowers failed to produce
pods and shanked off after 12 to 20 days. Also
parent flowers mated with the hybrids wilted
after 10 to 14 days.

Analyses of pollen viability performed in a
mixture of acetocarmine with glycerin revealed
around 20 per cent (19.6) of viable pollen in the
hybrids. Cytoembryological studies of hybrid
ovules proved that the sporogenesis and game-
togenesis were normal only in 1.8 per cent of
ovules; in the remaining ones macrospores and
embryo sacs disintegrated. The high sterility of
hybrids was mainly due to aberrations in the
processes of macrosporogenesis and macrogame-
togenesis.

13.2. Meiosis in Triploid and Allotetraploid Hybrids
between Beta vulgaris L. and Species of the
Section Patellares Tran. in connection with
Transmission of Genes. Helen Savitsky
(Salinas, U.S.A.).

Hybrids between B. vulgaris (sugarbeet) and
diseases-resistant Patellares species (B. patellaris
Moq., B. procumbens Chr. Sm., B. webbiana
Moq.) are usually inviable and sterile; viable
semi-fertile triploid and allotetraploid hybrids

are obtained. Hybrids between B. vulgaris and
Patellares species are segmental hybrids. Sterile
diploid hybrids show 0 to 5 bivalents (n=9).
Triploid and tetraploid hybrids form complex
associations. Triploids form tri- and quadri-
valents, while allotetraploids form quadrivalents,
pentavalents, hexavalents, and higher valency
associations. Configuration of some multivalents
and excess of chiasmata over the limits possible
for vulgaris-patellaris associations indicate the
occurrence of translocations and structural
changes of chromosomes. Each multivalent
association involves 1 or more chromosomes of
Patellares species, or translocated chromosomes
of parental species. First backcross generation
(Fi x B. vulgaris) resembles sugarbeet, but carries
some characters of wild species. Segments of
chromosomes and some chromosomes of Patel-
lares species (in trisome plants) are transferred
to bi generation. Outline of meiosis indicated
the possibility of genes* transmission in Vulgaris-
Patellares hybrids.

13.3. The Allopolyploid Hybrid between Fragaria
vesca and F. moschata. J. R. Ellis (London,
Great Britain).

Many attempts have been made to hybridize
the diploid Fragaria vesca with the hexaploid
F. moschata, the object being in most instances
to combine the everbearing habit of alpine forms
of F. vesca with the highly esteemed musk
flavoured fruit of F. moschata. A small number of
sterile hybrids have been reported and these have
been either tetraploids, pentaploids or hepta-
ploids, the latter two types having originated
from unreduced gametes in the diploid and
hexaploid species respectively. No hybrids have
as yet been reported to have originated through
non-reduction in both parents, an event which
would give rise directly to the allo-octoploid
hybrid.

From a series of colchicine treatments and
hybridizations, a moderately fertile allo-octo-
ploid hybrid line has been established. Pentaploid
hybrids obtained from crosses between auto-
tetraploid F. vesca and F. moschata. were
colchicine-treated to give decaploid hybrids,

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Section 13 — Plant Genetics and Breeding

which on subsequent hybridization with F. mos-

chata gave allo-octoploid hybrids. The sequence

of colchicine treatments and hybridizations

being:

F. vesca (2x) -> F. vesca (4x) x F. moschata

(6x) -> 5x hybrid -> lOx hybrid F. moschata

(6x) x lOx hybrid -> 8x hybrid.

The allo-octoploid hybrids closely resembled
the hexaploid parent in vegetative characters
and in being dioecious. In view of this morpho-
logical similarity, it is conceivable that the
controversial mid-nineteenth-century variety
"Belle Bordelaise", could have been a sim-
ilar allo-octoploid hybrid, having originated
through chance non-reduction in both parents.
In such circumstances this octoploid derivative
could have provided an opportunity for F. mos-
chata to enter into the evolutionary history of
some present-day varieties of F. grandiflora.

13.4. Natural and Experimental Festulolium Hybrids
and Their Derivatives. F. Wit (Wageningen,
The Netherlands).

Lolium perenne, L. multiflorum and Festuca
pratensis are important forage grasses. In
addition to their valuable qualities, they show,
however, some imperfections which are difficult
to eliminate by conventional breeding methods.
Intergeneric hybridization might be useful,
either through the transfer of single desirable
characteristics or through the production of
stable and fertile alloploids.

A study of some natural hybrid swarms from
L. perenne (2/2 = 14) F. pratensis (2/7 = 14)

showed that the predominating plants were
triploids (73-94 per cent, and belonged to two
distinct groups). Both are supposed to have
partly originated by fertilization of unreduced
egg cells of the male sterile diploid hybrid. They
may be represented by the genome formulae
LLF and LFF respectively.

As unreduced gametes are rare, these triploids
must exhibit high competitive abilities. They
are, however, unstable and partly sterile.
Corresponding hexaploids might represent stable
and fertile plants.

The development of successful alloploids
requires efficient methods to provide for the
widest possible range of genomic and genetic
recombinations. Results of breeding experiments
suggest a method starting with the production of
hexaploids LLFFFF. Such plants can easily be
obtained either by colchicining seeds from the
cross Lolium 2x x F. prat ent sis 4x, or by cros-
sing Lolium 4x and Festuca 8x. By backcrossing
to diploid Lolium large numbers of tetraploids

LLFF are obtained, which by a second backcross
produce triploid seeds LLF. By colchicine treat-
ment these give rise to hexaploids LLLLFF.
The triploids LLF are reasonably fertile, they
may be the proper plants for starting introgression
breeding.

13.5. Breeding Spineless Gooseberries using Ribes
nigrum as Donor Parent. R. L. Knight and
Elizabeth Keep (Maidstone, Great Britain).

Seedlings and mature plants of black currant
(Ribes nigrum) are entirely devoid of spines.
Sterile diploid hybrids (BG) between black
cuirant (BB) and gooseberry (R. grossularia,
GG) and the fertile colchiploids (BBGG) derived
from them bear weak nodal and internodal
spines when juvenile but are free from both
types of spine on their mature growth.

Crosses between diploid gooseberry, as female,
and these alloploids failed, except for one cross
from which sterile diploids were produced. The
reciprocal crosses set few seeds most of which
failed to germinate, but three triploid (BGG)
and one approximately triploid plant were
obtained. The spine development of these
four plants was similar to that of self-bred
progenies from BBGG allotetraploids.

Crosses of the triploid (BGG), as female, with
diploid gooseberries (GG) failed, or in one case
gave a single very weak distorted seedling.
Crosses between diploid gooseberries, as females,
and the triploid (BGG) set poorly. From over
2000 flowers pollinated, 62 plants were raised,
all of which closely resembled gooseberries in
their vegetative growth and all appeared to be
diploid. Root tip counts of eight of these
confirmed them to be 2/7 = 16. Some plants with
much reduced juvenile spines, and no mature
spines were present. These have not yet flowered
but they should show adequate fertility for
crossing with a range of gooseberry varieties
for the immediate selection of commercial
spineless types.

13.6. Amphihaploids and Amphidiploids obtained
by Hybridization between Aurea Forms of
Nicotiana rustica L. and N. tabacum L.
Igor Bolsunov (Fiirstenfeld, Austria).

Interspecific hybridization of N. rustica var.
aurea x N. Tabacum var. aurea is extremely
difficult. By operating crossings of different
cultivars and strains belonging to the aurea

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Section 13 — Plant Genetics and Breeding

types of N. rustica with such of N. tabacum, the
author succeeded in finding genotypes which
permit relatively easy crossing. With the help
of acenaphten treatment of Fi subjects a great
number of amphidiploids with partial fertility
could be obtained, offering thus the possibility
of observing many amphihaploid and amphidi-
ploid plants under normal field conditions. On
this occasion the inheritance of morphological
and some most important physiological and
agronomical characters was studied. It was
interesting to note the following particularities:
disappearance of the "aurea" character in Fi,
increased resistance of Fi hybrids to some
diseases and unfavorable environmental con-
ditions, a clearly expressed heterosis with
amphihaploids and diminution of this phenome-
non with amphidiploids.

Testing of smoking qualities of fermented
leaves of amphidiploids N. rustica N. tabacum
gave positive results, especially with regard to the
use of such raw material as cigar filler.

13.7. Incompatibility Among Species of Darwinia.

Barbara G. Briggs (Sydney, Australia).

Interspecific crosses in Darwinia (Myrtaceae)
have shown several cases of non-reciprocal
incompatibility acting at about the time of
fertilization. The twelve eastern Australian spe-
cies and subspecies may be divided into two
groups, within which the species are interfertile.
In one group the flowers are small and regularly
self-pollinate. The members of the other group
are also self-compatible but show considerable
outbreeding. Crosses between the groups are
fertile when an inbreeder is used as the female
parent. In the reciprocal cross, best studied in
the example D. fascicularis $ x D. bifiora $,
pollen of an inbreeder germinates on the stigma
and the growth of pollen tubes to the ovary is
unimpaired, but failure apparently occurs just
before fertilization. The combination of incom-
patibility and breeding behaviour prevents or
greatly restricts the formation of natural hybrids
between some combinations of species. The
genetical and physiological basis of the incom-
patibility reaction is unknown.

13.8. Incompatibility in Betula verrucosa Ehrh. and
Betula pubescens Ehrh. Max Hagman (Maisa-
la, Finland).

Self-incompatibility is found in both Betula
verrucosa and B. pubescens. The incompatibility

inhibition occurs in the style and is expressed
through retarded growth of the pollen tube.
Interspecific incompatibility of the same type
occurs when the two species are crossed. How-
ever, the incompatibility reaction is more pro-
nounced when B. verrucosa is used as father. In
the reciprocal cross many of the combinations
hitherto made show a certain percent of B. pu-
bescens pollen tubes with good growth in the
style of B. verrucosa. This agrees with earlier
results that more filled seed is obtained when the
species cross is made in the direction B. verrucosa
$ > B. pubescens $•

In the 25 combinations made with 5 trees of
B. verrucosa from the same stand there are 7
combinations indicating cross-incompatibility.
In 20 individuals from the distribution limit of
B. verrucosa in Northern Lapland and from
isolated islands in the southwestern Finnish
archipelago there was no tendency toward
increased self-compatibility.

Low temperature slows down the incom-
patibility reaction so that fertilization may
occur after selfing. The same effect may be
reached through pollination at a late stage of
development of the female flower. The knowledge
of these facts may help the forest tree breeder
carrying out inbreeding as a breeding programme
for birch.

There are serologically detectable differences
between the pollen of the two species. Individual
differences which could be used as a guide for
selecting compatible combinations are under
investigation.

13.9. Radiation-induced Chromosomal Aberrations
which Simulate Point Mutation of Incompatibil-
ity Alleles. James L. Brewbaker (Honolulu,
U.S.A.).

Self-incompatible flowering plants of the
gametophytic or Nicotiana type mutate with
comparative ease to self-fertile (SF) forms.
Mutants were sought in petunia following pie-
meiotic X-irradiations among 40 x 106 pollen
grains. A 16-fold increase in mutation was ob-
tained at 1000 r. Over 300 mutants were studied
cytogenetically. Most mutants were self-fertile,
and most of these reacted in crosses as if the
pollen activity alone had been lost ("pollen-
part" mutations). With few exceptions, these
plants carried supernumerary centric fragments on
which the S gene was located. Fragments varied
greatly in size, and could be shown to pair
with trisomes bearing the S locus. Subsequent
fragment loss was held to account for self-

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Section 13 — Plant Genetics unci Breeding

incompatible offspring (called "revertibles" by
some authors) among the mutants. Exceptional
SF mutants included several tetraploids, and
rare deletions. Colchicine-induced tetraploids
were selfincompatible only when homozygous for
S alleles (5 tested); all heterozygous tetraploids,
triploids, and S-gene trisomes were SF. The
following conclusions are drawn: Incompatibili-
ty mutations, in petunia at least, rarely if ever
involve intragenic change; aberrations which
lead to loss of pollen activity include duplication
(largely through centric fragments) of the locus
and deletion. The data do not support theories
of bipartite S gene structure or of revertible
mutations; they do encourage consideration of
the possibility that point mutations are not
expressed due to the probable multiple-stranded
structure of the chromosomal gene.

13.10. Pollen-Style Incompatibility in Oenothera.

Adolph Hecht (Pullman, U.S.A.).

Self-incompatible races of Oe nothera organensis,
O. rhombipetala and O. caespitosa are being
studied by the author and his students in an
attempt to discovei the mechanisms of the
incompatibility reaction. Unlike most other
plants that have a gametophytically determined
incompatibility, the pollen of these species is
inhibited at the time of its germination on the
stigma, and few if any pollen tubes in an in-
compatible mating even reach the style. When the
cut ends of styles were reunited in a moist cham-
ber and their stigmas pollinated with compatible
pollen, the cut surfaces resulted in only a slight
inhibition to the growth of pollen tubes from
the upper (scion) portion into the lower (stock)
portion of the style. When stylar tissues incom-
patible with the pollen were used as the stock
portion of the graft, there was an appreciable
inhibition of the pollen tubes, but some of them
did grow several centimeters into this otherwise
incompatible tissue. Compatible pollen tubes
passed readily from the cut end of a style into a
stigma lobe which was also compatible with the
pollen, but when a stigma incompatible with the
pollen was used in this position below a cut
style, the pollen tubes were completely stopped
at the stigmatic surface. These experiments
show that the incompatibility reaction in these
species is considerably stronger in the stigma
than in the style.

Work supported by National Science Foun-
dation Grant G-14056.

13.11. A Theory of the Control of Complex Unilateral
Incompatibility in Lycopersicon hirsutum.

Franklin W. Martin (Mayaguez, Puerto
Rico).

Unilateral incompatibility is the ability of
two hermaphroditic plants to cross in only one
of the reciprocal combinations. The type consid-
ered here is similar to self-incompatibility in
that both are due to inhibition of pollen-tube
growth in the style. In Lycopersicon hirsutum and
L. esculentum this phenomenon has 4 levels of
expression. The various compatibilities occur in
a step-wise sequence suggesting a single form of
behavior with various levels of manifestation.
Measurements of inhibited pollen tubes indicat-
ed two kinds of variability among 5 accessions of
plants tested: variability in strength of an in-
hibiting reaction in the style and of a stimulating
reaction in the pollen. The presence of different
balances of stimulating and inhibiting substances
could account for this hierarchy of incompatibi-
lity reactions. This theory is consistent with
knowledge of the occurrence of such substances.
Evidence for the occurrence of different balances
of such substances has come from 4 other sources.
First, measurements of pollen tube growth
within styles showed variances associated with
degree of heterozygosity of a single, partially
imbred parent. Secondly, segregation for degree
of inhibition of pollen-tubes occurred among
plants of a single family. Thirdly, crossing among
selfincompatible plants from different geogra-
phical sources sometimes destroyed the balance
resulting in pseudo-compatibility. Finally, the
inheritance of unilateral compatibility was
conditioned by many minor genes in hybrids.
The body of evidence suggests polygenic control
of the quantity of growth substances, modified
by environmental factors.

13.12. Changes in Pollen Proteins during Pollen
Tube Growth from the Incompatibility Point
of View. Jaroslav Tupy (Prague, Czechoslo-
vakia).

Proteins of the pollen with different S alleles
are serologically distinguishable (Lewis, 1952;
Linskens, 1960; Makinen, Lewis, 1962).
This fact supports the antigen-antibody hypo-
thesis of the incompatibility reaction. The ger-
mination and the growth of pollen tubes in in-
compatible styles is normal during first hours
with the gametophytic system of incompatibility.
In Nicotiana alata the incompatible tubes often
attain the length of as much as 4 cm without any
inhibition. That is why we can expect changes in

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Section 13 — Plant Genetics and Breeding

pollen proteins during the pollen tubes growth.

Pollen proteins of Nicotiana alata were se-
parated into at least 14 fractions by means of
gel filtration and chromatography on Sephadex
and DEAE-cellulose. After 15 hr artificial cul-
tivation under aseptic conditions new protein
fraction was found. More than one half of the
total proteins of pollen tubes have been released
into the medium. The same fractions, but in
relatively different quantities, have been found
both in the proteins of medium and in those of
pollen tubes. The new fraction occurred mainly
in the medium. After the administration of L-
proline-14C the highest specific activity in this
fraction was determined.

It is possibler, therefore, that the proper specific
proteins which are responsible for incompatibili-
ty reaction in species with a gametophytic system
of incompatibility are synthesized during the
pollen tube growth.

The corresponding data will be published in
full in Biologia Plantarum, Praha.

brids of an intermediate type. Such are P. spinosa
L. P. domestica L. hybrids (2 n = 40) and
P. ceiasus L. P. avium L. hybrids (2 n = 24).

When these low-fertile hybrids (Fi) were pol-
linated with the pollen mixture of several va-
rieties of one of parental species, F-2 consisted
of quite fertile and commercially valuable
plants with the characteristics of both species.

The second variation consists of the addition
of small amounts of own or alien pollen related
to maternal form to the pollen of an uncros-
sable species. This variation is based on the
fact that in the process of fertilization own or
alien pollen favourably affects the pistil, creat-
ing conditions which stimulate the growth of
pollen tubes and the fertilization with the pollen
of an uncrossable or hardly crossable species.

The analysis of morphological characteris-
tics and chromosome numbers in Fi hybrids
shows that preferable fertilization with own or
closely related pollen takes place in these crosses,
though in some cases this method helped to
cross two hardly crossable species, for example,
Ceiasus Besseyi Bail Primus cerasifera Ehrh,
(2 n = 16); C. Besseyi P. spinosa. (2 n = 24.)

13.13. Pollination with Pollen Mixture to Produce
Interspecific Hybrids of Plum and Cherry
(Prunus), K. K. Enikeyev (Moscow,
U.S.S.R.).

Soviet horticulturists use the method of pol-
lination with pollen mixture in interspecific
crosses.

This method was worked out by I. V. Michurin
in order to overcome incompatibility of different
fruit species. I. V. Michurin based this method on
the regularities of selective fertilization and
complex physiological interaction between the
components of pollen mixture and the pistil of a
maternal form.

The author has used two variations of the
pollination with pollen mixture and got the
following results:

The first variation consists of the pollination of
one species with the pollen mixture of several
varieties of another species which is almost or
entirely unable to cross with the former, it se-
cures the selective fertilization by means of the
most biologically adapted pollen grains of one
of the varieties of a paternal form.

As a result of pollinating Cerasus Besseyi
Bail with pollen mixture of several European
varieties of apricot (Armeniaca vulgaris L.) quite
viable and fertile hybrids have been produced.

The hybridization of several Prunus species
with different chromosome numbers in Fi
results in the development of low-fertile hy-

13.14. Transfer of Self-Compatibility from Solanum
verrucosum to Diploid S. tuberosum. L. A.

Dionne and K. M. Graham (Birmingham,
Great Britain, and Fredericton, Canada).

The development of homozygous diploid lines
of Solanum tuberosum has been complicated by
the extreme selfincompatibility of the diploid
forms of the series Tuherosa. Attempts to isolate
mutations for self-compatibility from the di-
ploid Tuberosa have not given much encourage-
ment to this means of solving the problem. How-
ever, it has proved to be relatively easy to transfer
the self-compatibility of Solanum verrucosum, a
Mexican diploid species in the series Demissa, to
S. tuberosum. By this means it has been possible
to establish breeding lines that combine a high
level of self-compatibility with earliness and good
resistance to Phylophthora infestans.

13.15. Aneuploids in Some Artificially induced
Polyploids of Cultivated Plants. Mechtild
Rommel (Zaragoza, Spain).

When investigating chromosome numbers of
artificially induced autotetraploid barley stocks
(Hordeum vulgare), autotetraploid sugarbeet
populations (Beta vulgaris) and wheat-rye
derivatives (Triticum durum Secale cereale)

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Section 13 — Plant Genetics and Breeding

many plants were found not possessing the
euploid chromosome number. While in diploids
loss of a chromosome seems to be lethal many
polyploid plants with off-numbers will survive.
But gain or loss of one or more chromosomes or
even the presence of a fragment apparently
disturbs plant development thus making these
plants unsuitable to grow as commercial crop
plants.

In autotetraploid barley (4.v =•- 28) euploid
plants produced progenies with 38-57 per cent
euploid plants only. Among poor seeds aneu-
ploids were much more frequent than among
well-developed ones. Aneuploid plants showed
great variation in morphological appearance
ranging from very poor to almost normal growth.
Fertility of aneuploids was much more reduced
than fertility of euploids.

In populations of autotetraploid sugarbeets
(4.v = 36) aneuploidy occurred in about 42-44
per cent of all plants investigated. Growth of
leaves and roots varied from very poor to almost
normal. Weight of aneuploid roots seldom
reached that of euploid ones.

In some strains of wheat-rye hybrids
(6x = 42) aneuploids were present but in lesser
number.

The occurrence of aneuploidy in autotetrap-
loids of homozygous and heterozygous plants as
well as in newly established amphipolyploids
enlightens the fact that production and/or
survival of off-number gametes and zygotes
must be primarily conditioned by induction of
polyploidy. Their presence necessarily leads to
reduce fertility in polyploids. In addition the
number of aneuploids in a plant progeny ap-
parently is depending upon the genotype used
and upon environmental conditions.

fertility can be related to the frequency differen-
ces of aneuploids. In 1962 aneuploid plants had
a reduction in fertility of appr. 18 per cent, as
compared to euploids. The fertility of the euploid
plants of the most thoroughly examined tetra-
ploid population had a fertility which was only
3.3 per cent below that of the corresponding
diploid population.

Meiosis has been studied in euploid plants
belonging to populations which differ in fer-
tility and frequency of aneuploids. In this con-
nection the types of diakinese configurations,
frequencies of bivalents and multivalent chro-
mosome associations, co-orientation of chromo-
somes at first metaphase and chromosome dis-
tribution at first anaphase have been studied.
The mechanism leading to differences in the
frequency of aneuploids will be discussed.

13.17. Different Fertility of Mitotic and Meiotic
produced 4x-Forms in Primula malacoides
Franch. Kurt Skiebe (Quedlinburg/Ffarz.,
Germany).

The fertility of Primula malacoides was stu-
died, both generated by colchicine induction
and 2 x x hybridization. Plants within the
types were crossed exclusively. Comparison is
made from Ci to Cs. The fertility of the meiotic
produced 4x forms exceeds significantly the
mitotic originated ones. Therefore in future 4x
forms from unreduced gametes only should be
used in polyploid breeding.

13.16. The Relation between Fertility and Cyto-
logical Properties in Autotetraploid Rye.

Knut Aastveit (Vollebekk, Norway).

Twelve years ago six autotetraploid popula-
tions of winter rye were produced by colchicin
treatment. The original tetraploid populations
have since been kept isolated. At the same time
new populations have been established by inter-
crossing and selection. In 1960, 1961 and 1962 a
number of populations were compared in respect
to fertility (seed set). Highly significant differen-
ces were found in several cases. It is concluded
that the fertility of autotetraploid rye is a heritable
character, which can be influenced by selection.

Populations differing significantly in fertility
are now under cytological investigation. As far
as our results go, the population differences in

13.18. Study of Pollen and Pollination in Rela-
tion to Partial Sterility of Autotetraploid
Rice. J. Bouharmont (Louvain, Belgium).

In autotetraploid rice, there is no correlation
between the rate of meiotic irregularity and the
level of sterility, but a number of factors take
part in the reduction of seed setting. An
important one is the relatively lower quantity of
good pollen shed on the stigmata. We have to
consider the quantity and the quality of the pol-
len produced in the anthers, the abundance of
pollination and the pollen germination.

After chromosome doubling, the number of
stainable pollen grains per spikelet is reduced by
about 30 per cent: the number of PMC's is
lowered (18 per cent) and some microspores
fail during their maturation (12 per cent). The
diploid pollen grains are larger than the haploid
ones (20 per cent) and their size is more variable:

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Section 13 — Plant Genetic and Breeding

the coefficient of variation is 7.3 per cent for the
latter and varies between 10.7 and 15.0 per cent
for the former.

Though the quantity of viable pollen in tetra-
ploid flowers is still sufficient, the number of
grains deposited on the stigmata is generally
lower than in diploid plants and a variable
percentage of spikelets are not at all pollinated.

The pollen germinates on the stigma as soon
and as well in tetraploid as in diploid plants.
Nevertheless, many pollinated flowers fail to
set seed; thus if the low pollination is one cause
of the partial sterility of tetraploid rice, it is
not the only one.

These data on pollination and the study of
other causes of sterility will be published later,
probably in La Cellule.

13.20. The Location of a Gene for Male-sterility in
Linkage Group I of Barley. J. Milohnic
(Zagreb, Yugoslavia).

Crosses have been made between male-sterile
(ms ms; Suneson) lines of H. vulgare and normal
(Ms Ms) H. distichum. The former species is
six-rowed (v v) and has a low number of rachis
internodes (rin rin). The latter species is V V
Rin Rin. F2 analysis shows that ms is linked to
the two chromosome I loci. The order is as fol-
lows (rec. per cent by product-ratio): v - 30.7 -
rin - 33.8 - ms. Rec. per cent between v and ms
is 48.7. It was also found that ms ms reduces
the number of rachis internodes in the Rin-rin
genotypes.

13.19. Pollen Morphology in Relation to Pollen
Germination and Seed Set in Tetraploid Red
Clover. Marie Bragdo, (Vollebekk, Norway).

Pollen morphology, judged by the aceto-
carmine-glycerine-method, and seed set were
studied in 1961 in 176 plants. The following
characters were observed:

Xi Per cent good pollen grains (well stained

and not shrivelled).
X2 Diameter of good pollen grains.
X3 Variation in diameter of good pollen grains.

y Number of seeds per head after open polli-
nation in the field.

AsXi was closely correlated with X2, the latter
was discarded. No correlation was detected
between Xi and X3. Y was positively correlated
with Xi, and negatively correlated with X3.

Some plants, selected for pollen quality ac-
cording to morphology, were propagated by
cuttings. In 1962, 15 plants of each clone were
planted in a field and pollen morphology stu-
died. There was a good agreement between the
percentages of good pollen grains in 1961 and
1962. A test of pollen germination on sugar-agar
showed this to be significantly correlated with
percentage of morphologically good pollen.
Thus, pollen morphology, judged by the aceto-
carmine-glycerine-method gave a fairly good
indication of the relative germinability. The
average of germinated pollen grains was lower
than the average of morphologically good pollen.

On account of the selfsterility in red clover,
the seed set cannot be much influenced by the
pollen quality of the same plant. The association
must be due to some other factor, with which
both are correlated, probably the quality of the
female gametes.

13.21. Tetraploid Inheritance of Monogerm Charac-
ter and Male-sterility in Beta vulgaris L. (Sugar-
beet). V. F. Savitsky (Salinas, U.S.A.).

1. Monogerm character (gene in) is inherited
in tetraploids on the basis of chromatid assort-
ment at an intermediate frequency of double
reduction (parameter a). Value of a is stipulated
by an intermediate frequency of crossovers
between the locus and kinetochore (parameter
e), and by an intermediate number of quadri-
valents formed (parameter a). Gene expression:
nulliplex nu produces monogerm fruits; sim-
plex Miiri3 and the rest of genotypes produce
multigerm fruits.

2. Gene a, responsible for mendelian male-
sterility, and gene m are located in different chro-
mosomes. Gene a is inherited on the basis of
chromatid assortment, but the value of its
parameter a is greater than the value of a for
the gene m. Gene a is located further from its
kinetochore than the gene m. Gene expression:
nulliplex a4 causes complete male-sterility;
simplex Aia3 and all other genotypes are male-
fertile.

3. Inheritance of cytoplasmic-genetic type of
male sterility in tetraploids indicated the pre-
sence of only 1 basic gene which controls pollen
fertility in cytoplasmic male-sterile races, not of
2 genes as often assumed in studies of diploid
heredity. This gene is inherited on the basis of
chromatid assortment.

13.22. (D.) Sterility in Broad Bean (Vicia faba L.).
D. G. Rowlands (Bedford, Great Britain).

The demonstration shows that sterility in
Broad Bean is not a simple system but operates at

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Section 13 — Plant Genetics and Breeding

different stages in the breeding system. Under
field conditions only 10-20 per cent of the total
flowers produced on a plant ultimately set pods,
and under controlled self-pollination this pod set
may be as low as 2-3 per cent.

In some genotypes, damage to the papillae of
the stigma during pollination by insects or
mechanical means is necessary for pollen
germination and pollen tube development. In
such genotypes, foreign pollen appears to have
a developmental advantage over pollen from the
same plant. Unsatisfactory pollen tube devel-
opment causes the flower to absciss.

When pollen tube growth is normal, the young
ovary develops until it reaches 20-22 mm in
length, but if fertilization is not effective, the
young ovary falls off at this stage.

After self-pollination of some genotypes, up
to 80 per cent of the pods initially developed may
reach a length of 40 mm. Pod development then
stops and the ovules within these pods de-
generate. Occasionally a single ovule develops
normally, in which case, degeneration of the
other ovules occurs at a much later stage.

Embryological studies show that this ovule
degeneration in Broad Bean resembles thesoma-
toplastic sterility in Medicago sativa as described
by Brink and Cooper.

13.23. Effectiveness and Specificity of Ionizing
Radiations and Some Chemical Substances in
Inducing Mutations in Winter Wheat.
V.V. Khvostova, V. S. Mojaieva and N. S.
Aigaes (Moscow, U.S.S.R.).

1. A comparison was made of the mutagenic
effect of gamma-rays in dosages of 10, 15 and
20 kr and of fast neutrons with energy 1-2
MeV in dosages of 350 rad (210 rad for neutrons
and 140 for gamma) and of 750 rad (425 rad for
neutrons and 286 for gamma) and ethylene imine
in concentrations of 0.01-0.04 per cent treating
air-dry seeds (at room temperature) of winter
wheat Triticum-Agropyrum hybrid- 186.

2. Taking into account the largest number of
progenies segregating mutants in M2, the most
effective mutagene proved to be ethylene imine in
concentrations of 0.01 : 50.8 percent of progenies
segregated mutants; on the second place stood
fast neutrons in dosages of 740 rad, producing
36.5 per cent of progenies with mutants, on
the third place were gamma-rays in dosages of
10 kr: 6.2 per cent of progenies.

3. The spectrum of the mutations, appearing
under the action of neutrons, gamma-rays and
ethylene imine proved to be different. The nar-
rowest spectrum was produced by the neutrons:

a total number of 208 mutants showed 80 per
cent of erectoids, squareheads and speltoids. and
only 20 per cent other changes. Among 81
mutants, which appeared after gamma-ray
treatment, the number of other types of mutants
amounted to 33 per cent. After treatment with
ethylene imine there were found 100 mutants, 34
per cent belonging to the above mentioned three
types, 66 per cent various other forms.

Thus, ethylene imine appeared to be the most
effective mutagene, producing at the same time
the most broad mutation spectrum.

4. Under the action of these mutagenes,
valuable agricultural forms are produced:
resistant to lodging, to fungal diseases, and
highly productive.

13.24. Mutation Research in Canary Grass.

D. E. Bremer-Reinders (Wageningen, The
Netherlands).

In studying the influence of chemical mutagens
and X-rays on the frequency and spectrum of
mutations in Phalaris canadensis (2w = 12)
comparable doses (seedling percentage surviving
treatment) were chosen.

Irradiation with 15,000 r decreased the num-
ber of seeds per spike in the Mi -generation to
about 50 per cent of the control. A corres-
ponding dose of ethylene imine decreased the
Mi-fertility to about 36 per cent of the control,
while the corresponding E.M.S. treatment
decreased the fertility of Mi to about 15 per
cent of the control.

Of 406 progenies tested after irradiation
(15,000 r) on an average 20 per cent contained
chlorophyll mutations. With ethylene imine this
was nearly 27 per cent in 78 progenies, while with
E.M.S. this amounted to nearly 30 per cent in
325 progenies. Related to the number of seed-
lings the percentage of chlorophyll mutants was
nearly 3 per cent with 15,000 r and 2.6 per cent
with ethylene imine, while it amounted to 7 per
cent with E.M.S.

The spectrum of chlorophyll mutants, after
chemical treatment, had more common chloro-
phyll aberrants, while in the case of irradiation
with 15,000 r more of the rarer mutant chloro-
phyll types and other morphological seedling
mutants occurred.

With 15,000 r and with E.M.S. there was a
correlation between the occurrence of chloro-
phyll mutants and major morphological mutant
types. In both cases (406 and 325 progenies
respectively) the chance that morphological
mutants will appear is more than twice as great
in M2-families with chlorophyll mutants than in

216

Section 13 — Plant Genetics and Breeding

families without chlorophyll mutants. In the case
of ethylene imine no correlation was found,
perhaps on account of the low number of proge-
nies tested (78).

13.25. A Contribution to the Effect of Acute Gamma-
Radiation of Jonathan Apple Scions. P. D.

Misic (Cacak, Yugoslavia).

In order to improve Jonathan apple, besides
the method of hybridization, acute gamma-radia-
tion of dormant scions has been used. The
applied doses of gamma-rays, ranging from
1500 rep (1 hr 7 min) to 10,000 rep (7 hr 20
min), showed the different effects on scion sur-
vival, length of growth and changes of leaves and
shoots.

There was no visible suppressing effect of the
lowest gamma dose (1500 rep) on scion survival.
The doses increasing from 3000 rep to the highest
one (10,000 rep), however, showed clear suppres-
sing effects (3000 and 4500 rep) or full lethal ones
(6000 and 10,000 rep).

Total new growth per scion showed no statis-
tically significant difference between control and
1500 rep, but the reductions in growth were
significant among 1500, 3000 and 4500 rep
respectively.

The induced changes such as disturbance of
ortostichy and appearance of rosette, pinnately-
serrate leaves, adventive shoots and bifurcation
of leaf veins became more frequent when the
dose increased. On the other hand, the per-
centages of induced dichotomy per total number
of shoots, leaf chlorosis and bifurcation of leaf
petiols were less frequent when the dose
increased. The doses of 1500 and 3000 rep,
however, produced rather high and approximate-
ly the same rates of induced dichotomy shoot
growth (19.5 and 19.4 per cent respectively)
per cm of total growth.

To summarize: the lower acute gamma-radi-
ation doses applied (1500 rep in 1 hr 7 min and
3000 rep in 1 hr 13 min) showed more numerous
primary effects.

13.26. Formation Process in Avena sativa provoked by
the Influence of Ionizing Radiation. G. M. Zak-

harova and I. E. Gloushchenko (Moscow,
U.S.S.R.).

It is known that under the influence of different

factors which affect negatively the development
of plants (low temperatures, high doses of
ionizing radiation, some chemical compounds)
there occur plants in the oat, progeny (Avena
sativa) with characters of wild oats (Avena
fatua). The results obtained by us while studying
the formation process in oats show the same
phenomenon.

The experimental work has been carried out
with two oat varieties: Pobeda (Victory) and
Dippe. They had white grains and awnless
spikes (or spikes with delicate awns). The dry
seeds of these varieties have been irradiated by
X-rays, the doses being 8000-13,000 r. Plants
obtained out of non-irradiated seeds have been
used as a control.

Cytological and field investigations have
shown that the chosen doses of X-rays affect
negatively the development of cultured oats.
By the further studies of irradiated forms of prog-
eny it has been established that the variety Dippe
in X3 had 4.3 per cent of plants with wild oat
characters and 1.4 per cent of plants with the
characters of both oats and wild oats; in x4 5.5 per
cent of plants with the characters of wild oats and
0.3 per cent of plants with oats and wild oats
characters.

The Pobeda in X3 had 9.4 per cent of plants
with wild oat characters and 0.2 per cent of
plants with the characters of oats and wild
oats; in X4 32.7 per cent of plants with wild
oat characters and 0.6 per cent with oats and
wild oats characters.

The newly-formed wild oat forms as a rule
are dwarfish and completely or partially sterile.
It should be noted that the plants with wild oat
characters had white spikes (maternal type).
It is important that the modified forms have
appeared only in X3 and in the progeny of the
plants which in Xi and X2 had coarse curved
awns. No wild oat forms have been noticed in
the control nor in the irradiated plant
progeny which had spikes with awns, no awns
or coarse awns.

At present it has been established that ionizing
radiation disturbs physiological and biochemical
processes and this no doubt changes different
biological properties of the organism. To all
appearance x-rays in the dose of 8000-13,000 r
provoke such important changes in the metabo-
lism that awnless oat plants or plants with
delicate awns give forms with coarse curved
awns. The latter have labile heredity, a fact
demonstrated by the presence in the progeny of
such plants of great diversity. As a result of the
disturbance of physiological harmony there
appear in the progeny of irradiated oat plants
new specific forms with the characters of wild
oats.

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Section 13 — Plant Genetics and Breeding

13.27. Variability of Anthocyan in Chimera Cabbage
Plants. A. S. Kruzhilin, I. E. Gloushchenko,
Z. M. Shvedskaja and L. K. Sokolova
(Moscow. U.S.S.R.).

1. The anthocyan colour of plants is a stable
hereditary character which is inherited both by
sexual and vegetative reproduction. Therefore
we have studied possible variability of anthocy-
ans in intraspecific grafting of cabbage forms of
different colour — i.e. in cabbage chimeras
(developed from the callus) and their seed
progeny.

2. It has been shown that under the influence
of non-coloured plants of the stock which do not
contain anthocyan, the content of anthocyan of
rubobrassicin type in the coloured scion plants is
considerably reduced. The analogical phenome-
non is seen in the chimera sprouts. In spite of
their cutting off the coloured chimera sections,
the content of anthocyan is very low. The same
is also true for their further vegetative propaga-
tion (by cutting) and for their sexual repro-
duction. Therefore the hereditary character of
the plants — their colour — is changed in chimeras
obtained by grafting. This shows that callus
tissues of the chimeras do not exist autonomous-
ly, but interact physiologically by means of
metabolism. Their properties modify in the
course of seed progeny development. Therefore
we believe the intraspecific cabbage chimeras to be
of the vegetative hybrid type.

3. As to the anthocyan content, the coloured
plants of chimera seed progeny and of sexual
hybrids of the same combinations are almost
alike. This indicates that the variability of
plants obtained by grafting and crossing is of the
same type.

4. Anthocyan is synthesized and accumulated
in leaves and stalks. It is not found in the roots,
even of the plants with coloured surface organs.

13.28. The Study of Intraspecific Cabbage Chimeras
and Their Seed Progeny. I. E. Gloushchenko
(Moscow, U.S.S.R.).

joined together (at the place of the cutting), the
adventive buds of hybrid nature are formed.
They have different amounts of genetical charac-
ters of grafting components. The highest
percentage of hybrid plants in our experiments
amounted to 30.7 per cent.

It has been shown by the research that the
plants obtained out of the callus tissues are
young as to their stage of development and are
biologically identical with those obtained out of
the seeds. The taking off of the developmental
processes occurs when the places of multicellular
callus tissue are formed. These places are charac-
terized by the higher basophily of the protoplasm,
which, as a rule, is an indication of cell youth.

The studying of seed progeny of intraspecific
cabbage chimeras has confirmed the hybrid
nature of the experimental plants. When crossing
of the initial components (control) produces in
the first generation only the plants of one type
which have intermediate inheritance of the main
characters (colour and form of stalks and
leaves), the chimeras of the first seed generation
possess a great variety of these characters
(segregation).

There has been established the following
regularity: when seeds are taken directly from the
grafted plants, the diversity of the progeny is
very great; when seeds are taken from the chime-
ra cuttings the variation of the progeny is not so
great. However, in the latter case the variation is
different from that of the crossing.

The second generation of the chimera grafted
group possesses greater diversity than the first
one.

When a callus occurs as a result of different
species grafting and then decapitation, there
are also formed adventive buds and sprouts of the
chimera type. The progeny of such sprouts
however have, as a rule, the characters of one
species.

Consequently the nature of intraspecific
cabbage chimeras which are practically one of the
forms of vegetative hybridization is different
from that of interspecific chimeras.

The nature of intraspecific cabbage chimeras
and their seed progeny has been studied by the
author during eight recent years.

It has been determined that in the progress of
callus tissues forming, there take part the products
of scion and stock protoplasts destroyed by the
cutting. The basic character of the callus is the
small-celled tissue which is formed out of
cambium as a result of the trauma.

When the callus occurs at the place where
two varieties belonging to the same species are

13.29. Alterations of Hereditary Traits in Solanum
melongena induced by Grafts with Solanum
nigrum. C. C. Mathon, M. Stroun and
J. Stroun (Poitiers, France).

The pupil plant is the "white round" egg-
plant, and the mentor plant a strain of black
nightshade.

The pupil is the epibiota and the mentor the
hypobiota. Controls are represented by homo-
grafts of the pupil variety. The technical con-

218

Section 13 — Plant Genetics and Breeding

ditions are as follows: for the pupil plant: of a
less advanced age than the mentor-plant and
total removal of leaves all along evolution; for
the mentor plant: maintenance of leaves and
absence of flower-buds which are cut off as soon
as they appear.

The influence of the black nightshade mentor
on the "white round" eggplant pupil became
apparent only in the offsprings of the third
generation of grafts. Out of 7 symbionts carrying
fruit at the third generation of grafts, 2 offer in
their descendency an alteration that so far
prevailed down to F3: the colour of the stem is
slightly purple. These anthocyanins are extreme-
ly thermolabile. Chromatographic analyses made
so far show the Rf of anthocyanins to be differ-
ent from those found in the black nightshade.

In these interspecific grafts we did not observe
any mentoi -oriented alterations. However, the
new traits that have become apparent in the
descendency of both modified plants are of the
same type, therefore, the influence of the mentor
may be assumed to work in both pupils in the
same direction.

Article to be published in: Comptes rendus des
seances de la Societe de Biologie de France, 1963 ;
Archives des Sciences de Geneve, 1963.

13.30. Alteration of Traits obtained in Solanum
melongena as a Consequence of Inter-variety
Grafts. M. Stroun and J. Stroun (Poitiers,
France).

The "Early Violet" variety was used as mentor-
plant and the "White Round" variety as pupil-
plant. The mentor-epibiont / pupil-hypobiont
grafting procedure is applied as follows: the
pupil-plant is totally rid of its leaves all along
its development, whereas the mentor-plant
retains its foliage but is rid of its flower buds
which are cut off. The graft is repeated on each
new generation until alterations appear. Sexual
crossbreeds were also made "White Round" § x
"Early Violet" $.

The 19 homografted "White Round"/"White
Round" controls did not show any alterations.

The influence of the mentor-plant "Early
Violet" on the pupil variety "White Round"
appeared only at the third generation of grafts.
Out of 24 fruit-bearing symbionts, 9 presented
various alterations in fruit and stem colour,
shape of fruit, or in a trait of the stamen. These
alterations resemble the traits of the mentor
variety. They are found again in the sexual
descendants of the modified symbionts, studied

so far down to F2. They often differ from the
characteristics conveyed by sexual crossings
between the two varieties used. There is a dis-
junction in Fi, only part of the alterations being
transmitted. Futhermore, new traits appear on
certain plants. To all appearances therefore,
grafts, as practised by the authors, seem to
influence the hereditary traits of a given variety.

C. R. Acad. Sci. 255, 561-563, 1962; Arch. Set.
Geneve, 1963, to be published.

13.31. To a Correlation of the Objectives in
Tree Breeding. C. Lazarescu and E. San-
duleac (Bucharest, Rumania).

Since trees constitute the most constant
vegetable covering of the earth, the approaching
of the problem of tree breeding from the point
of view of timber production — as that is
applied usually in forest genetics — is only a
single aspect of this question.

The multilateral task of trees in nature requires
a simultaneous correlation of the objectives in
their breeding: (a) fast growth and higher
quality of the timber; (b) resistance to damages
by biotical and abiotical factors and the achieve-
ment of a stable biological balance within the
biocenoze; (c) increase of the task for protection
of nature (soil, sources, rivers, mineral water
sources, etc.); (d) increase of their healthy and
aesthetical task in the crowded centres.

We refer here as an example to the breeding of
black locust in Rumania. In the best conditions
(light but rich sandy soils and more than 11 °C
annual mean temperature), the 25-30-year-old
stands of this species realize yearly mean growths
up to 18 m3 per hectare. As a first objective in
the breeding of black locust cultivated in this
country, we have in view to propagate the de-
scendants of the plus trees, which give maximum
growth and better properties of the wood.

Regarding the second objective, we have
identified a few hybridogenous populations ob-
tained in the cultivation (Robinia pseudacacia
R. viscosa and R. pseudacacia R. neomexi-
cana), which realize a more large biocenoze,
due to their more marked entomophyll character
and their increased resistance against frost and
disease. At the same time, these hybrids are
faster in growth and give a higher nectariferous
production (0.0700 g bloom instead of 0.0460 g,
at the 62-63 per cent concentration).

On the other hand, the black locust is fre-
quently used in erosion control and sand
fixation. The selection of the forms with a

219

Section 13 — Plant Genetics and Breeding

noted capability for land improvement can also
be correlated, under the climatical conditions
of our country, with the objectives of timber
production in this case.

The existence of many black locust cultivares
may be possibly an evident increase of the
aesthetical task of this species in the crowded
centres, due to the great variation of their habitus,
to the colour of the flowers and the different
time of the blossom. At the same time, it appears
possible to choose and propagate as aesthetical
forms in cultivation, only these cultivares which
correspond with the other specified objectives.
So we have: Robinia Decaisneana and the hy-
bridogenous populations R. pseudacacia R.
neomexicana and R. pseudacacia R. viscosa.

When we analyze the objectives of breed-
ing for each species of trees, we can see
that it is generally possible to correlate the
majority of the four objectives above, in order
to obtain more efficiency in the tree improve-
ment work.

13.32. Some Important Characteristics of Autote-
traploids of Tree Species induced by Colchicine
Treatment. Sin-Kyu Hyun and Chung-Suk
Kim (Suwon, Korea).

With a view to produce new germ plasm to be
used in tree breeding as well as to obtain triploid
forms which would result in vigorous growth
through hybridizing diploid with tetraploid,
induction of polyploidy has been conducted by
means of colchicine treatment for twenty-four
tree species.

In total 9300 colchiploids were produced as
the result of treating seed or apical growing
point with colchicine and those are now 4-7
years old.

The optimum colchicine dosage as evaluated
by the frequency of stable tetraploids was not
related to the optimum colchicine dosage as
evaluated by the frequency of abnormal seed-
lings. Apparently, the physiological condition
related to the development of tetraploid tissue
was of greater significance than the colchicine
dosage.

Some important characteristics of induced
autotetraploids so far observed are as follows:

1. In all induced tetraploids, the increased
stomata-size as well as the decreased stomata-
frequency which were observed at their seedling
age are maintaining up to the date (age of 7).

2. The frequency of gigas pollen was much
higher in An plants than in the corresponding
In plants.

3. The frequency of deformed pollen as well
as the pollen sterility were higher in An plants
than in the corresponding 2/; plants.

4. In both Hisbiscus syriacus and Robinia
pseudoacacia the elongation of pollen-tube was
slower in An plants than in the corresponding
In plants as observed on artificial media
(with Robinia) as well as in the styles (with
Hisbiscus).

5. The corolla of An plant was apparently
larger in Robinia pseudoacacia (Gigas type)
than in the corresponding 2n plants, but it
was not so with Hibiscus syriacus.

6. The induced tetraploids of Robinia pseudo-
acacia were classified into four types as follows
according to the forms expressed by the grown
plants.

(a) Gigas type

(b) Spinless and pendulous type

(c) Brush type

(d) Dwarf type

In the wild population of In Robinia pseu-
doacacia too, the similar four types of plant
form are recognizable.

In order to elucidate the relations between the
forms occurring in the wild population of Ro-
binia pseudoacacia and the forms acquired by
chromosome doubling by colchicine treatment,
karyotypic analysis of those plants are now
progressing.

13.33. Notes on Some Needle Characteristics of
Soft Pine Species and Hybrids. Howard B.
Kriebel (Wooster, U.S.A.).

Needle characteristics of 6 species and 8 hy-
brids of soft pines were studied. Many of the
needles were replicated samples from trees
analyzed independently by other workers.
Other samples were taken from arboreta in
Ohio and Illinois, and from hybrids of controlled
pollinations made in Ohio.

Needle characters studied included length,
relative cross-sectional area, margin, position
and number of rows of stomata, position of
resin canals, and number of resin canals.

Needle cross-sectional area was intermediate
in hybrids P. monticola V griffithii and P. flexilis
griffithii, but not in other hybrids. The P. mon-
ticola x peuce hybrid was intermediate between
the two parent species in number of needle
serrulations on the inner edge. Needles of P.
flexilis James had from 2 to 4 rows of dorsal
stomata (in previous studies P. flexilis was re-
ported to have 2 rows).

The following species and hybrids were found

220

Section 13— Plant Genetics and Breeding

to have ventral resin ducts in some needles, in
addition to those reported in a recent report by
other workers: P. flexilis (0 to 2), P. strobus
(0-1), P. ayacahuite (2 to 4), P. strobus X flexilis
(0-1), P. monticola ■ P. ayacahuite (0-1). A
ventral medial resin duct was found in a needle
of a P. monticola griffithii hybrid from Pla-
cerville, California.

Comparison of results with previous work
indicates that more research is necessary on all
species and hybrids of pines before definitive
descriptions are prepared. Evidently more
sampling is needed to avoid overlooking varia-
tion. Additional analyses are also needed be-
cause of variation caused by environment,
intraspecific genetic variation, or a combination
of both factors.

13.34. Some Biochemical Aspects of the Phenomenon
of Heterosis in Maize. T. Craciun, A. Ciofu,
M. Ivanescu, M. Ferrando, M. Birsan,
I. BJrnaure and M. Ionescu (Bucharest,
Rumania).

The present paper was written on the basis of
research work carried out by the Chair of Gene-
tics and Plant Breeding of the "N. Balcescu"
Agricultural College of Bucharest.

In the framework of the more complex prob-
lem of the development dynamics of some char-
acters and special features in simple and double
hybrids in Fi in connection with the nature of
the crossed organisms and of hybridization,
detailed research work was carried out on the
chemical composition in different phases both
in the parents and in the maize hybrids.

After the chemical analyses, the following was
found, inter alia:

In the first phases of growth the general ten-
dency was observed that the inbred lines have a
higher percentage of starch-protein than the
simple hybrids. The relationship starch-protein
is in favour of protein, namely higher in the
inbred lines and lower in the simpler hybrids.
The amount starch + protein is higher in the
inbred lines and lower in the simpler hybrids,
while the amount of cellulose and hemicellulose
is higher in the simple hybrids than in the inbred
lines.

At the end of July, in the milk-dough stage,
and taking in view precocity, the starch contents
of the simple hybrids have a tendency to equal
or to exceed the inbred lines. The protein con-
tents decrease and the relationship starch-pro-
tein is getting favourable to starch, generally in
hybrids.

13.35. The Phenomenon of Heterosis and Vitamin
Concentration in Maize Plant. W. N. Stole-
tov, Ye. N. Odintsova and M. Shinkovitz
(Moscow, U.S.S.R.).

In our investigations of the biological founda-
tions of heterosis the simple interlinear maize
hybrid and its two parent forms were used.

We observed the growth and the development
(in hothouses and under field conditions) as
well as the distribution of the vitamins of the B
group in root, stem, leaves and reproductive
organs (panicle and corncobs).

Also the leaves development and the dynamics
of vitamin accumulation in them were studied
(up to the stage of the appearance of panicle).

Using Odintsova's microbiological micrometh-
ods the following six vitamins of the B group
were determined: meso-inositol, biotin, pan-
tothenic acid, thiamin, pyridoxin, nicotinic
acid.

As was found, the vitamins of the B group are
concentrated mainly in the leaves and in the re-
productive organs — in panicle and corncobs.
Their total amount in these organs is very high.

The above six vitamins are present in a very
small amount in the roots and in the stems.
Often they are absent entirely in these organs.

Therefore the vitamin content in the leaf and
in the panicle can be used as the indicator of the
concentration of the vitamins of the B group in
the plant.

The phenomenon of heterosis correlates
to the sharply increased concentration of the
vitamins of the B group in reproductive organs —
panicle and corncobs. In the leaves the more
high concentration of the vitamins of the B
group was observed for only two vitamins—
biotin and meso-inositol.

The great ability to the accumulation of the
vitamins of the B group by the heterosis plant
in the process of its development, i.e. the ability
to the higher concentration of biocatalitical
substances is one of the important factors which
provides the most high accomodation by this
plant to the synthesis of plastic substances, to
the most powerful development of the vegetative
organs.

13.36. Observations on Heterosis in Zea mays L.

Helena Bankowska (Warsaw, Poland).

Twenty-one inbred lines of maize of American
origin have been intercrossed. Their Fi exhibited
heterosis. Selection and inbreeding of some of
the most vigorous plants in F2 and in succeeding
generations resulted in isolating stable inbred

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Section IS — Plant Genetics and Breeding

lines, exceeding Fi in vigour. To study the effect
of heterosis several features are analyzed :
height of plant, length and width of leaves,
length and number of internodes, number of
days from seeding to pollen shedding. From
the experimental data total sum of leaf surfaces
per plant was computed.

To establish the differences between the par-
ents, Fi and the stable vigorous line No 10
(cross WD W9) an experiment in randomized
blocks was performed. The differences in all
features are significant and statistically proved.
As concerns the heterosis for earliness, it was
stated that in case of mutual differences in
earliness of parents less than 7 days, their Fi
exhibited pronounced heterosis for earliness.
If the difference surpassed 10 days, Fi was asym-
metrically located between the parents and clo-
ser to the earlier parent. A significant positive
correlation has also been observed between the
number of days from seeding to pollen shedding,
expressed by mean value of both parents, and the
plant height of their Fi (/• = 0.905 + 0.034).

Heterosis for earliness can be explained by
presence of dominant genes reducing the num-
ber of days from seeding to pollen shedding.
Besides, additive genes exhibiting blending in-
heritance can participate in determining the
time of flowering. The vigorous growth is
probably due to interaction of complementary
genes, which assure more intensive growing
rate of longer duration in vigorous lines than
in parents.

13.37. Gene Action in the Inheritance of Quantitative
Traits. H. F. Robinson and R. H. Moll

(Raleigh, U.S.A.).

be presented. These results provide a basis for
evaluation and development of breeding proce-
dures as will be illustrated in a discussion of
reciprocal recurrent selection. The relevance of
the results to the explanation of the issue of
heterosis will be given.

13.38. Influence of Radiation on the Combining
Ability of Inbred Lines of Maize. D. L. Palen-
zona and R. E. Scossiroli (Pavia, Italy).

A problem which may bring more under-
standing on heterosis in corn, and may have
important implications in practical research,
concerns the possibility to change under radia-
tion the combining ability of inbred lines of corn.

We know the consequence of this phenomenon.
Some inbreds possess a general high combining
ability in crosses when final yield is considered,
while others show high combining ability only
with specific inbreds but low combining ability
with others. Is it possible to change under
radiation the combining ability from high to
low or vice versa? Is it possible using radiation
to improve further the combining ability of an
already good high combiner?

A series of inbred lines derived from an auto-
diploid strain were used to establish a pilot ex-
periment to test if radiation applied to the origi-
nal stock could be effective in changing its
combining ability when used as pollen parent
in crosses with a good combiner, the well-
known inbred WF 9.

The results obtained show that radiation may
change combining ability in crosses when yield
of single crosses is considered.

Mathematical approaches to the study of gene
action in maize have involved development of
procedures and experimentally estimating the
additive genetic, dominance and epistatic var-
iances for complex characters such as yield of
grain. The research of the past fifteen years has
provided a basis for concluding that (1) ad-
ditive genetic variance exists in open-pollinating
varieties in sufficient quantity to allow for prog-
ress in yield improvement from selection;
(2) overdominance is not the predominant type of
action of genes concerned with yield; (3) the
existance of appreciable epistasis has not been
demonstrated and (4) genotype environmental
interactions are very important in quantitative
genetic studies and in research on breeding
methodology. The results of the extensive re-
search program on quantitative genetics will be
summarized and implications of the results will

13.39. Response to Selection in Tangiiis Cotton.

Jose A. Giles (Lima, Peru).

A method based on phenotypic selection was
used in Peru for seven years to improve Tangiiis
variety (G. barbadense). Individual plants were
selected in the field and their fiber characteristics
analyzed. Selected plants were sown in progeny
rows. Progenies with best appearance were
chosen and put into yield trials and also propa-
gated in increased blocks. To start a new cycle
of selection, individual plants were selected from
progeny rows and increased blocks.

A selection differential of the average differen-
ce in phenotypic value between selected progenies
and overall progenies was obtained for number
of bolls per plant, lint percent, lint index, boll

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Section 13 — Plant Genetics and Breeding

weight, and fiber strength. These data were re-
corded for each season only at the end of the
seven year program. The results showed that
some improvement was obtained in number of
bolls per plant with an average increase of 4
per cent per year. There was no improvement in
the other characteristics. This may be due to
lack of genetic variability for these factors and
due to the fact that the progenies were selected
only on the basis of their field appearance. The
latter may explain why the only success was in
increasing number of bolls. Variability was
large for number of bolls; fair for lint percent;
and small for lint index, boll weight, and fiber
strength. After seven years the variability re-
mained practically constant for each character.
This method of breeding showed to be ineffi-
cient for making steady improvement once there
is a genetic population which has reached a
certain stage of progress. Procedures that can
utilize new recombinations and that can measure
accurately the genetic variability are suggested.

must be exploited in order to realize the ful
yield potential of Upland cotton in Uganda.
1. Comstock, R. E. and Robinson, H. F.
Chapter in Heterosis, Iowa State College Press,
Ames, Iowa.

13.40. The Average Degree of Dominance of Genes
determining Components of Lint Yield in the
BP52 Upland Cotton Variety. J. T. Walker

(Namulonge, Uganda).

1 . Estimates of the average degree of domi-
nance of components of lint yield of Upland
cotton were obtained from a field trial based on
a design by Comstock and Robinson (1952.)(1).
The F2 populations of hybrids between inbred
lines of the BP52 variety were used in back-
crossing and had previously been evaluated
in diallel crossing trials.

2. Only additive genetic variance was found
for the traits loculi per boll and lint per seed.

3. Genotypic interaction due to specific geno-
types caused dominance for seeds per loculus,
seeds per boll and earliness as measured by the
number of days to first flower.

4. The characters lint percentage and seed
weight showed generalized genotypic interaction
and apparent overdominance with transgressive
segregation, the interaction variance being dis-
tributed evenly throughout the material.

5. Findings are discussed in relation to pre-
vious inferences about the inheritance of yield
in cotton made as a result of selection index and
diallel crossing studies. It is suggested that in-
teraction at the genetic level may be involved
in the expression of lint yield heterosis.

6. It is concluded that non-additive effects

13.41. Genetics of Internode Length in a Wide Cross
of Pisum sativum L. Donald L. Mahoney
and Jules Janick (Lafayette, U.S.A.).

The inheritance of internode length was
analyzed in reciprocal Fi, F2, F3, and backcross
progenies of crosses between the field pea
(Black-eyed Susan) and the garden pea (Wiscon-
sin Perfection). Associative characters (total
number of nodes, total height, and number of
nodes to the first flower node) were also analyzed
and correlated with internode length.

The Fi exhibits positive hybrid vigor in height
and average internode length significantly greater
than either parent (BES — height, 156 cm, average
internode length, 6.3 cm; WP— 90 cm, 4.9 cm;
Fi— 186 cm, 8.9 cm). The total number of nodes
appears to be controlled by partially dominant
factors; in most cases, the Fi lies closer to the
fewer noded parent ( WP). The greater height of
the Fi is therefore attributable to heterosis in
internode length. Analyses indicate that the
heterotic expression of internode length in the
Fi through the F3 is best explained by the
dominance rather than the overdominance
hypothesis. Lines were obtained in the F3 whose
average internode length was equivalent to the

Fi.

Tetraploidy decreases the average internode
length in both Black-eyed Susan and Wisconsin
Perfection. Fertility in tetraploid peas is very low,
and crosses involving tetraploids and diploids
were unsuccessful.

13.42. The Somatic Variability of Plant Clones.

G. Haskell (Dundee, Great Britain).

Variability, the capacity for variation, is a
property of living matter. It may be genetic,
producing observable differences between indi-
vidual organisms, and somatic, embodying dif-
ferences in the same individual at different life
stages (Raymond Pearl).

Biometrical variability of the plant clone, a
group of individuals arising vegetatively from one

223

Section 13 — Plant Genetics and Breeding

parent, has been neglected. Yet many horticultural
crops are reproduced vegetatively, like apples
which are artificially budded on rootstocks; these
might in turn influence the free variability.
Strawberries are more naturally reproduced
from runners. These rot ultimately, so leaving
self-supporting ramets at the rooted nodes form-
ing an asexual population. Such clones provide
opportunity for assessing the variability of a
vegetative reproductive system not previously
analysed intensely.

Plant clones can be of all ages, and of all
vegetative stages removed from the original
parent. A clone may be diploid or polyploid,
homozygous or heterozygous, healthy or virus
infected. Some clones may even transmit plas-
magenes vegetatively through successive gener-
ations. The clone is subject to the rejection or
accumulation of somatic mutations of oligogenic
or polygenic origins; hence they may be homo-
somatic or hetero-somatic. Not surprisingly a
clone may show vegetative segregation.

The variability in ramet size is analysed for
octoploid virus-free cultivated strawberry clones
and those carrying plasmagene infection. The
internal variation is examined within grafted
clones of very old and new apples for the bio-
metrical character "stamen number". These
data are then assessed in the light of contempora-
ry views on genetic homeostasis, the "c" effect,
and the individual in the plant kingdom.

13.43. Genetic Variability in Isogenic Lines of Barley.
C. O. Qualset and C. W. Schaller (Davis,
U.S.A.).

Backcross-derived isogenic lines of Atlas
barley with two segments of chromosome
marked by independent genes governing the
length of the lemma awn are being used to study
types of quantitative gene action and the effect
of the awn on yield and various other characteris-
tics. The theoretical length of chromosome
associated with each of these loci retained from
the nonrecurrent awnless parent through the
backcrossing procedure has been calculated to be
about 5 crossover units. Several quantitative
characters are being studied to assess the
amount of genetic activity in these short segments
of chromosome. Many workers have found that
photosynthetic activity in the awn contributes
significantly to final kernel weight and hence
total yield. In this material the four homozygous
genotypes are phenotypically distinguishable by
the length and distribution of the awn. They are
designated full-awned (AABB), half-awned

(AAbb), quarter-awned (aaBB), and awnless
(aabb).

A study has been completed in which the four
genotypes were grown at five plant densities
(1/6 to 6 ft2/plant) in two years. Over both years
genetic variation was found for 5 of 8 characters
measured. For the quantitative characters
measured the genotypes were stable over the
various densities but interacted with years in
5 out of 8 cases. Significant additive genetic
variability was found at the A locus for kernel
weight, plant height, and spike density. At the B
locus only kernel weight and percent sterility
indicated additive effects. All of the genetic
variance for kernel number/spike was due to
interaction of the A and B loci. This interaction
resulted in a significant increase in total yield of
half-awned over full-awned in the first year and
equal yield in the second year in spite of a signifi-
cant decrease in kernel weight due to the reduced
length of awn in half-awned.

13.44. The Effect of Different Treatments on the
Variability of Polygenic Characters in Wheat.

S. Borojevic (Novi Sad, Yugoslavia).

A study has been made of the effect of mineral
nutrients, rate, mode and date of planting on the
variability of ear and kernel characters as well as
on the yield of wheat.

The absence of fertilizers, as well as the use of
nitrogen in the absence of phosphorus produced
the highest variability in all the characters studied.
Phosphorus in the absence of nitrogen did not
affect the variability of the length of ear and
number of spikelets but increased considerably
the variability of the number and weight of
kernels per ear. Potassium does not seem to affect
the variability. The lowest variability of the
characters studied and the highest yield have been
obtained when high doses of nitrogen, medium
ones of phosphorus and low ones of potassium
were applied.

Heavier stands lead to the increase in vari-
ability of the number and weight of kernels but
not of other characters. The date of planting
affected the variability in a similar manner. The
mode of planting did not exert any great influ-
ence on the length of ear and number of spikelets.
However, larger distances between rows resulted
in lower variability and higher mean values.

These findings have interesting bearing on the
exploration of the genetic capactity for yield and
indicate that from the increase of heritability
level in the number of kernels the most may be
expected.

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Section 13 — Plant Genetics and Breeding

13.45. Percent Dry Matter as an Index for the
Evaluation of Individual Plants and Strains of
Sugar Beets (Beta vulgaris L.). P. Bergen
(Tober, Canada).

Data will be presented which indicate that
percent dry matter of the petiole is under the
control of a genetic mechanism which also
influences the sucrose content of the root.
Percent dry matter of the petiole is positively
correlated with sucrose percentage in the root
and negatively correlated with root weight. The
progeny of plants selected for percent dry matter
of the petiole showed that significant changes
had been obtained for percent dry matter of the
petiole, percent sucrose, percent dry matter of the
root, root weight and sucrose yield. A further
experiment demonstrated that percent dry
matter of the leaves and petioles of young sugar
beet plants may be used to predict the potential
root yield and percent sucrose of sugar beet
varieties and strains.

13.46. International Sources of Resistance of Wheat
Tested in Respect to the Major Physiologic
Races of Puccinia recondita Rob. ex Desm. in
Yugoslavia. Momcilo Boskovic (Novi Sad,
Yugoslavia).

In order to select the best sources of resistance
to the prevalent physiologic races of Puccinia
recondita in Yugoslavia, i.e. 6, 20 and 77, as well
as some races of minor importance, such as 17
and 105, a large number of varieties and hybrids
of wheat was tested for resistance in the green-
house at the seedling stage of plants to the various
races, and in field tests at the adult stage of
plants. These experiments were conducted in the
years 1960, 1961 and 1962.

It has been observed that the races used
segregate many of the wheat varieties and hybrids
with a greater or lesser degree of resistance into
resistant variants of plants on the one hand and
susceptible ones on the other.

From international winter wheat rust nurseries
six hybrids with the highest resistance were
selected, in which segregation occurs only in
respect to one race in the greenhouse. A still
higher degree of resistance to all the races
examined, and that without segregation, was
found in Chinese X Ae.umbelhdata P-54-44-16
and B.Y., selected from international spring
wheat rust nurseries. Apart from the above
varieties and hybrids, some other valuable
material, selected on the basis of the present
investigation, has been presented in this paper
in the tables. The best hybrids selected are

intended for use as sources of resistance in our
breeding programmes.

It has been found that many sources of
resistance used in various countries show either
complete susceptibility or only partial resistance
when used in Yugoslavia, while only a small
number of these is fully resistant to our races of
the parasite.

13.47. Seedling Reaction to Leaf Rust Races of
Wichita Wheat Lines with Genes for Resistance
Transferred from the Differential Varieties.
E. G. Heyne and C. O. Johnston (Manhattan,
U.S.A.).

A series of lines of Wichita hard red winter
wheat has been developed for separating
physiologic races of Puccinia recondita Rob. ex
Desm. These lines have been obtained by trans-
ferring the genes for resistance from the eight
standard differential varieties. Wichita was the
recurrent parent and the backcross lines available
for study have reached the 4th to 6th backcross.
The resistance of Transfer, which has leaf rust
resistance derived from Aegilops umbellulata,
also has been transferred to Wichita. Tests with
physiologic races from eight important race
groups have shown that the backcross Wichita
derived lines have a similar reaction to the
standard differentials. The Wichita line with the
Hussar reaction gives readings more like those
originally described. Apparently the Wichita
background results in less variation to leaf rust
infection than when the factor for resistance is in
Hussar. These Wichita backcross lines have an
advantage in that they all have the same growth
habit and virtually are the same except for
reaction to various leaf rust races. In the present
material apparently only one major gene from
each donor parent has been transferred. For
example, the Wichita5 x Carina and Wichita5 x
Brevit do not have the resistance of Carina or
Brevit to race 9 of leaf rust. Seed of these Wichita
backcross lines are available for use by other
investigators.

13.48. Inheritance of Rust Resistance in Stem Rust
Differential Varieties of Wheat. W Norman
D. Williams and F. J. Gough (Fargo,
U.S.A.).

The inheritance of seedling resistance to
physiologic race 1 1 1 of Puccinia graminis f. sp.
tritici Eriks. & Henn. was studied in eight of the
stem rust differential varieties of wheat. Physio-

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Section 13 — Plant Genetics and Breeding

logic race 1 1 1 (the most widely avirulent of the
described physiologic races) was used because a
culture avirulent on a wide spectrum of varieties
would be expected to differentiate more re-
sistance genes than a virulent one. The Fi, F2,
F3, backcross-Fi, and backcross-F2 generations
from crosses of resistant tetraploid varieties
Acme, Arnautka, Kubanka, Mindum, Vernal,
and Khapli with moderately susceptible P.I.
192334, and from crosses of resistant hexaploid
varieties Marquis and Kota with susceptible
Little Club were tested with a single spore
culture of physiologic race 111. Two resistance
genes in Kubanka and three in each of the other
resistant varieties were indicated. All of the
resistance genes were dominant or partially
dominant. Genes for high levels of resistance
commonly appeared epistatic to genes for low
levels of resistance. In some cases, notably in
Vernal, the resistance genes were cumulative in
effect.

1. Cooperative investigations at Fargo, North
Dakota, of the Crops Research Division,
ARS, USDA5 and the North Dakota Agri-
cultural Experiment Station.

13.49. A Monosomic Analysis of Leaf Rust Resistance
in Five Varieties of Triticum aestivum L.

R. C. McGinnis and W. J. R. Boyd (Winni-
peg, Canada).

Five leaf rust resistant varieties of common
wheat (Africa 43, Frontana, Klein Aniversario,
Klein Titan, and Sinvalocho) were crossed to all
21 different monosomies of the susceptible
varieties Red Bobs and Rescue, as well as in
diallel in order to investigate the inheritance of
rust reaction and associate genes for resistance
with specific chromosomes. Chromosome counts
made from root-tip squashes of germinated Fi
seed prior to planting were used to identify
monosomic and disomic plants. The Fi's were
inoculated with race 9 of Puccinia recondita
Rob. ex Desm. at both seedling and adult stages
and rust reactions recorded. In the seedling
stage, resistance proved to be recessive in all
cases; segregation for resistance occurred in
only the critical chromosome lines. It was
possible, therefore, to associate genes for re-
sistance with specific chromosomes as follows:
Africa 43 — chromosomes IB and 5D; Frontana
— 2A and 5A; Klein Aniversario and Sinvalocho
5A, 4B and 5D; and Klein Titan— 4B, 4D and

5D. Resistance was found to be dominant at the
adult plant stage.

F2 populations from monosomic Fi plants
together with disomic controls were grown and
also inoculated at seedling and adult stages. The
data showed that the inheritance of adult plant
reaction is complex and expression of reaction
is influenced greatly by environment. No satis-
factory genetic hypothesis could be proposed for
the different varieties on the basis of the available
data. Chromosome 5A of all varieties appeared
to carry a gene for adult plant resistance and is
particularly important in Frontana while chro-
mosome 6B appeared to be important to all
varieties except Frontana.

13.50. Genetic Studies on Geographical Distribution
of Barley Varieties with Special Reference to
Uzu or Semi-brachytic Form Native to Japan.

Ryuhei Takahashi (Kurashiki, Japan).

The present writer has already shown that
about 80 per cent of barley acreage in Japan is
now occupied by a group of varieties which
possess in common a recessive mutant gene
called uzu (uz). They are characterized by short,
thick culms and compact heads with short awn,
which are especially preferred by the Japanese
farmers. A further study was made to gather
more evidence for the prevalence of this type in
Japan. For this, effects of the genes, Uz and
uz, on yield and its components were compared
using 24 normal and uzu isogenic paired lines.
The results indicated that uz was slightly less
favorable than Uz in an average genetic back-
ground. However, productivity of uzu relative
to normal line was found to vary considerably
with genetic background. Thus, possibility of
breeding higher yielding uzu variety by selecting
good genetic background was suggested. Re-
sponses of several selected isogenics to three
different levels of fertility disclosed that uzu
barley was adaptive to heavy manured con-
ditions, but was inferior to the normal one at the
lower levels of fertility. It may be possibly
concluded that progress in barley breeding and a
marked trend of heavy manuring have fostered
the spread of uzu barley, replacing the formerly
existing normal type varieties.

13.51. Breeding Lodging Resistant Winter Barleys
from "Uzu" Parentage. John M. Poehlman
(Columbia, U.S.A.).

Lodging resistant winter barleys with short,

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Section 13 — Plant Genetics and Breeding

thick culms, dark green color, dense spikes and
short awns, described as semibrachytic or
"uzu", possess a recessive gene that has a pleio-
tropic effect on the plant, shortening stature,
rachis internodes, and awns. One strain, C.I.
7439 from Korea, when compared with Mo.
B-475 at different fertility levels was less pro-
ductive, the higher grain yield of B-475 re-
sulting from more tillers and larger seeds. C.I.
7439 set more seeds per spike and had a straw/
grain ratio of 1.46 vs. 1.88 for Mo. B-475.

When C.I. 7439 was crossed with Mo. B-475,
the Fi was tall and had lax spikes and long awns
like B-475. The F2 segregated into three phenoty-
pic classes — tall, lax-spike, long-awns ; tall, dense-
spike long-awns ; short, dense-spike, short-awns—
with minor variations in height and spike density
within each class. Segregation was conditioned
by one major gene affecting spike density in
addition to the "uzu" gene. Effects of the spike
density and "uzu" genes on several plant
characters were measured by comparing sets
of partially isogenic lines derived from different
F2 plants. Plants with the recessive spike den-
sity gene had reduced plant height and seed size
in addition to the reduction in rachis internode
length. Plants with recessive "uzu" genes had
lower grain yields, reduced height, smaller
seed size, increased sterility, and later heading
date than "non-uzu" counterparts. In the "uzu"
types primary selection for high tiller capacity
and larger seed size will be needed to obtain
maximum yield. Superior winter hardiness was
observed in all three phenotypic classes.

13.52. A Genetical Study of "Cold Test" Reaction in
Seed Corn. Shu Ting Chang (Hong Kong).

The maize material involved in this investiga-
tion included inbred lines, single and double
hybrids, and open-pollinated progenies of
single hybrids, grown on the Wisconsin Univer-
sity farm at Madison in 1957 and 1958. Re-
presentative ears of each of these classes were
harvested at successive stages of maturity, the
moisture content of the kernels ranging from
60 to 18 per cent. In order to simulate preharvest
frosts as they might occur in the field conditions,
the ears were exposed to freezing treatments
of varying intensity and duration, under con-
trolled conditions in a specially equipped freezing
cabinet.

The following conclusions can be drawn
from these studies:

1. Significant differences between inbred lines
and between hybrids of which they are the
parents are shown to exist in respect to

tolerance to freezing injury as well as to
pathogenic fungi attacking theerminating
seed.

2. In general, hybrids are more tolerant to
frost injury effects on germination and to
attack by soil-borne seed-decaying patho-
gens than are their parental inbred lines.

3. The ability of seed corn to resist damage
to germination by freezing treatments is
not necessarily correlated with inherent
resistance to attack by soil-borne seed-
decaying organisms. The tolerance of
hybrids to freezing injury was not as strong-
ly affected by the maternal parents as was
resistance to attack by soil-borne organisms
under "cold test" conditions.

4. The effect of age on freezing seed viability
under "cold test" conditions is significantly
greater for inbred lines than for hybrids.

1 3.53. Resistance to Low Temperatures in Connection
with the Stages of Organogenesis of the Ear
in Some Genotypes of Wheat. Misic Todor
(Novi Sad, Yugoslavia).

The winter hardiness of six genotypes of wheat
in dependence on the stage of organogenesis
of the ear (21, 50, 58, 70, 91, 104 and 1 12 days
after emergence) has been examined in a field
test, conducted after the split plot method in
conditions with and without a snow cover, as
well as in a laboratory experiment. A regularity
was found to exist in the gradual increase in the
percentage of winter killing from the early
stages of organogenesis of the ear to the later
ones, which was particularly remarkable in
genotypes with low winter hardiness. The winter
killing resulted in a significant decrease in grain
and straw yields, 1000 kernel-weight and hecto-
liter weight of grain. This decrease is in accord
with the percentage of winter killing in depen-
dence on the stage of organogenesis of the ear
in a given genotype.

13.54. Inheritance of Frost Resistance by Wheat.

A. Popovic (Kragujevac, Yugoslavia).

At the Plant Breeding Institute in Kragujevac
the frost resistance of 15 Fi, 7 F2 and 4 F3
hybrid combinations of wheat were studied.
The highest level of frost resistance was by hy-
brids which originated from steppe x steppe-
like varieties. The combination "Minhardi x
Bankuty 1201" had 100 per cent of survived

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Section 13 — Plant Genetics and Breeding

plants under cold chamber temperature — 12°C
for 30 hr.

By crossing steppe-like varieties with medite-
ranean (mainly Italian) varieties we got the hy-
brids with 5.0-93.3 per cent of cold resistance.
Among Italian varieties San Pastore showed the
highest effect on frost hardiness. The hybrids
from this variety had 53.3-93.3 per cent survived
plants. The cultivars like Produttore, Leonardo
and Mara have lower frost resistance. Espe-
cially the cultivar Mara demonstrated a high
grade of susceptibility to frost.

Italian varieties intercrossed gave the Fi hy-
brids with 1.6-5.0 per cent of alive plants.

F2 hybrids were tested under — 14, — 16 and
— 18°C in the course of 16 hr and showed dif-
ferent reactions up to the severeness of test. By
— 14°CF2 progenies were intermediate between
their parents. The last two temperatures resulted
in high winter killing.

From hardier parents came more frost-
resistant plants in F3 generation. Some F3
progenies combined the spring character with
good winter hardiness.

Breeding wheats with still greater hardiness
is being continued and it is expected to pro-
duce high yielding material with satisfactory
frost resistance.

1 3.55. The Transforming of Winter Italian Wheats
With Low Cold-resistance into Spring Wheats
in Moscow Region. R. D. Glavinich (Moscow,
U.S.S.R.).

In the course of 1958-1962 an experiment was
carried out on changing the heredity of soft
Italian wheats with low cold-resistance (30
varieties) which are characterized by their
non lodging and with high agrotechnics give
40-45c on 1 hec.^1)

The work has been carried out in two direc-
tions: (1) to obtain winter wheats with high cold-
resistance for Moscow region and (2) to obtain
spring forms out of the same initial material.

We have used the following method of chang-
ing Italian wheats with low cold-resistance into
wheats with high cold-resistance. By twofold
autumn sowing (18-20 October) under the in-
fluence of spring-summer conditions we have
changed winter forms with low cold-resistance
into spring forms. Then by optimal sowing
(25-31 August) we have transformed them into
winter forms with high cold-resistance. By
this method we have changed 16 varieties out
of 30, but only 6 of them have become real win-
ter forms for Moscow region (80-100 per cent
of their F3 survived winter of 1961-62).

The vegetative period of new winter forms
in F3 did not differ from the standard winter
varieties and in two forms was 4-7 days shorter.
The absolute weight of 1000 grains of new winter
wheats was more than 40 g, the weight of the
standard being 38-39g. The yield of these forms
averaged 44c on 1 hec, the yield of the standard
being 36-37c on 1 hec. All six winter forms were
morphologically alike. Three of them (Kva-
derna, Forlani and Klavatino) are the per-
spective forms for the Moscow region.

After three years of spring sowing (1959-1961)
9 real spring forms have been chosen out of 30.
88-98 per cent of them ripened in 1961 (spring
standard variety Moscowka). The vegetative pe-
riod of new spring forms was 2-5 days shorter
than that of the standard. The absolute weight
of 1000 grains was 1-4 g bigger than that of the
standard. Some of the new spring forms (For-
tunato, 1373, Forlani) are of interest for selec-
tion.

1. 1 centner (c)
10.000 m2.

50 kg. 1 hectare (hec) =

13.56. Molecular Structure of DDT Analogues in
Relation to Gene Action in Barley. G. A.

Wiebe (Beltsville, U.S.A.).

The insecticide DDT kills certain barley
varieties and not others following foliar applica-
tions of this chemical during the seedling stage.
Inheritance studies show this reaction is con-
trolled by a single gene and that resistance is a
recessive character. Biochemical tests show that
DDT enters the leaf tissue of resistant and sus-
ceptible plants in approximately equal amounts.
Since the reaction appears to be highly specific,
tests were undertaken to determine the reaction
of a series of DDT analogs to this gene. On the
basis of incomplete tests of a group of 50 analogs,
8 were found that reacted similarly to DDT.
In comparing the chemical composition and
structure of those analogs that gave a differential
kill with those that did not, one finds that the
structure of the molecule is of greater impor-
tance than its composition. This finding is in
line with the growing concept in molecular
genetics that the specificity of gene action may
be governed in part by the secondary and ter-
tiary structure of its primary product, an en-
zyme. For example, in the present case, where
ordinary p,p' DDT will kill one variety of
barley and not the other, the o,p' DDT form
will kill neither variety. The specific action of
DDT on barley has not been determined. It

228

Section 13 — Plant Genetics and Breeding

can be stated, however, that in general, those
analogs showing toxicity to insects also are
toxic to barley, although there are exceptions
to this rule.

13.57. Outlines of the Cacao Selection and Breeding
Programme in San Thome. J. C. Ascenso
(Lisbon, Portugal).

The cacao breeding programme in San Thome
includes single plant selection in the local popu-
lation, introduction of hybrid seed and selected
parents and hybridization work utilizing clones
selected locally and abroad.

13.58. (D.), Evolutionary Maize Breeding. Alexander
Grobman (La Molina, Lima, Peru).

Recent information has become available on
evolutionary rates of improvement of maize
grain yield components, through studies of
archaeological and living maize ears in Peru
stratified over a period of nearly 2500 years.
It has been possible to establish differences in
evolutionary rates of change of quantitative
ear characters between "tripsacoid" and "non-
tripsacoid" maize races. The former have a
steeper rate of improvement of ear length,
which becomes evident after the introduction
of "tripsacoid" maize germplasm in certain
geographic areas.

Modern breeding evidence points also to the
yield enhancing effect of the introduction of
tripsacoid germplasm into intervarietal hybrids
of maize studied in the highland region of Peru.

Phylogenetic relationships have been estab-
lished on the other hand between more than
200 races of maize in Latin America, based on
morphological, genetical, cytological, and other
kinds of characters. Tentative degrees of teosinte
and Tripsacum introgression into these races
have been estimated.

On the basis of all this evidence, breeding
schemes may be divided in different areas of
Latin America to better utilize heterosis in
intervarietal and interracial hybridization as a
first step in the improvement of yield of grain
of maize.

13.59. Parthenogenesis in Polyploid Populations.

O. J. Eigsti (Chicago, U.S.A.).

Polyhaploid individuals appear in popula-
tions supposedly all polyploid. Parthenogenesis

accounts for the origin of polyhaploids. These
variants cause the entire population to produce
more and more seed each generation. The
discovery of a parthenogen was made from self
pollination of a supposedly tetraploid Citrullus
vulgaris. Subsequent observations confirmed
continuing origin of polyhaploids during each
generation and the data support the conclusion
that a frequency of 1 seed per thousand tetra-
ploid carry polyhaploid embryos. Such indi-
viduals are not easily recognized in field popula-
tions. Pollen from polyhaploids produces di-
ploid hybrids instead of triploid hybrid seed in a
field where such seed production is the goal. The
practical problem created must be recognized
and compensation made to eliminate the ulti-
mate and unfavorable influence upon the pro-
duction of seedless watermelon fruit. Similar
origins of polyhaploids among populations of
Polygonatum commutatum, a naturally occurring
polyploid species, causes confusion taxonomical-
ly within the glabrous forms that make up the
P. biflorum complex. Propagators of tetraploid
cultures and studies dealing with evolution
among polyploid species must recognize the
role played by parthenogenesis. More and more
cases are being discovered since this discovery
was made.

13.60. Cytoplasmic Replacement through Andro-
genesis in Maize. Sherret S. Chase (Sycamore,
U.S.A.).

In maize, as in many other species, cytoplasm
is transmitted by the female gamete only.

Following a suggestion of the author, the
Nebraska inbred line N6 has been converted
(Goodsell, S. F. Crop Sci. 1, 227-228, 1961) to
the "Texas-sterile" cytoplasmic phase by utilizing
the rare androgenetic individuals (paternal
monoploids) resulting from crossing N6 as male
to a diploid "Texas-sterile" cytoplasm stock.

The present report is concerned with the at-
tempt to increase the effective rate of androgen-
esis by utilizing a specially synthesized "Texas-
sterile" tetraploid, Emerson-brown, marker
strain as the cytoplasmic donor. Initial results
indicate that crosses of inbreds to this strain do
yield higher frequencies of androgenetic indi-
viduals and, even more interesting, that an ap-
preciable portion of the androgenetic individ-
uals are diploid.

Cytoplasmic transfer through androgenesis
has the advantage over the customary back-
crossing technique of leaving the chromosomal
system intact.

229

Section 13 — Plant Genetics and Breeding

13.61. Inheritance and Utilization of Five Dwarfs in
Pearl Millet (Pennisetum glaucum) Breeding. W
Glenn W. Burton and James C. Fortson
(Tifton, U.S.A.).

Over 39,000 parent, Fi, F2, and backcross
spaced plants of hybrids involving five dwarf and
four normal inbreds (Gahi-1 lines) of pearl
millet were grown in replicated randomized
blocks in 1962. The dwarfs ranged from 32 to
60 in. in height, one-half to three-fourths the
height of normal inbreds. Most dwarf x normal
Fi hybrids were significantly taller (up to 38 per
cent) than the normal parent. Fi hybrids be-
tween four dwarfs were as tall as normal x
dwarf hybrids proving that these dwarfs car-
ried different recessive genes for dwarfness.
One dwarf x dwarf hybrid was little taller than
the taller dwarf parent, suggesting that both
carried the same dwarf genes. Inheritance of
dwarfness appeared to be conditioned largely
by one or two recessive genes. Evidence that
the normal inbred in some instances introduced
modifying factors was observed.

Where pearl millet is grown as a cereal,
converting existing tall varieties to dwarfs can
reduce lodging and facilitate harvesting. Dwarfs
can be used to reduce the cost of producing
hybrid seed of tall forage millets. Experience
indicates that a field planted to a mixture of
equal numbers of seeds of four different dwarfs
will by natural crossing produce seed, 75 per
cent of which will be Fi hybrids. These hybrids
will be tall, will carry hybrid vigor (up to 50 per
cent), will crowd out the inbred seedlings, and
will give forage yields comparable to 100 per
cent hybrid seed. Cytoplasmic male-sterile
dwarfs recently developed can be used as
females to facilitate single-cross seed production
and will give tall hybrids if normal or different
dwarf males are used.

Cooperative investigations at Tifton, Ga., of
the Crops Research Division, ARS, USDA,
and the University of Georgia, College of
Agriculture Experiment Station, Coastal
Plain Experiment Station.

times made that ssp. spelta originated as a simple
deficiency for Q.

A line with chromosome 5A of ssp. spelta
substituted into ssp. vulgare var. Chinese Spring
was used for testing the effect of increased dosage
of the spelta allele q. Squareheadedness was
chosen as the character that most clearly
distinguishes the vulgare from spelta phenotype.
Five doses of q resulted in squareheadedness,
while six doses caused even further compaction
of the spikes. Evidently q is not a deficiency
nor an amorph but is an allele which has an
effect similar to that of Q but of a lesser degree.
Relative to Q, q is a hypomorph, and five doses
of q corresponds to two doses of Q. The threshold
level for squareheadedness lies between four
and five doses of q.

Being qq, spelta should be closer to vulgare in
phenotype than is speltoid, which is deficient for
the q locus. That the reverse is true is attributed
to the effect of modifying genes. Modifiers are
also assumed to account for the ambiguities of
dominance that are found in the Fi between ssp.
vulgare and ssp. spelta.

Evidence has been obtained for the existence
of duplicates of q on the homoeologous chromo-
somes, 5B and 5D that are similar in effect, but
probably not identical, to q.

13.63. The Nature of Supergenes in Polyploid
Wheats. D. Zohary, M. Feldman and Z.
Brick (Jerusalem, Israel).

It is proposed that supergenes differentiating
between the principal types of hexaploid wheats
(i.e. spelta or Q factor, sphaerococcum or S
factor, compactum or C factor) are alien chromo-
somal segments that have been incorporated into
the polyploid wheats by means of distant in-
trogression from several genera of the Triti-
cinae. Such mode of origin explains why these
factors are genetically compound and why
they lack homology in the respective chromo-
somes of the primitive wheats (i.e. show null-
allelic situation). These supergenes are confined
to the polyploid level since polyploidy buffered
the incorporation of alien segments.

13.62. The Nature of the Spelta Gene q. Mikio
Muramatsu (Columbia, U.S.A.).

Deficiency of the vulgare gene Q on chromo-
some 5A (IX) in hexaploid common wheat,
Triticum aestivum ssp. vulgare, results in a
phenotype called speltoid because it is very
similar to ssp. spelta. The assumption is some-

13.64. Induced Mutations at the "Q" Locus in
Relation to the Phylogeny of Hexaploid
Triticum Species. M. S. Swaminathan (New
Delhi, India).

Induced mutations leading to the loss or du-
plication of the free-threshing gene Q located

230

Section 13 — Plant Genetics and Breeding

in the long arm of chromosome 5A were studied
in sub-species vulgare, compaction and sphaero-
coccwn of T. aestivum {2n = 42). In vulgare,
the loss of Q leads to the origin of speltoid char-
acteristics and an overdose of Q results in sub-
compactoid and compactoid phenotypes. Vari-
ous defects in floral organs and rachilla elonga-
tion as in vavilovi occur only in the absence of
Q. Orderly flower morphogenesis is thus one
among the many traits brought about by Q.
In compactum, the loss of Q makes the plant
grow taller and the ear less dense, in addition to
making the glumes tight thereby rendering
threshing difficult. A phenotypic resemblance
to a speltoid form of vulgare is thus generated,
though true speltoids are obtained only when
both Q and C are deleted from compactum.
An over-dose of Q in the presence of C gives
rise to hyper-compactoid ears. Similar phenoty-
pic consequences are observed in sphaerococcum
when Q is absent or is present in a tetrasomic
condition. Thus, the absence of Q alters radically
the key characteristics of sub-species vulgare,
compactum and sphaerococcum of T. aestivum.
Q has not so far been found to occur either
spontaneously or in irradiated populations in
the non-free-threshing hexaploid sub-species
spelt a, vavilovi, macha and zhukovskyi. If Q
is added to these sub-species by crossing them
with Q containing sub-species they also lose
their key characteristics. Q is thus a most interest-
ing locus from the phylogenetic angle, besides
being of immense importance from the agro-
nomic and adaptive standpoints.

13.65. (D). Genome Manifestation of Wheat in
Aegilops Cytoplasm. Hitoshi KiHARA(Misima,
Japan).

In order to investigate the effect of alien
cytoplasm on genome manifestation, genome
complements of 4x and 6x wheat have been
introduced by successive backcrosses into the
cytoplasm of Aegilops caudata or Ae. ovata.
The backcrosses have been carried out 14 times
for the most advanced materials and only twice
in the least advanced ones.

Influence of the alien plasm has been noticed
in morphological and physiological wheat
characters. It was most remarkable in the floral
organs. When their genomes were introduced
into caudata cytoplasm, two varieties of 6x
and four of 4x wheat showed complete or
incomplete pistillody and all became male sterile.
Fourteen other varieties or strains became also
male sterile but no pistillody occurred. Those
were seven varieties of 6 x wheat, three of 4 x
wheat, three strains of synthesized 6x wheat

and one Triticale. A 6x wheat, Triticum com-
pactum var. No. 44, differed from the others in
being normally male fertile. In general, genome
manifestation of 4 x wheat in this respect was
more influenced by caudata cytoplasm than that
of 6x wheat. This was critically shown in the
difference between 4x wheat (pistilloid) and
the hexaploids (male sterile but not pistilloid)
synthesized from the former as one of the parents.

Genomes of four 4 x wheats were introduced
into ovata cytoplasm, and all became male
sterile. Pistillody did not occur in any strains.

Effect of the alien cytoplasm was also noticed
in the increased occurrence of haploid and
twin seedlings. The extent of the increase varied
greatly with the genotype.

13.66. Mutable Alleles at the R Locus in the Soybean

Leonard F. Williams (Columbia, U.S.A.).

In the soybean (Glycine max), four alleles are
known at the R locus, R (black seed-coat pig-
ment), rm (brown base with concentric black
stripes), rx (black with brown hilum), and r
(brown). R and r have been described. rm and rx
are in use by soybean breeders. In the USDA
World Collection of soybean lines, R and r are
most common, rm is uncommon, and rx is rare.
Varieties and genetic types with these genotypes
breed true, and no mutation has been reported
at this locus in established lines. Dominance is
complete and in the order: R, rm, rx, r. In cer-
tain crosses of rm with r, rx sectors appear on
a few Fl and F2 plants, and a few F2 plants oc-
cur that are completely rx. Linkage associations
indicate that the mutants are derived from rm
rather than r. Crosses between true-breeding
/x lines and mutant rx lines gave no segregation
in F2, indicating that the mutant is identical to,
or very similar to rx. In crosses with R, rm
and r, the mutant rx and original rx give similar
segregations in F2. However, certain crosses of
mutant rx with R and r give a small proportion
of /x plants sectored with rm and a very small
proportion of rm plants. One mutant rx line has
been maintained for 15 generations without
change, but others tend to reverse mutate to
rm. No mutants to or from R or r have been ob-
served in these crosses. These results indicate
that the mutability of rm and rx is intrinsic, but
the degree is influenced by other genetic factors.

1 3.67. Mutational Tendency of Different Genes with
the Same Phenotypic Expression. L. B. Mazoti
(Llavallol, Argentina).

In a maize pedigree studied throughout 20

231

Section 13 — Plant Genetics and Breeding

generations, 4 mutations have been produced:
vp YW (I); vp YW (II); vp YW (III); vp YW
(IV) (the three last ones were spontaneous),
each one conditioning the viviparous character
or premature germination of the embryo. The
former mutations, with the exception of the last
one, are not allelemorphs. The 4 mutations
behave as simple recessive genes in the various
genetic environments tested.

In this pedigree apparent cases of homozygous
double crossing-over appeared, (theoretically
probable) without interference, once in a million
cases. Mosaics in aleurone color due to instabi-
lity of the A2 gene. Progeny with necrosed
grooves.

To be able to interpret the conjunction of these
phenomena in closely related individuals, we
believe that there are interactions between cer-
tain loci with metabolic states or with molecules
of variable localization of specific mutagenic
action.

The mutant genes vp which determined a
specific mutational tendency were saved, and
crossed with lines from other stocks; they be-
haved as simple genes of monogenic mendelian
segregation. The specific mutational tendency,
even only in a limited number of generations,
and in exceptional lines of a species, could clear
up some of the phenomena of evolution. On the
other hand, families with a certain advantageous
mutational tendency could probably be used in
breeding objectives.

13.68. On a Spontaneous Mutation of the Cauli-
flower-head Type in Medicago sativa L. A. Pa-
nella, F. Lorenzetti, A. Mariani and G. M.
Briganti (Perugia, Italy).

On a wide program carried out at the Plant
Breeding Department, University of Perugia,
Italy, regarding methods and techniques applied
in alfalfa breeding, in single plant selfed proge-
nies, the appearance of a spontaneous mutation
of the cauliflower-head type was observed.

A morphological and cytological research was
made on the mutant material along with a
study of the hereditary behaviour in three subse-
quent selfed generations.

Morphological and cytological description of
the mutants and data gathered on the segregat-
ing material are discussed.

13.69. A New Inhibitor of Aleurone and Plant Colour
in Maize. N. K. Notani and Chandra Mouli
(Bombay, India).

A new inhibitor (proposed symbol h) of aleur-

one color has been discovered in maize. Its mode
of action is somewhat different from the only
other known inhibitor — C1. Whereas C1 C1 when
reciprocally crossed with homozygous colored
aleurone stocks (AA CC RR) gives only color-
less aleurone, h h does so only when used as
the female parent. In the reciprocal cross, hh
only partially inhibits pigment formation. Oc-
casionally a sector of lighter colored kernels
has also been noted on such cobs.

The hh stock also has the property of inhibit-
ing plant color when crossed to intense — red
{A A BB PIP1) plants. The extent of inhibition
varies from partial to complete. Comparison
between the results from reciprocal crosses has
shown slight differences in inhibition.

13.70. The Heterogametic Sex in Dioecious Flower-
ing Plants. J. N. Hartshorne (Manchester,
Great Britain).

Dioecism in the angiosperms has probably
arisen independently on many occasions. Of the
13 species or groups of species where the sex-
determining mechanism is known, all except
one have heterogametic males, although the
animal kingdom shows that female heterogam-
ety is an effective alternative. There are three
possible explanations for this preponderance
of male heterogamety. The first is that by coin-
cidence an excess of species of the one type
happen to have been investigated. The second
is that dioecism based on male heterogamety
evolves more readily than the alternative. The
third is that although both systems are equally
likely to arise, male heterogamety confers an ad-
vantage which increases its chance of persisting.

Evidence supporting the third possibility
comes from Correns's demonstration that com-
petition between two sorts of pollen leads to
an excess of females in the progeny. Since only
females produce seed, and a single male can
fertilize several females, this arrangement could
help to ensure survival of the species. Experi-
ments to test for Correns's certation effect, using
natural agencies of pollination, are being car-
ried out with dioecious species of Melandrium,
Mercurialis, Rumex and Silene. Results so far
do not confirm Correns's observations, but show
that:

(a) the quantity of pollen reaching the stigma
has no effect on the sex-ratio of the prog-
eny;

(b) sex-ratios either do not deviate signifi-
cantly from 1 : 1 or in some species show
a significant excess of males.

232

Section 13 — Plant Genetics and Breeding

13.71. Sex Variability of Grape Vine Hybrid Off-
springs. M. Neagu (Bucharest, Rumania).

The development of characteristics and fea-
tures with interspecific vine hybrids was studied in
the experiment plot for genetics and selection of
the Genetics Chair, Agricultural College, Bucha-
rest.

Sexual offsprings were used, obtained by cros-
sing the native with foreign varieties belonging
to different geographical and ecological groups
{Proles pontica and Proles occidentalis). Mother
varieties such as Negru moale, Cabernet Sauvi-
gnon, Pinot gris, Italian Riesling, St. Emilion,
Gordan and Aligote have normal hermaphro-
dite flowers.

Sex differentiation of the offsprings studied so
far proved to be a developmental phenomenon,
subject to the heredity of genitors historically
formed during ontogenesis and phylogenesis on
the one hand, and to the environmental factors
on the other hand. The percentage of individuals
with hermaphrodite flowers and that of individ-
uals with female flowers deviates from the clas-
sical ratio 1:1.

Each hybrid combination shows different
segregation ratios between individuals with her-
maphrodite flowers and individuals with func-
tional female flowers. Thus, the percentage of
individuals with hermaphrodite flowers is lower
(38.9 per cent) with the hybrid combination
Braghina ■ St. Emilion, while the same per-
centage is higher (63.6 per cent) with Braghina
and Pinot gris.

Likewise, the mother variety influences these
ratios in different ways. The hermaphrodite per-
centage ranges between 53.5-53.7 when Negru
virtos is mother variety, while with the Braghina
combinations the percentage is highly superior,
ranging between 55.9 and 63.6.

The combination Braghina x Aligote pro-
duced an individual with male flowers as well.

The optimum growth conditions enables the
blossoming of a big number of plants during
the third and even during the second year after
seeding. That is very important both for the
grape vine selection and for rational selection of
genitors, in order to obtain a high percentage
of plants with normal hermaphrodite flowers.

13.72. The Effect of Ceitain Chromosome II Genes
on Fruit Size in the Tomato. L. Butler (Oakville,
Canada).

Chromosome 2 of the tomato contains the
loci of 26 known genes. It also has regions which
control fruit size. Two Fa's which were segregat-
;ng for tall/dwarf, smooth/peach, round/pear,

simple/compound inflorescence, green/suflava,
and green/diluta were grown. Individual fruit
weights, measurements, and locule numbers
were recorded for each plant. The mean fruit
weight per plant varied from 18 g to 196 g.
The order of the genes on the chromosome is
d,p sufdil o s. Analysis of these data showed that
peach and diluta had no effect on fruit size.
Dwarf, pear and compound reduced fruit size
by 7.0,18.5, and 7.7 g respectively. Suflava,
in spite of its reduced chlorophyll, increased
fruit size by 11.1 g. The decrease brought about
by dwarf and compound is not the result of
pleiotropy . The effect of the pear locus on fruit size
mediated by two factors: locule number and
fruit shape. All pear-shaped fruits have 2-4 lo-
cules as a result of either the dominant pleiotropic
effect of this locus, or of a closely-linked domi-
nant gene. In these crosses there is a linear re-
lationship between locule number and weight;
each locule adds 6.5 g to the weight of an indi-
vidual fruit. Pear shape also reduces weight be-
cause the same number of cells in the locule wall,
if arranged in a pear shape instead of a sphere,
decrease weight from 100 g to 80 g. The effect of
suflava is probably pleiotropic because diluta
and suflava are both mutants of Condine Red.

13.73. Leaf-Shape Differences in the Tomato.

J. A. Jenkins (Berkeley, U.S.A.).

Four recessive (dwarf, potato, entire, trifoliate)
and three dominant (Lanceolate, Mouse ear,
Curl) genes in the tomato modify leaf shape.
None of these genes has a noticeable effect either
on the initiation of leaf primordia or on the
structure of epidermal or mesophyll layers. They
do, however, markedly alter the length-width
relationships of mature leaves. Moreover, in
comparison with the normal, which has odd-
pinnately compound leaves with large terminal
segments and three or more pairs of laterals,
each of the mutant phenotypes has fewer lateral
leaflets and frequently a more entire margin.
Most, if not all, of these genes have pleiotropic
effects. Among the progeny of hybrids produced
by crossing the mutant lines with each other,
two hybrid combinations (potato-entire and
potato-trifoliate) were similar to heterozygous
lanceolate in having entire leaves with an almost
complete absence of lateral leaflets. Thus, similar
phenotypes may be produced by different gene
combinations. Studies of morphological, anatom-
ical and biochemical differences between lines
that differ by single genes and by combinations
of these genes are in progress.

233

Section 13— Plant Genetics and Breeding

13.74. A Novel Type of Gamete Elimination in
Tomatoes. Charles M. Rick (Davis, U.S.A.).

Unusual segregations are encountered for genes
on chromosome 4 in certain mating combi-
nations, always involving var. Gondine Red
(GoR) as one parent and Pearson (Psn) or related
vars. as the other. Although normal recessive
F2 ratios are obtained in other combinations,
the proportion of mutants is 51 per cent for afl,
70 per cent for wi, and 77-81 per cent for wA
when entering the cross on the Psn chromosome
and 12 per cent for di, 10 per cent for e, and
0.6 per cent for ful when on the GoR chromo-
some. All Fi's have normal phenotype and are
cytologically normal in all respects. In a distorted
h'4 F2, all of 96 + segregants proved to be
heterozygous and 96 per cent of the F3 families
segregated in the same abnormal fashion. The
cause of the disturbance can be transmitted
equally well from either sex. The CoR factor is
inherited in the fashion of a single gene as
ascertained from 1 normal : 1 disturbed F2
segregations from the cross C0R/+ X wA (Psn).
CoR/Psn hybrids show considerable abortion
of pollen and ovules, although the extent to
which abortion is associated with disturbed
segregation is still not clear. The data discount
dominance reversal, cytoplasmic influence, and
zygotic elimination as causes of the disturbance,
render unlikely paramutation or other mutation-
al phenomena, and suggest gamete elimination
affecting both sexes. Although no model is
entirely compatible with all observations, the
best fit is provided by a gamete killer of comple-
mentary determination and with nearly complete
expression. Map distances from a killer with
100 per cent penetrance would be 9-12 for wA,
16 for ul, 29 for afl, 1 for fid, 32 for e, and 35 for
di. These spatial relations are in the right order
and roughly approximate map distances known
for fid, e and di.

Research partly supported by grant GM 06209
of the U.S. Public Health Service.

13.75. Spontaneous Origin of a Balanced Lethal
Condition in a Synthetic Oenothera Hybrid.

H. T. Stinson (Ithaca, U.S.A.).

The Fi hybrid between the complex heter-
ozygous circle of 14 angustissima strain of
Oenothera parviflora and the 7 paired Douthat
4b race of O. argillicola forms a circle of 14
chromosomes at meiosis. The hybrid has a half
lethal system; the a parviflora (cup) complex is

transmitted through the egg only, whereas the
argillicola complex (a), being lethal-free, can be
transmitted by pollen and egg. Consequently,
the circle of 14 apa hybrid should yield equal
numbers of apa and aa plants on selfing. This
expected behavior has been observed in numer-
ous F2 families, although an excess of apa segre-
gates is sometimes seen. In one pedigree history
in which apa individuals were continually
selfed, aa segregates failed to appear in the F3
through F2 generations. Also, no aa segregates
appeared in progenies from the crosses F3, F4,
F5 apa $$ X argillicola get- The reciprocal
crosses (argillicola $$ X ocpa^), however, yiel-
ded only aa segregates. The a complex of the true
breeding apa hybrid is thus apparently no longer
transmitted through the egg. Substitution crosses
confirm that the aberrant behavior of the apa
hybrid resides in the a complex. In F7, the last
generation grown to date, seven aa segregates
were present among 806 offspring. Recipiocal
crosses revealed these aa segregates to be male
fertile but female sterile. This unexpected result
suggests that the failure of the a complex to be
transmitted through the egg is due to an egg
lethal (incompletely penetrant) rather than to
megaspore competition.

13.76. Multiple Effect of Fertilization in Floral
Plants. I. M. Polyakov (U.S.S.R.).

The widespread conception of fertilization
considers this process only as syngamy (double
fertilization in floral plants). Gamete combi-
nation is considered to be of random character
(with some exceptions).

Physiological and biochemical aspect of
fertilization is regarded as one that has no
direct effect on formation and modification of
hereditary disposition of the progeny. According
to this conception fertilization is nothing but a
"bridge" that helps the germinal plasma to pass
from one progeny to another.

There is a considerable number of facts now
showing that physiology of fertilization has a
direct effect on heredity, variability and vitability
of progeny. The main facts are connected with
finding the effect of polyfatherhood. This effect is
expressed both in general stimulating and some-
times specific modifying action on behalf of that
part of the pollen, which physiologically "par-
ticipates" in the process without participating in
double fertilization (in some cases the pollen
may belong to other species). This fact also
manifests itself in changing the character and
range of offspring variability when pollination
changing conditions are changed (especially

234

Section 13 — Plant Genetics and Breeding

under limited pollination). In connection with
the aforesaid, well-known facts of metaxenis
are becoming more important.

Our trials with 15 species of floral plants
(especially with Nicotiana and Zea mays species)
together with the investigations carried out by
other authors showed the double role of the
effect of polyfatherhood.

On one side physiological "combined action"
of pollen tubes maintains typical hereditary
disposition, on the other side the effect of poly-
fatherhood through changing conditions of
pollination and fertilization (change of qualita-
tive and quantitative composition of pollen, its
physiological state and so on) may lead to the
change of normal heredity.

It may be also considered that syngamy is not
only a simple combination of both parents'
heredity, it can lead to the development of new
formations as a result of metabolic interactions
of two gametes.

In trials with corn it was shown that the effect
of polyfatherhood concerns not only the first
progeny, in some trials this effect reaching even
the fourth generation. Thus, the constant
interrelation of fertilization processes and genetic
phenomena was deeper than it had been sup-
posed.

In our trials we tried also to find out physiolog-
ical and biochemical foundation of the effect of
polyfatherhood. In 1953-1962 we recommended
and used the new method for studying fertili-
zation processes with the help of the pollen
labelled with radioactive isotopes S35 and P3'2.
For the first time these tests were direct physiolog-
ical evidence that pollen without taking direct
part in double fertilization is active in metabolic
processes of growing seeds. We have partly
studied these processes and tried to give their
quantitative characteristics.

The further development of metabolic theory
of fertilization and the study of two most impor-
tant aspects of fertilization — polyfatherhood
and selectiveness will be of great significance
for genetics.

13.77. New Methods in Cotton Hybridization.

D. V. Ter-Avanesyan (U.S.S.R.).

of our work at the Central Asia Experimental
Station of the All-Union Research Institute of
Plant Breeding. There was a task to work out the
method of cotton hybridization using the pollen
of heterochromosomic species and even of
genera of Malvaceae family.

As it is known, in the case of hybridization of
G. hirsutum L. cotton species with G.herbaceum
L. and G. arboreum L. (African-Asiatic species)
having twice less chromosomes in their cells,
pollen tubes of the latter, as a rule, do not
fertilize even if they reach embryo sac of diploid
form. At the same time the pollen of the Mal-
vaceae family (Hibiscus esculentus, H. rosa
sinensis, H. cannabinus and Malva neglecta)
does not germinate on the cotton stigma at all.

But in spite of the evident genetical incom-
patibility of chromosomic gametes we succeeded
in using them when working out two breeding
methods. The first method is as follows: at the
growing period of maternal cotton there were
2-3 buds left on the plant, the rest being
removed. This method stimulated the accumu-
lation of nutrients in flowers. 10-20 pollen grains
were placed on the stigma of emasculated flower
followed by unlimited pollination with the pollen
of a paternal form (in this case of an alien species)
for 3 hours.

The second method with similar results was
the following: in that very evening emasculated
flowers were pollinated with unlimited quantity of
paternal pollen. Next day unlimited quantity of
parental pollen was again placed on the stigma of
the same flower, and at noon (about 20 hours
after emasculation) there was pollination with the
mixture of paternal and maternal pollen in
different amounts.

The first and second generations from seeds
developed by the described method, gave some
interesting plants. The progeny considerably
differed from maternal plants. It got the charac-
ters of non-crossing cotton species and changed
completely its qualitative and quantitative
characteristics being affected by the pollen of
Malvaceae plants. These characters became
hereditarily fixed, and they were transmitted to
progenies. The suggested methods can be used
for cotton hybridization. They will improve our
possibilities of using diversified Gossypium genus
in cotton hybridization.

Achievements in biological science reached in
the last decade are well known. It should be
noticed, however, that as profound is our knowl-
edge with regard to a living cell, so poor it is in
the field of biology of pollination and ferti-
lization. The experimental data remain unex-
plained so far, and they are strictly empirical.
Here are some interesting results of a long period

13.78. The Use of Heterosis, Polyploidy and Male
Sterility in Sugar Beet Breeding and Seed
Growing in the U.S.S.R. N. Negovsky
(U.S.S.R.).

On the ground of extensive use of modern

235

Section 13 — Plant Genetics and Breeding

breeding methods (hybridization, selection,
training on high-fertile soil, etc.) many sugar beet
varieties have been developed and are grown in
the U.S.S.R.: Uladovskaya 752, Verkhnjachs-
kaya 031, Verkhnjachskaya 038, Ivanovskaya
1745, Lgovskaya 059, Ramonskaya 06, Ramons-
kaya 023, Ramonskaya 065, Bijskaya 032, Kir-
ghizskaya 056, Pervomaiskaya 028, Mezhotnens-
kaya 080, and others. Home varieties of mono-
germ sugar beet are sown on the vast territory.
They are: Byelotserkovskaya Odnosemyannaya
1, Yaltushkovskaya Odnosemyannaya 2, Kir-
ghizskaya Odnosemyannaya 8. A number of
monogerm varieties are under State Variety Trials.

In conditions of intensive development of the
U.S.S.R. sugar beet growing, the development
and introduction of varieties and hybrids
(including monogerm) and the use of heterosis
in the first generation is of prime importance.

Together with developing of diploid varie-
ties and hybrids, the development of polyloid
varieties is of great interest, the latter including
a large number of triploid hybrids.

A large number of polyploid varieties developed
in the U.S.S.R. are under variety trials and in
the near future they will be released.

According to the data of the State Variety
Trials the best monogerm triploid hybrid
obtained at Byelotserkovskaya Breeding Station
in 1962 overcame the diploid monogerm variety
of this Station for sugar yield by 3.7-7.9 centners
per hectare in different localities.

To increase the productiveness of polyploid
varieties we conduct a serious task on the
development and use of male sterility in the
process of breeding and seed growing.

In order to reproduce and stabilize sterility
zero-pollinators are selected, inzucht (inbreed-
ing) reproduction being accompanied with
regular culling of half-sterile and fertile forms.

The best hybrids developed on the ground of
sterility will be used in production in the nearest
future.

(under single irradiation) is a dose of 7-8kr
and with such a dose only 9-25 per cent of
irradiated plants survive. Under irradiation of
seeds with a dose of lOkr the germination power
was 80-90 per cent, while the germination power
of the control seeds was 60-70 per cent.

Most seedlings having grown from irradiated
seeds began to flower much earlier than the
control seedlings. Thus in the control two
years after sowing, 30-50 per cent of the seedlings
flowered, while the percentage of flowering in
the irradiated seedlings was 75-85 per cent.

It was observed also that genetic variability in
irradiated seedlings was greater than in the
control.

In colouring of flowers in the control it could
be observed that the colour of flowers of the
paternal and maternal type oi of one of them
inherited most often (for example, domination of
deep-red colour). Irradiated seedlings (the dose
0.5-2kr) in the first generation (Yt) showed
great diversity in colour and shape of flowers
(up to 5-6 groups). Amoug irradiated seedlings
was observed the appearance of shortish and
dwarfish forms.

The diversity of irradiated seedlings in hybrids
obtained from geographically remote forms was
especially great.

Among the seedlings having grown from
irradiated seeds, I, together with G. E. Kazari-
nov, selected much more interesting seedlings
from the point of their ornamental perspective
than among the control hybrids.

In 1962 we selected for further research and
studies, 140 seedlings valuable for their colour
and corrugated, fringed and crocus-like flowers.

(!) kr=1000 roentgen.

13.80. Induced Mutants and Their Suitability to
Hybridization Breeding in Barley. Friedrich
Scholz (Gatersleben, DDR).

13.79. Influence of Ionizing Radiation on Genetic
Variability of Gladiolus Seedlings. IV. Drya-
gina (Moscow, U.S.S.R.).

Hybrid seeds of fifteen varieties of the gladio-
lus varying not only in color and shape of
flowers but in geographical origin were exposed
to radiation.

Dry seeds were exposed to Co60 before sowing.

The doses: 0.5,2,5, and lOkr 0)were tested.
Experiments showed that gladiolus seeds are
more radiation-resistant than their bulbo-tubers.

The critical dose for gladiolus bulbo-tubers

Experimental induction of gene mutations is a
promising tool in plant breeding, particularly for
specific improvements, e.g. as to lodging resist-
ance, early maturing, and the like. No lengthy
backcross procedure is necessary as unavoidable
if non-adapted material is used as source of a
desired character. The induced mutants are
especially suitable to hybridization breeding.
By crosses between various mutants many new
combinations of characters can be produced.
Thus several breeding objectives may be realized
simultaneously.

Model experiments were performed in con-

236

Section 13 — Plant Genetics and Breeding

nexion with the Gatersleben mutation work with
barley. As cross parents distinct mutant lines
of high yielding capacity were used, belonging to
the following groups: (a) stiff-strawed erectoides,
(b) early maturing, (c) naked-grained, (d)
smooth-awned. If two of the characters were to
be combined, e.g. a b, isolation of the desired
double recessives followed in F2 generation.
These were used for a second crossing step, e.g.
(axb) ■ c or (axb)x(cxd), if three or all the
four characters were to be combined. The most
promising progeny lines of numerous isolated
F2 plants were tested in yield trials for several
years. The yielding capacities of these combi-
nation lines on an average are at about the level
of the parent mutants and original varieties.
These forms are, consequently, distinguished by
two, three or even four desired specific characters
and sometimes by increased yields too. Each
of them seems to be more usable, in at least one
respect, than the original varieties or any of the
parent mutants.

13.81. Inbreeding and Hybrid Studies in Marrow-
stem Kale Biassica oleracea L. var. Acephala
D.C. T. D. Johnston (Aberystwyth, Great
Britain).

In this naturally outbreeding species it was
found that inbreeding causes a marked depres-
sion in yield of leaf to an average at I3 of 50
per cent of the original population. The depres-
sion was due to decrease in leaf size and not to
reduction in number per plant.

On hybridization mean yield was restored and
diallel cross analysis of the data for six inbreds
and their hybrids showed additivity of gene
effects on leaf number with no heterosis effects,
whereas leaf size showed marked heterosis.

All possible double crosses were also produced
from the Fi hybrids in an attempt to determine
a means of predicting the best combinations
for commercial exploitation. As would be ex-
pected from this genetic situation the number of
leaves per plant could be forecast with useful
accuracy from the values for constituent in-
breds or Fj hybrids. Leaf size could not how-
ever be so predicted. Although the mean leaf
size of the three possible double-cross combina-
tions obtainable from a given set of four inbreds
showed a significant correlation with the mean
of those inbreds, the range of values among the
combinations was very large and reference to Fi
data did not indicate which permutation would
be best of the three.

Thus, as no correlation was found between
number of leaves per plant and their size, it

appears that to obtain the highest yielding
double crosses severe selection can be carried
out among potential inbred parents for leaf
number, but for leaf size no useful preliminary
selection can be performed.

13.82. The Genetic Control of Growth Rate in
Tomato. W. J. Whittington and W. E. Peat
(Sutton Bonington, Great Britain).

A diallel cross experiment was carried out in-
volving Lycopersicon pimpinellifolium and sev-
eral varieties of L. esculentum in order to in-
vestigate the genetic control of relative growth
rates. One investigation followed the relative
growth rates for total dry weight and leaf area
in Fi and F2 generations; another followed
growth rate for leaf number in Fi, F2, Backcross
and F3 generations. Growth rates were calculated
as bi' and b2' from polynomial regression equa-
tions fitted to the log measurements of the data
and as initial growth rate from the exponential
period of growth. The results for dry weight and
leaf area were similar in that bi' was found to
be inherited with complete dominance. Although
there were significant differences between cros-
ses in values of b2', the large environmental com-
ponent of variation masked the genetic effects.
There was heterosis for initial relative growth rate
in the crosses involving L. pimpinellifolium and
negative heterosis in two of the crosses between
varieties of L. esculentum. This was probably
due to gene interaction. For leaf number bi'
and initial relative growth rate were found
to be inherited additively with varying degrees
of dominance.

The results showed that the inheritance of
growth rate could be studied successfully by the
methods of biometrical genetics and also that the
successive measurements of growth with time
allowed a greater understanding of the genetic
and physiological data obtained. The experiment
continues that described previously/1'

1. Kheiralla, A. I. and Whittington, W. J.
Genetic analysis of growth in tomato. I. The
Fi generation. Ann. Bot. 26, 489-504.

13.83. Effect of Growing Conditions of Fi Soft Spring
Wheat on Variability in F2. T. Ya. Zaru-

BAILO (U.S.S.R.).

There were made crosses between soft spring
wheat varieties with different duration of their
growing periods.

237

Section 13 — Plant Genetics and Breeding

The first progeny of hybrids (Fi) from these
crosses was grown in various conditions: some
Fi plants from each combination were sown in
spring as usual, the rest in late fall (early win-
ter) (on the 5th of October). The second off-
spring (F2) was sown in spring and was grown
in the same conditions. It appeared that growing
conditions of Fi affected the characters of F2
plants to a considerable extent, especially their
segregation (variability) according to the dura-
tion of their growing periods, especially when
parental forms (varieties) greatly differed in this
respect. In this case the following phenomenon
was observed: when Fi was sown in spring F2
plants entered their heading stage simultaneously
with the early-maturing parental form or earlier,
and finished it simultaneously with the late-
maturing parental form. Most F2 plants had
passed through their heading stage by the time
the earlier parent finished it.

If Fi was sown in late fall (early winter), the
behaviour of F2 plants was quite different. The
beginning of their heading also coincided with
the beginning of that of early-maturing parental
forms, but when Fi plants had already passed
this stage, the number of F2 plants with devel-
oped heads was about 20 per cent.

Most hybrid plants formed their heads
simultaneously with their late parent and the
considerable number of plants (about 20 per
cent) formed their heads later than their parental
form. This latter part of hybrid plants formed
their heads for a very long time and finished it
a month later than the late matured parental
form. In this case only 3 per cent of plants
reached their tillering stage before winter
dormancy, i.e. they became winter crops.

When parental forms did not considerably
differ in the duration of their growing periods or
if both of them were comparatively early-matur-
ing, early winter sowing of Fi also led to the
obviously prolonged period of heading of F2
plants, but this period was not so long as it
was in the first case. There were no winter crops
here.

Growing conditions of Fi also affected the
variability of other characteristics of F2 plants,
head productiveness in particular. When Fi
was sown in early winter F2 plants of all combi-
nations in our experiments were characterized
by higher productivity index as compared with
Fi sown in spring.

In that way growing conditions of Fi can affect
the characteristic of F2 to a considerable extent.
This can be explained by the fact that the stability
(conservatism) of the heredity of Fi plants is
shattered and therefore they are easily trans-
formed being affected by changing growing
conditions.

13.84. About Bieeding of Sprouting Resistant Cereals .
Herbert W. Mueller (Bernburg/Saale, Ger-
many).

Beginning in 1953 we crossed the resistant
"Wjatka-rye" with "Petka" and "Champagner".
293 ears were artificially pollinated and tested.
In Fi, sprouting was dominant. Only 2.7 per
cent of the kernels were as resistant as the "Wjat-
ka-rye". This was partially caused by the low
weight of the kernels. The average was 24 per
cent lighter than the average of the parents.
After crossing of barley the result in Fi was, that
there are reciprocal differences between the
crosses of the sprouting resistant "Neuga" and
the fast sprouting sorts "Mansholts" and
"Nordland". The differences are caused by
the matroclin parent, probably a result of the
covering layers of the kernel. The Fi in average
was equal to the better sprouting parent.

The spring wheat combination "Koga" x
"Bernburg 38626" was tested with 42 ears in
Fi, and sprouting was as quickly as "Koga".

In following years the combinations were
tested starting with the F2. The descendants of
the Fi- kernels were examined with the Eosin-
test. The reaction of the populations was not
uniform, but the effectiveness of an F2-selection
was very unsatisfactory. The correlation values
of the selected resistant descendants in F2 to
reaction in following generations were only:

rye r = 0.09 wheat r = 0.12

oat r = 0.10 barley r = 0.12

The following conclusions are possible:

1. The heritability of sprouting is regulated
by many genes.

2. The effectiveness of a selection in F2 is so
small, that for breeding purposes there is no
applicability.

The intensive testing of 10 winter barley popula-
tions on the other hand showed the possibility
of negative results of F2-selection.

Comparing the populations selected in F3,
higher correlations between the selected parents
and their descendants were possible. Averaging
all combinations we get following values:

winter barley r = + 0.46

winter wheat r = + 0.38

spring wheat r = + 0.48

oat r = + 0.39

The values are satisfactory for cereal breeding.

The correlations of F4-se!ections were some-
what higher but there are many technical
difficulties to select a greater number of popula-
tions and to test their descendants. It would be
more economic to test a greater number of
populations in F3.

238

Section 13 — Plant Genetics and Breeding

13.85. Breeding Drought-resistant and High-yielding
Spring Wheat. V. P. Kuzmin (U.S.S.R.).

Success of breeding depends on correct deter-
mination of plants' characters according to the
character of environment that forms, preserves
and intensifies these characters.

Central Kazakhstan has a peculiar arid cli-
mate: long cold periods in spring, abruptly
followed by hot dry weather in the beginning
of summer. Rains are typical for the middle of
summer, while the second period of drought
and early frosts are typical of autumn.

The main sources of moisture for plants are
their deep subsoil reserves and rains in the mid-
dle of summer.

Thus, to overcome drought and to make full
use of moisture, wheat breeding was aimed at
developing various varieties with different
development and growth rate, i.e. with proper
growth activity during the cold spring period
(early variety type) and with desirable growth
dynamics during summer rains. As for morpho-
logical characters the greatest attention was
paid to the development of the root system.

The general method of work consisted of
hybrids training by environmental conditions and
selection in usual field conditions.

Good results were obtained from Winter and
Spring wheat breeding: higher resistance to
spring frosts and drought. Hybrid training in-
cluded different sowing dates: very early, middle
and late.

Breeding stock selected according to its ability
to produce a great number of good seminal and
crown roots was sown on limited fertile, moder-
ately warm and humid soil. This soil correspon-
ded to usual field conditions. Plant varieties de-
veloped by this method had a good root system
that did not degrade but became even stronger.
As a result the plant productiveness increased.

Some Spring wheats were bred by this method;
they show in commercial trials the highest
yielding ability and annual stability of output
on the territory with peculiar arid climate.

13.86. Controlled Modification of Heredity of Non-
winterhardy Varieties of Durum Wheat, Pea
and Two-row Barley into Winterhardy Crops.
V. F. Khitrinsky (U.S.S.R.).

T. D. Lysenko has worked out theoretical
foundations of controlled modification of plant
heredity by training. In the Genetic Division
of the Ail-Union Institute of Plant Breeding and
Genetics (Odessa) we conduct experimental
work on controlled modification of heredity of

durum wheat, pea and two-row barley into
winterhardy varieties.

1 . 19 strains of winter durum wheat (7>. durum
Desf.) developed by training were investigated
at our Institute during the competitive variety
trials in 1962. The trials showed that as a result
of winter training non-winterhardy wheat chang-
ed hereditarily into winterhardy one. It changed
some physiological characters, including the
vernalization and light stages which became the
same as they are in common winter wheats. The
transformed durum wheat is safely wintering in
the field and endures low temperatures — up to
— -20°C. It is a good yielder (40-42 centners per
ha) resistant to drought, diseases and lodging
and its grain is of good quality. It was sown on
4 hectares in the autumn of 1962.

2. As a result of autumn training 22 non-win-
terhardy Pisum arvense varieties (originating
from the U.S.S.R., Western Europe, Asia and
Africa) are purposedly transformed into winter-
hardy varieties. They acquired a new property-
winterhardiness. They have changed the duration
of growing period, habitus, colour of seed and
some varieties, even the form and size of seed.
Winterhardy pea is blooming and fruiting 2-3
weeks earlier than the non-winterhardy pea and
the yield of green mass is considerably higher in
the first case.

Two varieties of non-winterhardy Pisum sati-
vum plants were transformed into a winter-
hardy variety by the same method.

3. Experiments on controlled modification of
heredity of spring and winter crops included two-
row barleys: Hordeum distichum L. var. nutanus
Schiibl. and var. medicum Korn. There were
commercial varieties among them — Odessky 9,
Odessky 14 and others. As a result of training
we have controlled hereditary modification of
morphological, biological and valuable economic
characters adequate to autumn conditions. Win-
ter and alternate forms of two-row winterhardy
barley were developed from spring non-winter-
hardy two-row barley plants. The Odessky 17
commercial variety produced the average yield
of 34 centners per hectare and 97 strains of
transformed two-row barley yielded 37.3-56
centners. 130 best strains of transformed two-
row barley were sown on 1.5 ha in autumn of
1962.

In conclusion it is necessary to note that using
plant training in particular conditions, it is
possible to modify and control their heredity
developing new varieties with desirable charac-
ters.

The method of controlled transformation of
spring non-winterhardy plants into winterhardy
ones has become a valuable method in the So-
viet plant breeding.

239

Section 13 — Plant Genetics and Breeding

13.87. Genetical Differences in the Sensitivity Against
2,4-D auxin-herbicide in Tissue Culture. Bela
Faludi (Budapest, Hungary).

The effect of auxin-herbicide 2,4-D consists of
the induction of tumorous growth in the resting
meristematic cells in the stem of dicotyledonous
plants. In order to study the sensitivity against
the herbicide it is conducive to exclude the en-
vironmental non-genetical differences and the
morphological factors of uptake and transport as
well. For this purpose the tissue culture method
offers a good opportunity. The proof of varietal
differences apart from its theoretical signifi-
cance might form the basis of the selection de-
creasing the sensitivity of cultivated plants
against the herbicide.

In tissue cultures of potato tubers (Solarium
tuberosum) we tested 12 varieties, and found
considerable differences in the extent and char-
acter of tumorous growth.

We subjected five varieties of characteristic
behaviour to a detailed biometrical investiga-
tion to examine the extent of variance among the
varieties, among the individual plants within
the variety and to compare it with the variance
of the method applied. The investigated varieties
were the following in the order of their sensiti-
vity: Margit, Aquila, Ella, Delta, Gul Baba. The
extent of multiplication of the fresh weight of the
cultures amounted in two weeks time: 0.8;
1.0; 1.5; 1.6; 3.2. The variances in the F-value
were for varietal differences 1041, for intra-
varietal differences 70, and for differences be-
tween tissues of individual tubers 83 respectively.
On the basis of these data we may conclude
that differences among varieties are highly sig-
nificant concerning the exclusively genetically
determined resistance. The variance in the same
variety was not significant. These data indicate
that prospects for clone selection are not favour-
able. On the other hand the prospects of the
varietal selection are promising to obtain resist-
ant potato varieties against the 2,4- D herbicide.

13.88. Some Vine Clones Resistant to Plasmopora.

P. Coutinho (Lisboa, Portugal).

The extent of damage caused by Plasmopora
viticola in most regions of Portugal is very im-
portant.

This paper refers to attempts made since 1945
with regard to this subject.

The adopted criterion of selection is described.

At present we are studying 110 clones of F2
of interest on account of their resistance.

There are another 18 clones already in an

advanced stage (experimental field scale),
so far the most interesting being clone C.27, a
cross of "Jaen X Azal branco".

Finally some of their productivity and quality
characteristics are mentioned.

13.89. Mechanism of the Loss of Fusarium
Wilt Resistance in Hybrid Bananas. D. L.

Richardson and D. J. Hutchison (La Lima,
Honduras).

In the classical method of banana breeding a
Fusarium wilt susceptible triploid clone "Gros
Michel" is used as the female parent. The major-
ity of functional eggs produced by this clone are
triploid, resulting from a meiotic restitution of
the chromosome complement. Genes conferring
wilt resistance are introduced through the use
of wilt-resistant diploid male parents and the
resulting tetraploid hybrids are screened for
commercial usefulness. Many cases are now
recorded where resistant segregates have been
grown for years before giving rise to susceptible
sub-clones, but no satisfactory explanation of
this loss of resistance has been proposed. That
resistance genes are found in only a single dose
in these autotetraploids, presents a situation
where the somatic loss of chromosomal material
containing resistance genes could result in the
appearance of wilt susceptible sub-clones,
Cytological studies indicate that this explana-
tion is probably correct.

13.90. Breeding of Tobacco for Resistance to Ordi-
nary Tobacco Mosaic (Marmur tabaci Holmes).

Serapion J. Bayubay (Los Baflos, Philip-
pines.).

Crosses between mosaic resistant varieties
Ambalema and Ky 52 (White Burley) and the
susceptible but otherwise desirable varieties
Marogui, Simmaba C, Vizcava and Romero
were made in the Central Expt. Station, Manila,
in March 1950. Including reciprocals, 16 cross
combinations were obtained.

The F2 seeds and seeds of the parental plants
were planted in rows with the corresponding
parents planted beside each hybrid and were
inoculated with the mosaic disease by rubbing
mosaic infected leaves over the surface of each
of the three young top foliage of the plants.
Bagged F2 seeds from each line that had no mo-
saic were again planted in progeny rows the fol-
lowing season and F3 plants were evaluated for
their agronomic characters and degree of

240

Section 13 — Plant Genetics and Breeding

resistance to the mosaic disease. F3 seeds of the
selected plants within a line were bulked.

Evaluation of resistance in the late segregating
generations was made by counting the number
of mosaic infected plants one month after trans-
planting and thereafter counting of mosaic
infected plants was made every week until
harvesting of leaves.

For a single entry, selection for mosaic resis-
tance and other desirable characters from genera-
tion to generation was performed following the
bulk method, assuming that continuous selling
leads to homozygosis.

Experimental Results. The Fi hybrids of the
reciprocal crosses were identical and were
intermediate in most characteristics between the
parents, especially with respect to height,
position and shape of leaves.

In all cases the majority of the Fi plants were
very susceptible to the mosaic disease under
artificial inoculation indicating that recessive
factors control mosaic resistance. Selected mosaic
resistant F2 plants, assuming that one pair of
factors controls resistance should have produced
all resistant plants in the succeeding generations,
but the fact that they did not, like the resistant
parent, suggests that resistance is controlled by
multiple recessive factors.

The segregating hybrids showed significant
difference in their degree of resistance in all
four years of testing. The hybrid lines Amsim
(Ambalema ■ Simmaba) and Simam (Simmaba
x Ambalema) were the most resistant followed
by Maram type B (Marogui x Ambalema);
Amar type A (Ambalema x Marogui) and Vi-
zam type A and B (Viscava x Ambalema).

Generally, hybrid lines were significantly
more resistant than the susceptible parents,
and as resistant as the introduced parents.

13.91. Method of Crossing Geographically Remote
Forms in Winter Wheat Breeding. P. P. Luky-

ANENKO (U.S.S.R.).

graphically remote forms of soft wheat supple-
mented with repeated crosses and controlled
individual selection led to the development of the
new, for the North Caucasus highly productive
winter wheat varietal type: Bezostaya 1 variety
(Lutescens). It is one of the first Soviet intensive
varieties that is distinguished among all winter
wheats for its short stem, lodging-resistance,
productive heads, weak susceptability to sriple
and brown rust, high yielding ability and good
milling and baking quality of grain.

Wheats of rather remote geographical and
ecological origin which belong to varieties
cultivated in various countries and continents
Argentine, Italy, Japan and others) had
become components of the genealogy of Be-
zostaya 1 variety.

Many years of the State Variety Trials showed
Bezostaya 1 to be one of the most productive
varieties among winter wheats in main winter
wheat growing areas of the U.S.S.R. (except
regions of severe climate). This variety yields over
50-60 centners per hectare. Having unprece-
dented yielding ability this wheat variety is
characterined by high grain quality and features
of "strong" wheats. Bezostaya 1 is the first
winter wheat in the Soviet breeding which is
widely cultivated not only in the Soviet Union
but in some foreign countries (Hungary, Ru-
mania, Bulgaria, etc.).

The report describes in brief the adopted
method of breeding; the initial breeding stock;
correlation coefficient that determines the main
trends of breeding; the method of individual
selections in hybrid populations and particularly
selection for brown rust resistance. The report
touches on a problem of the further rise in wheat
productiveness in humid areas by means of
developing varieties with still lower stems and
highly productive heads. These varieties have a
complex of biological characters on which their
adaptability to local conditions depends. The
report presents a brief survey of obtained
material and perspectives of winter wheat
breeding for high productiveness.

The lasting use of the method of crossing
geographically remote forms which had been
first suggested by I.V. Michurin proved highly
efficient in winter wheat breeding at the Krasno-
dar Agricultural Institute. In combination with
controlled individual selection this method
permitted to create regularly new varieties with
the complex of necessary economic and biological
characters and to raise the upper limit of the yield
of the developed varieties. As a result the yielding
ability of varieties doubled and during a short
period rose from 21.1 up to 50.6 centners per ha.

Interspecific hybridization of selected geo-

13.92. A Homozygous Heterozygote. Benjamin
H. Beard (Brawley, U.S.A.).

After all recombinations have been achieved,
varietal improvement by plant breeding methods
would appear impossible. Generally speaking
additional chromatin indicates an evolutionary
advance, i.e. as the chromosome number
increases the organism becomes more compli-
cated. Drastic changes such as the addition of
one or more chromosomes probably require

241

Section 13 — Plant Genetics and Breeding

many generations before complete compatability
is achieved. Another addition of chromatin is a
duplication. Stern showed that duplication of
certain genes in Drosphila leads to the production
of a larger quantity of the substance normally
produced by that gene. Wallace and Vetukhiv
showed an advantage for heterozygous Drosphila
if there is a mechanism for maintaining hetero-
zygosity. Two alleles in a disease conditioning
allelomorphic series such as found by H. H. Flor
in flax and J. G. Moseman in barley may give
resistance to two or more races of a parasitic
organism. A priori reasoning leads one to
believe that a duplication of a specific locus offers
many possible means for improving a species.
In a self-pollinating organism, a true breeding
heterozygote might be possible.

Detecting an irradiation-produced duplication
of a specific locus may be possible by genetic
techniques. The proposed method involves
irradiating crossed barley seeds and following the
pollen sterility of F1X1 and phenotypes of
F2X2 plants. The desired aberration, an unequal
reciprocal translocation between comparable
arms of homologous chromosomes should give 50
per cent pollen sterility. The progeny from such
plants can be studied for segregation. Alleles
showing partial dominance may express the
hybrid phenotype in the homozygous duplicated
condition.

13.93. On Natural Periodical Change of Inbreeding
and Crossbreeding in Plants. J. P. Mirjuta
(Novosibirsk, U.S.S.R.).

Many papers on selective fertilization have
appeared, in a number of which it is stated that
when applying a mixture of pollen from the
variety itself + other varieties of the same species,
the strain-population is mainly fertilized by its
own pollen, whilst deep inbred lines are to a
greater extent fertilized by pollen of the other
varieties.

In our own experiments on corn and spinach,
we compared two pollen mixtures, viz. (1)
variety itself + other variety, and (2) plant
itself W + other variety. The strain-population
plants selected related pollen to a higher degree
from mixture (2) than from mixture (1). With
deep inbred lines the reverse is true. In addition
it was found for deep inbred lines that selection
for own line pollen is higher than selection for
own plant pollen but lower than selection for
initial variety pollen.

This means that in not-inbred plants, pollen
selection is directed towards inbreeding (adap-
tation to natural inbreeding), whilst during the

course of inbreeding a shift towards the opposite
takes place, because deep inbred plants select
unrelated pollen, that is they are directed
towards crossbreeding (adaptation to natural
crossbreeding).

It should be noted that natural inbreeding is
widely spread even among such crosspollinators
as corn, spinach and hemp. Consequently,
deep inbred plants should be present in strain-
populations as the result of natural inbreeding.
We found that the corn variety contains 15 per
cent of plants at a level of inbreeding where
other variety pollen is favoured.

1. Or brothers

1 3.94. Some Biochemical and Physiological Properties
of Plant Reciprocal Hybrids. S. I. Issaev and
V. V. Vartapetyan (Moscow, U.S.S.R.).

Numerous investigations on morphological,
physiological and biochemical properties of
reciprocal hybrids of woody plants (apple-tree)
as well as other plants (tomato) were carried on
since 1935 in I. V. Michurin Horticulture
Institute and since 1955 in Moscow State Uni-
versity, Chair of Genetics.

The choice of the maternal plant often deter-
mines the difference in properties of the
hybrid generation derived from the same pair of
initial forms.

Initial varieties influence more actively the
expression of properties of the hybrid generation,
when they are used in the initial pair as maternal
plant.

Hence it was demonstrated, that crossing of
frostresistent (northern varieties) and frost-
sensitive (southern varieties) of apple-trees
produced more frost-resistant hybrid Fi, in the
case where the northern variety was used as
maternal plant.

Content of sugar and vitamin C in fruits of
reciprocal tomato hybrids was higher in the case,
where the variety with high level of these sub-
stances was taken as maternal plant. The choice
of the maternal plant influenced also the degree of
heterosis. Heterosis of a certain property in-
creased, when the maternal plant of the pair
was characterized by the same property.

Special experiments were carried out with
grafts and the reciprocal crosses on these. These
experiments showed that additional influence of
the maternal plant on the properties of the
progeny could be explained mainly by metabolic
action of plant plastic substances on the embryo,
which developed on this plant.

242

Section 13 — Plant Genetics and Breeding

The obtained data could be valuable in a
scientific as well as in a practical sense, namely
for selection and seed-growing.

13.95. Effect of Genetic Albinism on the Photosyn-
thetic Utilization of Light. Istvan Gyurjan
(Budapest, Hungary).

The efficiency of photosynthesis in plants, and
also the distribution of 14C02 over the different
compounds are greatly influenced by the intensity
and the wavelength of light. Photosynthetically,
light affects in the first line the amount of pig-
ments which, in its turn, influences the extent to
which carbon dioxide is incorporated, and
influences moreover the further part of carbon
and oxygen.

The assimilation of 14CG*2 by albino maize
seedlings, particularly sensitive to light, has been
studied at different intensities of light. The
activity of the alcohol-soluble fractions (amino
acids, organic acids, sugars) revealed a linear
connection with the concentration of pigment at
the employed intensities of light (5, 100, 1000 and
10,000 luxes). The rate at which 14C02 was
incorporated by alcohol-soluble compounds in
the range of optimum light intensity for albino
(from 100 to 1000 luxes) proved to be much
higher in albino than in normal plants. The rate
of incorporation became lower in strong light
(10,000 luxes) on account of the destruction of
pigment in the albino leaves.

Qualitative and quantitative analysis of
autoradiograms made in respect of the alcohol-
soluble fractions revealed the fact that activity
of the organic acids became higher in albino
leaves, while the activity of amino acids charac-
teristic of photosynthesis (alanine, glycine,
serine, aspartic acid) as well as that of sugars
appeared to be higher in normal leaves. This
difference was wider at higher intensities of
light. The qualitative differences make it proba-
ble that deviations in the light tolerance of nor-
mal and albino leaves affect the mechanism
through which 14CC<2 is incorporated. This mani-
fests itself in the fact that — apart from affecting
the amount of pigments — light does not uni-
formly influence the different ways of carbon-
dioxide incorporation.

13.96. Different Types of Carotenoid Abnormalities
in Albino Leaves. Agnes Faludi-Daniel
(Budapest, Hungary).

A considerable part of chloroplast mutants of

higher plants possess the ability to synthesize
chlorophyll. These mutants have the genetic
block elsewhere in the metabolic pattern.
Investigating the carotenoid content and synthe-
sis of albino leaves in maize and barley we found
abnormalities characteristic of individual chloro-
plast mutants.

We have shown that leucine is an effective
early precursor of carotenoid synthesis of the
leaves during chloroplast formation. Conse-
quently albinos with abnormal leucine content
might have disturbances in the carotenoid
synthesis. Another type of albinos contains
carotenoids with partially saturated double
bonds, lacking the ionon ring too (phytoen,
phytofluen, Z -carotene). A further type of
chloroplast mutants produces carotenoids
with normally conjugated double bonds but
the process of ionon cyclization is blocked
(lycopene).

The qualitative and quantitative deviations in
the carotenoid content are related to an in-
creased lability of the protein-pigment complex
of the chloroplasts. It was found that the lack of
the ionon ring decreases the amount of stable
carotenoid-protein complexes by about 30 per
cent. If the lack of ionon ring is associated with
the unsaturation of double bonds the amount of
stable protein pigment complexes decreases by
45 per cent below the normal level.

On the basis of our data we are able to set up
a hypothetical scheme concerning the localization
of genetic blocks in the albino mutants with
deviating carotenoid content.

13.97. Variability and Inheritance of the Protein Level
in Plants. S. Barbacki (Poznah, Poland).

Research of several years' duration into the
variability and inheritance of the protein level
in barley and lupin has pioved that this character
changes its value in dependence on the following
factors: climatic conditions (in main rainfall),
soil conditions, term of sowing, spacing, ma-
nuring (in main with nitrogenous fertilizers) and
time of harvest. The genetic background is also
important as it determines the metabolic type.

The protein level cannot be studied independ-
ently from the environmental factors and other
qualities of the plant. The type of development,
earlier or later ripening, the accumulation of
other compounds — in the first place of carbohy-
drates— are all of importance in this case. In
considering these elements it is possible to
distinguish genotypes greatly differing by their
reaction to the environmental conditions,
carrying a tendency to accumulate lesser or
greater amounts of protein. Crossing of various

243

Section 13 — Plant Genetics and Breeding

genotypes brings about different kinds of
segregations in the offspring which result among
others in the arising of transgressive forms.

The elements of "crude protein" are also
peculiar to the genotypes. Various forms of
nitrogen are differently represented in them.
Owing to the particular importance of the level
of certain forms of nitrogen, in main of exogen-
ous amino acids, in the nutrition of man and
animals, it may be of practical value to study the
tendency of their accumulation.

13.98. Biochemical Interspecific Differences within
the Genus Lathyrus. J. Przybylska (Poznah,
Poland).

Within the genus Lathyrus there were found
clear interspecific differences in the composition
of free amino acids in seeds. Individual species
are characteristic of an accumulation of different
peculiar new free amino acids (a, y-diamino-
butyric acid, homoarginine, (3-(-2-aminopiry-
midine-4-yl) alanine and other substances with
amino acid properties unidentified as yet) which
suggests the existence of different pathways of
nitrogen metabolism. It can be assumed that the
amino acids accumulated in seeds and peculiar to
the individual species are not metabolic by-
products; they are rather forms of storage, or
storage and transport of nitrogen. These com-
pounds are sure to have originated as a result of
genetically conditioned changes in the activity
of enzymes involved in the amino acid transfor-
mations. At the present moment comparative
studies are being carried out on the role of
individual peculiar non-protein amino acids
in the nitrogen metabolism of several species of
Lathyrus.

13.99. Genetical and Morphological Analysis of the
Regularity of Right- and Left-handedness in
Wheats. Hinako Suemoto (Kyoto, Japan).

Right-handed and left-handed leaves (di-
rection of folding) and spikelets (direction of
first floret) alternate regularly (are "concordant")
along stem and spike. "Discordant" leaves and
spikelets are often found below the 3rd or 4th
foliage leaf and above the 10th spikelet.

The degree of regularity or the intensity of
polarity at a given position is measured by the
"Concordance proportion" (C). Concordant and
discordant leaves or spikes are scored 1 and 0
respectively. The mean per position for a number
of shoots is C, with binomial standard error.

Averaging over positions one obtains c, the
mean concordance proportion, which a.o.
serves as a measure of species differences in R/L-
handedness.

Each species of Triticum and Aegilops has its
own specific value of c and the specific curve of
C. A certain relation between c-value and the
genome constitution is found. The results of the
polygenic analysis on two Einkorn species
(T. monococcum var. flavescens and T. aegilo-
poides var. boeoticum) support the view that a
polygenic system takes part in the species
difference of c-value.

The R/L-polarity is induced at about the time
of germination, and is maintained throughout
the developmental stages and disappears,
according to the genotype of the plant. But the
type of c-curve and the direction of the polarity
is modified by the various artificial treatments.
The deficient nutrition and X-ray irradiation
causes a reduction of c-values of developing
organs. The gravity affects the induction of the
R/L-polarity at the time of germination. This
sensitivity is specific of the genotype. Further-
more, growth-hormone treatment modifies the
R/L-polarity induced by the gravity.

These results will be reported in this paper.

13.100. Pathogenicity of Radiation-induced Mutants
in Two Important Phytopathogenes of Rice.

Yoshito Yamasaki, Takashi Suwa and
Nobuo Murata (Tokyo, Japan).

Nutritional deficiency mutations were induced
by u v X-, and y -irradiation in Xanthomonas
oryzae and Piricularia oryzae, pathogens causing
bacterial leaf blight and blast respectively in rice,
and changes in pathogenicity of mutants were
investigated with the view to make clear the
significance of mutations for nutritional charac-
teristics in specialization in pathogenicity in
each of these organisms.

In Xanthomonas, mutants requiring histidine
(4), nicotinic acid (2), and tryptophan (2 out of
6) were as pathogenic as wild strains whereas
those requiring arginine (2), leucine (2), isoleucine
and valine (4), threonine (1), and tryptophan
(4 out of 6) showed a marked decrease in
pathogenicity.

In Piricularia, mutants with requirement of
NH4-nitrogen (6), glutamic acid, etc. (1), glycine
(1), leucine (2), methionine (1), cystine or
methionine (1), cystine, methionine or thiosul-
fate (1 out of 2), histidine (1) tryptophan or
nicotinic acid (1), nicotinic acid (1), inositol (1),
and choline (1) remained pathogenic and those
with requirement of phenylalanine (2), adenine

»44

Section 13 — Plant Genetics and Breeding

or hypoxanthine (2), and cystine methionine or
thiosulfate (1 out of 2) proved to be of decreased
pathogenicity.

In the latter organism, restoration of patho-
genicity by supplementing infected tissue with
the nutrient required was observed in one of
adenineless strains and a close correlation be-
tween the nutritional characters and the loss of
pathogenicity was confirmed, though trials with
the other adenineless and the two phenylalanine-
less mutants have been unsuccessful, suggesting
other mutations for decreased pathogenicity
operating in these strains.

Experiments are under way to induce rever-
tants with respect to the nutritional deficiency
and observe whether they restore the pathogeni-
city.

13.101. Initial and Adaptive Tolerance of Ustilago
scitaminea and U. maydis on Various Concen-
trations of Sodium Arsenate. Elisa Hirsch-
horn (Llavallol, Argentina).

102 monosporidial cultures of U. scitaminea
and 70 of U. maydis, from different origins,
were tested in various concentrations of sodium
arsenate, in order to determine the limits of ini-
tial tolerance and the limits of adaptability to
increased concentrations.

U. scintaminea. The initial tolerance ranged
from 1.1 to 3.5 mg/ml. The following results
are obtained for initial tolerance and adaptive
tolerance (number of strains between brackets).
1.1-H.l (15, no adaptation); up to 2.2->3.0-4.8
(62); up to 1.75-M-8 (16); up to 3.5^7.0-8.0
(9). Adaptation was transitory, as found after
4-7 retransfers on arsenate-free medium. Te-
trad analysis showed for initial tolerance (toler-
ance: no tolerance) 0 : 4 and 2 : 2. The
acquired tolerance, however, was not transmitted
sexually.

U. maydis. Initial tolerance ranged from
4.0 to 10.0 mg/ml. All 70 strains first adopted
to 20.0 and then 59 strains to 50.0 and 11 strains
to 65.0. Transitory adaptation was found in 60
strains, though only after 20-25 retransfers on
arsenate-free medium. However, 6 strains which
adapted to 65.0, did not loose adaptation during
4 years.

Tetrad analysis showed for initial tolerance
(tolerance: no tolerance) 4 : 0, 2 : 2, 3 : 1 and
1:3. Sexual transmission of acquired tolerance
was found in 2 strains (adapted to 20.0 mg/ml),
with tetrads (tolerance: no tolerance) 0 : 4,
2 : 2, 3 : 1 and 1 : 3. The nature of adaptability
is considered.

13.102. (D.) Changing of Tomato Heredity under the
Influence of Light and Low Temperatures.
L. Rastunkova and R. Glavinich (Moscow,
U.S.S.R.).

Under the simultaneous influence of light and
low temperatures we have succeeded in changing
the heredity of the late Moscow region tomato
variety No. 7, created by R. Glavinich by means
of vegetative hybridization, as well as of the
middle variety The Best. The two varieties
have round red tasty fruit, but in Moscow condi-
tions often give many green fruit. The tempera-
tures of the experiment have been + 2 and — 5 and
the day length 10 hours. The germinated seeds
have been kept in low temperature conditions
for the time from 6 to 10 days.

The influence of light and low temperatures
on the tomato plants of these varieties decreased
the vegetative period of the experimental plants
as compared to the control plants as well as to
the plants of the standard variety Gruntovoj
Gribovskij. Beside that, the experimental plants
had different fruit size to compare with the control
ones. The experimental fruits have become smaller
but more numerous. Some of the experimental
plants had changed colour of the fruit, as well as
their form. They were elongated and of crimson
colour.

All these acquired characters have been in-
herited by the second and the third generations
and some of the plants of No. 7 and The Best
varieties can be used as good seed material.

13.103. Ionizing Radiation in the Evaluation of the
Genetica! Constitution of Some Characteristics
in Plants. A. Tavcar (Zagreb, Yugoslavia).

1. From genus hybrid (Triticum aestivum x
Secale cereale) > Triticum aestivum a homozy-
gous Tr. aestivum (2« = 42 chromosomes with
hairless upper internodium of the stem was se-
lected. In R2 gen. from seed irradiated with 5000
to 7500 r of gamma-rays, some speltoids with
hairy upper internodium, similar to the male par-
ent Secale cereale, have developed. During 8
generations of nonirradiated seed only Tr.
aestivum of hairless upper stem were observed.

2. In (Vicia Faba maior x V. F. minor)
homozygous V. F. maior and V. F. minor plants
were selected. Some of their seeds were irradiated
with gamma-rays. From their V. F. maior seed
also some V. F. minor and from their V. F. minor
some V. F. maior mutants were selected. From
seeds of the parents used for hybridization after
irradiation no changes from V. F. maior to
V. F. minor or vice versa were observed.

245

Section 13 — Plant Genetics and Breeding

3. In F2 of Vigna sinensis hybrids: black x
white seed coat, segregated plants with (a)
black, (b) white, (c) black with small brown
dots, (d) brown with large white sector seed coat.
Through selection homozygous plants with the
mentioned seed coat colours were obtained.
After irradiation of black seed of parental ge-
notype in R2, somatic segregation of the same
seed colours as in F2 hybrids, plus some reddish
seeds ocurred. From the irradiated white seed of
the parental genotype only plants with white seed
coat have developed. The parental genotype of
white seed is recessive for the mentioned
characteristics.

4. In year 1927 a natural mutation of decus-
sated position of leaves and branches on tassels
was first observed in one maize inbred. Now,
between many dozens of genetically very differ-
ent inbreds of Zea mays which have developed
from seed irradiated with gamma-rays only in
one inbred, a plant with decussated position of
leaves and branches on tassel has developed.
The mentioned inbred line has probably genet-
ical ability for the development of the mentioned
characteristics.

5. From seed of inbreds irradiated with 2000
and 3000 r the pachytene chromosomes in R2
were spread through the whole cell and
this facilitates the study of their structure in
comparison with the nonirradiated genotypes
where the pachytene chromosomes are clustered
round the nucleolus.

Conclusion: From the mentioned examples it
is possible to conclude that ionizing radia-
tion can be very helpful in evaluation of
the genetical constitution of some characteris-
tics in plants.

13.104. Induced Biochemical Mutants in Corn.

B. Andor (Godollo, Hungary).

In 1958 an open pollinated variety (Fk) and
different lines have been irradiated at dosage
rates of 10 and 15 kr according to the usual
methods.

Sensitivity to radiation proved to be different:
most resistant was the previous heat-treated
(aneuploid) material. Most sensitive were several
strains with high combining ability. Analysing
the variability of protein content in the progeny
of controlled ears, it ranged from 8.2-13.2 per
cent. The mean values of the X2 plots of lines
showed a variation range from 8.3 to 16.1 per
cent. In the X3 generation we succeeded in ob-
tain forms with hereditarily higher protein con-
tents from 6 lines (2.2-3.9 per cent). Germina-
tion tests performed on the strains in the labora-

tory under exposure to low temperature (8°C)
proved that the stock is suitable for the breeding
of cold-resistant corn varieties.

13.105. Inheritance of Alkaloids in Interspecific
Crosses in Lupinus. Edmund Nowacki (Poz-
nah, Poland).

Among the fully successful interspecific cros-
ses of lupines, e.g. L. arboreus x L. polyphyllus,
L. arboreus X /,. argenteus, L. hartwegi x L.
elegans, L. mutabilis x L. ornatus and other
species, the inheritance of alkaloids was studied.
In the mentioned species there occur the following
main alkaloids: sparteine (I), lupanine (II),
and hydroxylupanine (III). The aim of this work
was to investigate the genetical and metabolical
relation shipof alkaloids. There was also breeding
of alkaloidless fodder plants and on the other
hand high sparteine-containing winter hardy
plants for pharmaceutical purposes.

The observed segregation in progeny of hy-
brids between I and II containing plants was
of a type that could be explained only when as-
suming the conversion of I to II. Those results
were confirmed by feeding of labeled alkaloids.

13.106. The Culture of the Eggs of Plants. G. W. R.

Walker and H. F. Dietrich (Edmonton,
Canada).

The microslide culture of unfertilized eggs dis-
sected from the ovaries at floral induration into
hanging drops suspended in liquid paraffin has
been used with Hordeum sativum, Tradescantia
paludosa and Milium tigrinum. Readily identi-
fiable spherical eggs are obtained from H. sativum
but no success has been achieved with the other
species. The effects of chemical additives to the
culture medium, including kinetin and ATP,
have been studied by continuous observation un-
der phase-contrast. Progressive changes following
kinetin administration are interpreted by the
authors, in the light of data from anther-culture
as probable manifestations of kinetin-induced cell
division. The administration of ATP results in a
rapid and coarse vacuolization of the egg.

13.107. Breeding Aspects of Embryo Size Variations
in Wheat. A. Fasoulas (Thessaloniki, Greece).

The significance of embryo size variation in
wheat was studied within varieties, among

246

Section 13 — Plant Genetics and Breeding

varieties and to a lesser extent among species.

In order to evaluate the importance of embryo
size variations within varieties, it was necessary
to eliminate the influence of endosperm using
two different approaches. The first was to isolate
large and small embryos, place them on a suitable
culture medium and look for differences among
the derived plants. The second was to compare
plants derived from seeds of the same weight
which however had embryos of different sizes.
This last approach became possible after a care-
ful study of the means by which embryo varies
in relation to the endosperm at different places
in the spike.

It was found that kernels of the same weight
have not necessarily the same size of embryo.
This depends mainly on the position of the kernel
in the spike and on the variety.

The differences among large and small em-
bryos consist primarily of differences in the
number or degree of development of basic pri-
mordia, namely, those of the seminal roots,
of the three first leaves and of the tiller at the
base of the coleoptile.

Large embryos in comparison to small embryos
gave plants with greater (a) number and length
of seminal roots, (b) number and length of the
first leaves, (c) number of tillers, (d) weight of
green matter, and (e) number of spikelets per
spike.

Varieties show differences in the size of embryo.
Varieties with comparatively large embryos
were found to have a greater competative ability
in mixtures, a greater number of seminal roots,
and a greater tillering capacity.

The tendency was for varieties with relatively
large embryos to fit better on soils of lower fer-
tility and lower water-holding capacity.
The opposite was true for varieties with small
embryos. This tendency is also supported by the
observation that Triticum durum wheats which
were found to have on the average embryos
of greater size than Triticum vulgare wheats
show better adaptability in drier regions.

In this work are discussed in detail the bear-
ings of the results on the breeding of wheat, as
well as the importance of endosperm in relation
to the biological significance of double fertiliza-
tion.

13.108. (D.) Pictorial Technique in the Genetical
Analysis of Horticultural Crops in Scotland.

A. B. Wills (Dundee, Great Britain).

rescence. The ideogram method of Anderson and
Schregardus allows a graphical comparison of
variation in internode patterns and so provides
a partial analysis of habit. The method is adapt-
able and can be used for the comparison of
genotypes within species or genera. Diagrams and
preserved plant material will demonstrate the
method used at Mylnefield in the analysis of
tomatoes and Ribes species, with particular
emphasis on adaptation in these crops.

Evidence will be presented that tomatoes of
determinate growth, with either shorter inter-
nodes or fewer internodes, probably have evol-
ved from determinate types. Further evolution
has given rise to dwarf varieties with fewer,
shorter internodes and dwarf bushy types in
which apical dominance is suppressed.

The selection of suitable material and construc-
tion of the ideograms of woody perennials pres-
ent special problems. The methods devised to solve
these problems in the genus Ribes will be illustra-
ted. It is believed that differences between ideo-
grams of black currant varieties reflect adapta-
tion to different environments, whereas the
heavy pruning practised on red currants leads
to similar ideograms in many varieties.

; is a iunaamental characteristic of
plants which is partially dependent on the inter-

ittprn nf thp vpuptativp shnnt anH infln-

Habit is a fundament

piaius wnicn is paruany aepenaeni on ine inter-
node pattern of the vegetative shoot and inflo-

13.109. Inheritance of Awnedness and Glume Colour
in Some New Vulgare Wheat Varieties. A. A.
Omar, A. K. A. Selim and S. H. Hassanein
(Cairo, Egypt).

1. The inheritance of awnedness was studied
on the F2 and F3 generations of eighteen crosses
including nine varieties. This character was found
to be affected by three pairs of genes. The gene
Bi for apically awnleted, B2 for short awned,
the combinations Bi, B2 for awnless and the
recessive genes bi, b2 for fully awned type. The
gene A was suggested as a promoter for tips
and awns and its recessive allele (a) has no
effect. The interaction of these genes gives rise
to new types as B1-B2-A- for awnless beak,
Bi-b2b2A- for long apically awnleted ap-
proaching awnleted and bibiB2-A- for short
awned approaching fully awned.

2. The parental varieties were arranged in
three classes: awnless, apically awnleted and fully
awned. The awnless class includes the variety
Ramona with the genes B1B2A. The awnleted
class includes the varieties N.A.101 and hatcher
with genes Bib2a. The fully awned classes in-
volve the varieties Giza 144, Giza 145, Giza 147,
Giza 139, Lee and Mida with the genes bib2a.

3. The inheritance of glume colour was studied
on the F2 and F3 or eight crosses including six

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Section 13 — Plant Genetics and Breeding

varieties. This character appeared to be gov- white. The brown class includes the variety

erned by two pairs of genes. The gene Gci Ramona with the genes Gci gC2, the light brown

for brown colour, the gene GC2 for yellow col- class contains the variety N.A. 101 with the

our, and the combination GC1GC2 for light genes GC1GC2 and the yellow class involves

brown colour, while the recessive genes gCigC2 the variety Giza 139 with the genes gciGc2.

give white colour. The white class includes the varieties Giza 144,

4. The parental varieties were arranged in Giza 145 and Giza 147 with the recessive genes

four classes, brown, light brown, yellow and gcigC2.

248

SECTION 14

ANIMAL GENETICS AND BREEDING

14.1. A Possible Case of the Genetic Assimilation of
Behaviour. Neville Moray, Kevin Connally
and Paul Arnold (London and Sheffield,
Great Britain).

In 1961 Clutterbuck and Beardmore reported
that Drosophila melanogaster reared on media
containing peppermint oil, to which they were
aversive, became less aversive to the adulterant
over several generations.

In a replication of this experiment with further
controls a stock of wild-type Drosophila me-
lanogaster were artificially selected for aversion
or non-aversion to peppermint. These two groups
and two unselected groups were divided into two
and reared either on normal food or on food
adulterated with peppermint oil. The offspring
from each group were tested at each generation
for their aversion to peppermint. In the artificially
selected groups on normal food the degree of
aversion showed only a very slight upward trend,
the unselected control remaining almost con-
stant. The line "Unselected on peppermint" also
showed little change.

In the lines which had been subjected both to
artificial and natural selection results were
striking. Non-aversive flies reared on peppermint
(artificial and natural selection in co-operation)
showed a marked decrease in aversion, resulting
in the almost complete extinction of the line.
Aversive flies reared on peppermint (artificial and
natural selection in opposition) also became less
aversive over six generations.

When this line was released at F6 onto normal
food, the degree of aversion remained the same
as those flies still left on peppermint. We suggest
that this effect bears similarities to the genetic
assimilation of a character which does not have
a threshold, as discussed by Waddington.

14.2. Genetic Transmission of Alcohol Preference in
Mice. John L. Fuller (Bar Harbor, U.S.A.).

Four inbred strains of mice and 6 interstrain
Fa hybrids were given access to 6 different con-
centrations of alcohol over a 6-day period. A
measure of preference was obtained by adding 1
(for elimination of negative values) to the loga-
rithm of the alcohol concentration correspond-

ing to the midpoint of an animal's total accumu-
lated fluid intake from all solutions. Homoge-
neity of variance was demonstrated in the
group scores. No significant sex differences
were found. The mean scores of the pure strains
were: C57BL/6J, 1.74; C3HeB/FeJ, 1.61;
A/J, 1.39; and DBA/2J, 1.08. The C57BL/6J and
DBA/2J strains had significant general combin-
ing effects in their hybrids. However, specific
combining effects were greater indicating the
importance of non-allelic interactions in the
determination of the phenotype of the hybrids.
The variation between the hybrids in genetic
control of alcohol preference (or aversion) sug-
gests a similar variability in its physiological basis.

14.3. Factors Affecting Mating Competition in
Mice. L. Levine (New York, U.S.A.).

Albino males of strain, ST/Jax, were paired
with pigmented males of strain, CBA/Jax,
and placed in cages with single albino
females, St/Jax. The albino males fathered 83
per cent of the offspring. Cage size does not
appear to affect the results. When pigmented
females, CBA/Jax, were used, the albino males
fathered 70 per cent of the offspring.

Single albino males were taken at birth and
reared with pigmented litters. At weaning, each
albino male was paired with one of its foster
brothers, and both were placed in a cage with
a single albino female. The albino males fathered
93 per cent of the offspring. When single pig-
mented males were fostered to albino litters
and treated as above, the albino males fathered
50 per cent of the offspring.

Albino and pigmented males were fought in
round-robin fashion. In two separate series,
eleven matched males from each strain were
used. In the first series, the fights were concluded
at the end of the first submission or after thirty
minutes if no submission occurred. In the second
series, the fights were observed for thirty minutes
regardless of number of submissions. In the first
series, the initial fights included 5 albino vic-
tories, 3 pigmented victories, and 3 no-victories.
In the second series, the initial fights included
6 albino victories, 3 pigmented victories, and 2
no-victories. Thereafter in both series, the pig-

249

Section 14 — Animal Genetics and Breeding

merited males won no less than 9 fights per ses-
sion and eventually won all 1 1 fights each time.

Supported by NSF Grant GB-312.

14.4. Behavioral Effects of Single Genes in Mice: an
Investigation with the Use of Ethological
Methods. J. H. F. v. Abeelen (Nijmegen,
The Netherlands).

Until now screenings of known genes and
their influences on behaviour have been carried
out only to a very limited extent. The same holds
for the appliance of ethological methods in be-
haviour genetics, i.e. the drawing up of
ethograms. In the present study a combination
of these two approaches was attempted. For
this purpose the behaviour of the mice was
analyzed in a few tens of behavioral elements
that can be seen performed by a solitary male in
an observation cage, several special elements by
two males placed together, and a number of
elements by one male and one female placed
together. The screened alleles were: yellow,
pink-eyed dilution and jerker. Environmental
factors were equalized for all subjects as far as
possible. Frequencies of the defined elements
performed by mutants were compared with those
from non-mutants from the same strains. Also
qualitative comparisons were made.

In spite of the endocrinological effects attribut-
ed to the yellow allele no evidence was found for
a behavioral effect of this allele.

Homozygotes for pink-eyed dilution showed
significantly less staring at observer and lifting
one paw (intention-reconnoitering) and perhaps
less reconnoitering also; more grooming and
shaking the fur are indicated. Thus these ani-
mals show an autistic type of behaviour, probably
because of a more or less impaired vision.
Other explanations are possible but must remain
open at this time.

Qualitatively the behaviour of the jerkers dif-
fered in many respects from normal behaviour.
Quantitatively: feeding, fighting and sexual
behaviour were disturbed the least, exploratory
and comfort behaviour very clearly.

By the use of ethograms, strain differences
may be discovered quickly. Full details will be
published in Genetica.

evolutionary mechanisms of reorganization of
the reproductive function in mammals from
monoestrousness to polyestrousness.

It has been shown that the selection of the
silver foxes (Vulpes fulvus Desm) kept under
farm conditions for mating at the beginning of
the breeding season has no effect and cannot
change the time and the number of reproductive
cycles during the year. The heritability of the
mating time in these animals during the breeding
season is very low. Nevertheless, some reorga-
nization of the reproductive function has taken
place in the evolution of domestic animals; the
majority of modern forms has lost its monoes-
trousness and the strictness of seasonal repro-
duction which is characteristic of their ances-
tors; they became polyestrous. The causes which
called forth such a hereditary reorganization of
a stabilized function have not been revealed.

In our investigation we have shown that in
regulation of the reproduction time of foxes the
cerebral cortex is implicated. Its properties, in
particular, are manifested in the type of the de-
fensive behaviour of animals; the foxes with
calm type of behaviour mate earlier in the breed-
ing season than the aggressive ones and their
litter sizes are significantly higher. This correla-
tion is not only phenotypical but genotypical as
well. The differences in the type of behaviour of
animals are based on the genotype and it gives
the possibility to select animals according to this
character. Ten year's selection carried on by the
authors for receiving foxes of calm type of be-
haviour called forth in some females the reor-
ganization of the reproductive cycle: there were
recorded obvious signs of estrus in 4 females out
of the breeding season— in September and Oc-
tober. During the breeding season (February)
these females were mated and gave litters, but
their estrus was prolonged up to 18-20 days
instead of 5-6 days as it must be usually. In
females selected for calm type of behaviour
some changes of the fur structure were noted as
well.

The change of a character with law heritability
became possible by selecting a correlated charac-
ter which regulates the main selected character.
The reorganization of the reproductive function
realized in the evolution of domestic animals
was probably a result of unintentional selection
of animals "for domestication".

14.5. The Ways of Reorganization of the Reproduc-
tive Function in Seasonly Reproducing Mam-
mals. D. K. Belajev and L. N. Trut (Novosi-
birsk, U.S.S.R.).

This work is devoted to the investigation of the

14.6. Parity and Mendelian Segregation. L. C.

Strong and F. N. Johnson (Springville,
U.S.A.).

Luxoid has been reported in several species.

250

Section 14 — Animal Genetics and Breeding

In 1956, Strong and Hardy reported on a new
luxoid condition in mice. It arose in a mouse
of the F5 after crossing a polydactylous mutant
of the Brpb subline, of the NHO/St strain, to
C57/St. (This condition has been designated
first.) The condition, according to Strong and
Hardy, was similar, but differed in some respects,
from previously reported luxoids. In 1962,
Forsthoefel reported that there were, in fact,
differences between Strong's luxoid and those
reported by Carter and by Green.

The present communication will introduce a
concept of parity and its effects on the segrega-
tion of genetic traits. Mice were bred through-
out their reproductive life with some having as
many as thirteen litters. Points were plotted
for each parity for these traits: percent having
normal feet, percent having either poly on one
hind foot (Poly 1) or on both hind feet (Poly 2),
percent having some form of luxoid, and percent
of total defective mice. It was learned that the
percentage of animals having some defect did
not change with increased parity. However,
if the percent of mice with some form of luxoid
and percent of mice with Poly 1 or 2, were
compared with parity, trends were apparent.
To get a straight line regression was applied
giving a positive slope to the luxoid curve
and a negative slope to the polydactylous curve.
To determine the significance of the curve, a
test was applied to the regression coefficient.
A positive coefficient of the luxoid slope (r =
0.895; d.f.-ll) was significant at the <0.01 level
and the negative coefficient of the polydactylous
slope (r == -0.792; d.f. -11) was also significant
at the <0.01 level. Thus, it is concluded that in-
creased parity does not effect the total number of
defects but does increase the severity of the
morphological defects.

14.7. Two Unusual Mutations Affecting Pigmenta-
tion in the Mouse. H. Glenn-Wolfe (Bar
Harbor, U.S.A.).

A mutation to pink-eyed dilute (/?') that oc-
curred in the Production colony of C57BL/6J
has been maintained continuously on the same
inbred background by cross-intercross to C57BL/
6J. Breeding tests established its allelism, but
not its identity with existing pink-eyed dilute
(p). An unusual feature is the sporadic occur-
rence of mosaic coat-color patterns in mice of
genotype p'p\ These vary from a few dark hairs
at only one place on the body to heavily mottled
animals. Eye color in these same mottled animals
ranges from pink eye to full color, and often
shows bilateral asymmetry. Mosaics have pro-

duced wild type progeny in addition to mosaic
and normal pink-eyed dilute progeny when
test-crossed to non-mottled pp or p'p' animals.
In outcrosses of C57BL/6-/?y x \29jRe-pp,
deviants varied from light mottled to wild type
(8 observed in approximately 3000 classified).
Besides effects on pigmentation, />' has been
shown to affect the superovulation rate.

Five mutant animals, borne in two different
litters, appeared in the cross C57BL/6J $ x
C57BL/6J-A//'wh + $• Mutants are pale yellow
with white spots. Breeding tests showed the mu-
tant gene to be located at the microphthalmia
locus and yellow-spotted animals to beM/wh w/sp.
An unusual feature of /«/sp in heterozygous
(m/sp+) or homozygous (/?7/sp/w'sp) form is the
absence of any visible expression distinguishing
it from wild type. Allelic interaction between
/w/sp and MiKh or mi is being investigated,
particularly effects on pigment granule size and
distribution in selected hair types.

14.8. Inheritance of Leucocyte Counts in Rats.

W. G. Downs (Cockeville, U.S.A.).

In an effort to obtain a strain of rats having a
consistent leucocyte count, studies have been
conducted on the method of inheritance of
these counts. Animals were from our own strain
of Wistar Sprague-Dawley, out of a Wistar
male crossed to four Sprague-Dawley females,
then very closely inbred for fifteen or more
generations.

Originally, total leucocyte counts varied
between 6800/mm3 and 17,800/mm3. Initially,
sixteen crosses were made between animals
with a very low count and those with a very high
count. Of the 207 of these offspring, 192 had
counts between 7900/mm3 and 15,700/mm3.
Of these latter animals, 16 crosses were made,
and 197 offspring resulted. Of these 178 varied
between 9000/mm3 and 14,200/mm3.

In three successive generations 16 crosses
were made, in each instance discarding approxi-
mately the 10 per cent of animals at higher and
lower extremes. The last, or fifth generation,
resulted in 190 offspring whose leucocyte count
varied between extremes of 9600/mm3 and
10,900/mm3, being fairly evenly distributed be-
tween these figures, though with a tendency to
concentrate around a mean of 10,400/mm3.
Considering the inherent error in blood-cell
counting, these figures are believed to have
attained the initial objective. As the limits of the
total count narrowed, so did the differential,
as between lymphocytes and granulocytes, the
fifth generation having a ratio of 67 per cent

251

Section 14 — Animal Genetics and Breeding

lymphocytes to 30 per cent granulocytes 5.
A polygenic type of inheritance is hypothe-
sized with not less than four, and possibly more,
pairs of factors.

14.9. Adrenocortical Variation in Mus nwscuius.
F. M. Badr and S. G. Spickett (Cambridge,
Great Britain).

Study of the adrenal cortex of the house mouse
reveals wide variation in the ratio of adrenal
weight to body weight both between long stand-
ing inbred strains and between laboratory bred
wild strains. This variation is, in part, the ex-
pression of variation in adreno-cortical zona-
tion. Different strains show variation in the levels
of circulating corticosteroids and in the levels
of excreted steroid metabolites. This is due to
variation in adrenocortical synthetic activity
as determined by in vitro incubation, and in the
A 4 hydrogenase activity of the liver, and adrenal
cortex.

Studies of hybrids indicates that this variation
is, in large measure, of genetic origin.

14.10. Genetic Studies on the Resistance of Polyoma
induced Mouse Tumour Cells to Polyoma
Virus Infection. David Gershon and Leo
Sachs (Rehovoth, Israel).

Resistance to challenge infection with poly-
oma virus (PV) was studied in 88 tissue culture
clones from PV induced mouse tumors, to de-
termine whether there exists in these mammalian
cells any similarity to the immunity to superin-
fection found in lysogenic bacteria. There
was no spontaneous virus production by any
of these clones. Cloning of two resistant un-
cloned mass populations yielded about 50 per
cent susceptible clones, while cloning of two
susceptible clones yielded 7 and 37 per cent
resistant clones. A segregation of resistant from
susceptible clones was observed through two
cycles of recloning. Although the resistant unclon-
ed populations segregated susceptible clones in the
first cloning cycle, second cycle cloning of resistant
clones yielded only resistant clones. In contrast
to lysogenic bacteria, susceptible cells thus
segregated back to resistance in the absence
of new virus infection. Treatment of two resis-
tant clones with acridine orange did not render
them susceptible. Both resistant and susceptible
clones produced tumors in mice, and possessed
the PV induced cell antigen. There was no ap-
parent correlation between differences in the

chromosome number of the tumor cells and their
response to challenge infection. The results will
be discussed in relation to the possible me-
chanisms that determine resistance and suscep-
tibility of PV induced tumor cells to PV chal-
lenge infection.

14.11. Selection and a Maternal Effect. Nigel
Bateman (Edinburgh, Great Britain).

Individual, family and within-family selections
are often regarded as alternative methods, merely
differing in efficiency, for selecting the same
genes for improving the genotypes of indi-
viduals. The possibility that family selection
especially, and to a lesser extent individual
selection, could give qualitatively different
results from within-family selection by procuring
heritable maternal effects semes to have been
mostly overlooked. The importance of such
effects for 5-week weight in the mouse is de-
scribed.

Both sexes were selected on individual 5-week
weight. After ten generations heavy mice weighed
34 g and light mice weighed 17 g. Their difference
amounted to 32 per cent of the accumulated
selection differential. Differences between off-
spring of the worst individuals and the selected
lines averaged 39 per cent of selection. Presuma-
bly on account of maternal effects, reciprocal
hybrids differed by 43 per cent of the weight
difference between parental lines. The responses
of the selected lines and of offspring of the
worst individuals could be accounted for if
one-quarter of the maternal effect stemmed from
maternal 5-week weight and maternal genotype
(selected through family differences which con-
stituted two-thirds of the superiority of selected
individuals) determined the remainder.

At the beginning of the experiment a simple
1: 2: 3 relationship obtained between weights
at 3, 5 and 9 weeks of age. Unexpectedly, the
selection on 5-week weight alone left this
relationship unchanged in both selected lines
though the hybrids did not conform. In fact,
the inherent growth curves for the selected lines
had changed, but were masked by changes in
the maternal effects.

14.12. The Spermatozoa of Mouse-Strains selected
for Body Weight. R. A. Beatty (Edinburgh,
Great Britain).

When n strains of mice are crossed in all
possible ways, the n2 types of progeny fall into

252

Section 14 — Animal Genetics and Breeding

a table, with (say) the rows, M, representing the
n kinds of male parent, and the columns, F, the
female parents. Analysis of variance gives mean
squares for the M, F and MF interaction effects.
Alternatively, after adding the M and MF
interaction effects. Alternatively, after adding the
M and F marginal totals (strain by strain) a mean
square a for "additive variation" (parental
effects averaged over sex) is obtainable; after a
corresponding subtraction of the totals, the
differences can yield another mean square c for
"maternal effects" (differences between strains
when used as male parent and as female parent (0.
The MF interaction is further divisible into
"mean dominance," etc. In a diallele cross
(material of D.S. Falconer and R. C. Roberts)
of four large strains of mice selected for high
body weight, spermatozoan dimensions studied
were: head area in optical projection; maximum
head breadth; midpiece length. There was much
significant additive variation, with a tendency
towards a greater male parent effect than female
parent effect. The (log) within-male variance in
spermatozoan dimensions showed significant
female parent effects and non-significant male
parent effects. Difference between parental
effects were sometimes reflected as significant
"maternal effects". No significance was at-
tached to "mean dominance" or any other items
within the MF interaction. In a diallele cross of
three small strains of mice there were no genetic
differences in spermatozoan dimensions. There
was no correlated response to selection; sper-
matozoan dimensions of small strains in general
were similar to those of large strains. A detailed
paper will be submitted to Genetical Research
(Camb.)

1. See B. I. Hayman, Biometrics 10, 235-244,
1954.

14.13. (D) Genetics of Reproductive Mechanisms and
of Color Variations in the Freshwater Planarian
Dugesia lugubris. M. Benazzi and Lentati
G. Benazzi (Pisa, Italy).

Dugesia lugubris presents a diploid amphimic-
tic biotype and some polyploid pseudogamous
ones, namely: a triploid biotype with ameiotic
oocytes, and a triploid biotype with hexaploid
meiotic oocytes; the female germ line of the last
biotype becomes hexaploid through a chromo-
some doubling. The male germ line is always
diploid with normal meiosis in all biotypes.

The genetic background of chromosome
cycle of the above mentioned biotypes was

experimentally analyzed by crossing amphimic-
tic diploid specimens, used as females, with
pseudogamous polyploid ones. The results
obtained have shown that asynapsis, chromo-
some doubling in female line and pseudogamy
may be transmitted through the sperm, and
therefore must be considered as controlled by
chromosomal genes. However, the inheritance
of such characters is very peculiar, since the
oocytes may be both synaptic and asynaptic,
diploid and polyploid, amphimictic and pseudo-
gamous in the same hybrid; this variable
expression of the characters is not easily framed
in formal genetics.

Starting from diploid, asynaptic and am-
phimictic Fi it was possible, through repeated
crosses or backcrosses, to obtain the 2nd, 3rd
and 4th generations, which are triploid, tetra-
ploid and pentaploid, respectively.

Moreover such experimentally produced poly-
ploids allow the study of the genetics of the pigmen-
tation by crossing differently colored specimens
(white and dark respectively). A gradual increase
or decrease of the color intensity occurs at each
successive generation, related to the increase or
decrease of the chromosome sets derived either
from the white or the dark parent. This result
postulates a cumulative action of the color
factors, which can be quantitatively analyzed.

14.14. Linkage Map of Sex-chromosomes in the
Fish, Oryzias latipes. Toki-o Yamamoto
(Nagoya, Japan).

Artificial control of sex differentiation as
advanced in Oryzias, where normal sex-deter-
mining mechanism isXX=$ andXY=cJ, renders
it possible to obtain estrogen-induced XY $$
ad libitum. Of 57 sons of induced XrYR$ x XrYR
<$, where R stands for orange-red colour and
/• for its recessive allele, only two proved to be
YRYR. YRYr males, however, are as viable as
normal males. All evidence points to the suppo-
sition that there is an inert section (-) in the
regular YR and a "viable" section ( — ) in the
ordinary Xr as well as Yr.

The linear order in the normal male is ( x )
0.2 r— (+) in the Xr or (y) 0.2 R— (— ) in the
regular Y and that in the induced XrYR female
is (x) 1.0 r— ( + ) or (y) 1.0 R— (— ), where
(x) and (y) stand for non-homologous sex-
differentials and the numerals represent observed
recombination values. To estimate recom-
bination value of the interval r — (+) or R — ( — ),
the sum of all fractions relevant to viable YRYR
of induced XrYR$ x XrYV is taken as the
numerator and the sum of all fractions of

25;

Section 14 — Animal Genetics and Breeding

viable orange-red (R) sons as the denominator
and the ratio is equalled to 2/57. The recom-
bination fraction of the interval r — ( + ) in the
induced XrYB§ is assumed to be five times as
high as that in the normal XrYR^ as that in the
interval ( x) — r. The established linkage map of
YR in the normal male is (y) 0.2 R 1.2 (— ) while
that in the induced estrogen-induced XrYB$ is
(y) 1 .0 R 6.0 ( — ). A full account will be published
in Genetics (U.S.A.) in the near future.

A strain has been fixed for extra-abdominal
legs in some 30 per cent of the flies, but the
character does not segregate after outcrossing.

The coiling direction of the hypopigium and
the orientation of the asymmetrical sclerites
completing the male terminalia seem to be
pleiotropic effects of the same gene and inde-
pendent developmental processes.

Close inbreeding leads quickly to sterility.

14.15. (D.) Formal Genetics of the Housefly.

M. G. Rubini- Franco (Pavia, Italy).

14.16. (D.) The Inheritance of Insecticide-Resistance.

M. T. Lanna (Pavia, Italy).

The complement of the housefly (Musca
domestica L.) is n = 6; somatic pairing is
generally very good in metaphasic plates from
supra-aesophageal ganglia; colcemide can easily
be supplied with good results both to larvae and
to adults by ingestion or to larvae by injection.

Variability of paleo- and neo-arctic popu-
lations shows great similarities in abdominal
pigmentation and load of minor wing venation
abnormalities, all having incomplete penetrance
and variable expressivity.

Complex mutant phenotypes with lethal
effects are common; they are of little value for
formal genetics, requiring cumbersome breeding
methods; good mutants have allowed identi-
fication of the five autosomal linkage groups.
The complement includes big X Chr. ; however
no diaginic mutant has been found so far. The
loci of the II linkage group show occasionally
holandric inheritance (flies from Australia and
Florida); cytological evidence of two distinct
Y-chromosomes has been obtained.

Dominant mutants are rare and doubtful,
apart from some genes for insecticide resistance,
giving intermediate hybrids.

Three not allelic recessive genes causing
similar yellow eyes are known mutant larvae
developing with normal ones and can result in
various pinkeyed flies.

Genetically determined eyelessness or de-
formed eyes are fairly common, but can hardly
be fixed in true-breeding strains; narrow Bar-like
eyes are recurrent in some strains but their
inheritance is still obscure.

Among mutants affecting appendages differ-
entiation special mention is deserved by
(1) aristapedia which turns the arista into tarsal
segments and generally reduces bristles; (2)
antennapedia, which is irregularly dominant and
of erratic expressivity and (3) tarsi -fusi (fused
tarsi).

The response of houseflies to DDT can be
measured either by the time required for the on-
set of well-defined symptoms of intoxication
(knockdown) or by the probability of survival
to dosages directly applied to the fly (topical
application).

The first approach is adopted when samples are
confined into containers having the walls coated
or impregnated by the toxicant and the knock
down times are recorded; the second approach
is followed when dosage/ response regression
lines are needed; either methods has its short-
comings and its points in favour for genetical
research.

Tolerance levels for DDT generally show
great variability within populations, and response
to selection is prompt and speedy.

Three different genes are known, all of the
II linkage group, which independently cause
DDT-resistance ; all provide clear F2-segregations
and in the following generations behave ac-
cording to expectation of monofactoriality.

Genetical research based on the study of
knockdown times or on that of dosage/mortality
correlations have provided parallel results, but
the evaluation of dominance differs somehow.

Dieldrin resistance is usually measured by
topical application and has been found widely
distributed among laboratory strains of various
origin, some of which can positively be assumed
as never having had any experience of dieldrin.

Hybrids from susceptible and resistant flies
are intermediate; the F2S give clear indications of
simple inheritance, with free recombination
with linkage groups II and V on which genes for
DDT- and for OP- resistance respectively are
located.

Free recombination of Dieldrin resistance
occurs also with IV Chr. markers.

254

Section 14 — Animal Genetics and Breeding

14.17. The Genetics of Insect Resistance to Insecti-
cides. R. Milani (Pavia, Italy).

About seventy independent strains of resistant
insects belonging to twenty species have already
been studied genetically.

Toxicological and genetical studies agree
indicating specificity of resistance mechanisms
for groups of chemically related toxicants and,
to some extent, within groups.

Most of the information on inheritance comes
from resistant strains developed under pest-
control condition rather than under controlled
selective pressure on laboratory populations.
For many species the studies on the inheritance
of resistance are the only property genetically
analyzed.

Resistance and susceptibility generally are
discrete properties, inherited as simple genetic
differences; resistance is almost always dominant
or partly dominant on susceptibility, as would be
expected for a property selected through
survival to sudden extreme adverse factors. The
very few instances of recessive resistance known to
the present author through the literature refer
to forms of resistance developed to toxicants
different from those which have been exerting
pressure and genetically independent from the
resistance primarily caused by selection.

Selective pressure with Organophosphorous
(OP) insecticides on the housefly and on mosqui-
toes appears to cause resistance also to DDT; it
has been shown for a strain of flies and one of
Culex tarsalis that independent genes are
involved. Other instances of multiple resistance
have been genetically resolved in their com-
ponents in various species. Linkage relations of
various genes for resistance and genetic markers
are on records for Musca domestica, Aedes
aegypty, Anopheles albimanus, various Droso-
phila species, Blattella germanica.

High dechlorinating activity is always joined
by DDT-resistance, both properties being
jointly inherited as a simple mendelian character.

Low-aliesterase activity is often coupled with
OP-resistance in the housefly; they are then
inherited as a unit.

Cholinesterase activity is inherited jointly
with DDT-resistance in Culex tarsalis, but, like
aliesterase activity, independently from mala-
thion resistance; in the same species carboxyes-
terase activity and malathion resistance have
been inherited jointly.

The full text has been accepted for publication
in a special issue of the Bulletin of the World
Health Organization.

14.18. Artificial Allotetraploids (2n Bombyx moii
L. + 2n B. mandarina Moore) in Silkworm and
Their Bisexual Reproduction during Three
Successive Generations. B. L. Astaurov and
V. N. Vereiskaya (U.S.S.R.).

As reported earlier (Proc. X Inter. Congr.
Genet., 1958), by crossing mixoploid partheno-
genetic females (3« + 6n) with normal 2/7-males
autotetraploids could be easily obtained in the
silkworm B. mori L. Females An display normal
fertility, whereas autotetraploid males are
sterile. Repeated attempts to obtain a bisexual
autotetraploid race failed. The usual presence
of several tetra- or polyvalents in 4/7-males
meiosis suggests the aneuploidy of spermatozoa
to be a cause of sterility. Consequently am-
phidiploids produced by crossing domestic
B. mori L. (//= 28) with wild B. mandarina
Moore (Ussury and Shanghai races, n= 28)
could be expected to be fertile. 220 crosses of
partheno-females An-mori x <§ 2n mandarina
gave many offspring 3// (2« mori+ln mandarina)
of both sexes. Analysis of 595 males proved
their and their sisters triploidy. By heat acti-
vation of unfertilized ova extracted from 532
3/7-females many parthenogenetic 3/7-daughters
were produced in 52.9 per cent of batches.
Percentage of hatching made 6.4 per batch,
reaching 63.0 per cent. A portion of partheno-
females was expected to be mixoploids 3/? + 6//.
After reduction of 6/?-oocytes (An mori -(-2//
mandarina) in case of preferential homologous
pairing, 3/7-pronuclei (2/7 mori-\- In mandarina)
must be formed. When fertilized with mandarina
haplosperm, amphidiploid (2« mori + 2n man-
darina) progeny of both sexes is expected.
1049 crosses of hybrid partheno-females 3/7 and

3/7

- x $ 2/7 mandarina were performed and 1439

larvae obtained. Percentage of batches with
hatched larvae (17.9) and total percentage of
hatching (0.36) are close to those observed in
matings of partheno-females 3// mori x <§
2n mori, yielding autotetraploids. Sex ratio in
235 V-instar caterpillars was 1.00$ : 1.05^.
Cytogenetican alysis proved their tetraploidy and
presumably — amphidiploidy. Phenotypically they
resemble 2/7 mori x mandarina Fi-hybrids. 103
males were repeatedly mated and 51 of them
(49.5 per cent) proved to be partially fertile.
Hatching percentage fluctuates from 0 to 35.8
per batch, total one being 1.1 per cent. Poor
fertility indicates the probable segmental allo-
tetraploidy. Up to 1963 three successive gener-
ations of allotetraploids were reared: 1° —
primary "amphidiploids" (Ai); 2° — "amphidi-
ploids" A2 and /?i — hybrids from back crosses
$4/z mori x <§ An "amphV; 3° — progeny of

255

Section 14 — Animal Genetics and Breeding

$ An Ri x cS 4« -Ki and °f ? 4/i wo/-/ x J An R\
crosses. Presently we have 274 batches of the
4th allotetraploid generation, a part of which is
expected to yield larvae in spring. In all, from
1353 matings 2263 allotetraploid larvae were
produced; 452 were reared up to Imago stage. In
314 crosses An X An percentage of hatching
fluctuates from 0 to 39.5 per batch; total
hatching making 1.0 per cent 54.3 of males proved
to be partially fertile. In all generations sex
ratio remains close to the normal one.

To our knowledge, it is the first case of bisexual
reproduction of artificial allotetraploids in
animals.

14.19. The Present Status of the Formal Genetics of
Triboiium castaneum, T. confusum, Latheticus
oryzae and Gnathocerus cornutus (Tenebrioni-
dae). Alexander Sokoloff (Berkeley, U.S.A.).

Linkages established for castaneum: Chromo-
some I (=X) from left to right in the order given:
spotted elytra (sp); lethals 2 and 4 (:2, H);
divergent elytra (dve); lethal-3 (13); pygmy (j>y);
miniature-appendaged (ma); red eye (r); paddle
antennae (pd); truncated elytra (te); red-
modifier (MT); lethal-1 (:1). II: pearl eye (p);
pink eye (ppk); pegleg (pg). Ill: black (b);
light ocular diaphragm (lod). IV: Bar eye (Be);
sooty (s); deformed legs (dfi). V: microcephalic
(mc); jet(j); split (spl). VI: Microphthalmia A/o).
VII: blistered elytra (ble); chestnut eye (c);
curved appendages (ca); fused tarsi and antennae
(Fta); short antenna (Sa). VIII-X: Vacant.

Unassigned: cut prothorax (cp) and juvenile
urogomphi (jit) (which are linked to each other);
fused antennal segments 1-3 (fas-l, fas-2, fas-3);
abbreviated appendages (aa); squint (sq);
extra urogomphi (eu); prothoraxless (ptl);
engraved metasternum (em); incomplete meso-
sternum ( ms); warped ely tra ( we) ; pointed elytra
(pe); short elytra (sh); dent (dt).

For confusum: Chromosome I (X): Striped
(St); eyespot (es); labiopedia (Ip); reduced
antennae and elytra (rae); lethal-1. II: pearl (/?);
ebony-2 (e-2); pegleg (pg). Ill: black. V: ebony.
VI ;I blistered. IV, VI, VII, IX to be filled with
following: short elytra (sh); light ocular dia-
phragm (lod); ruby spot (rus); ruby (Ru); dirty
pearl eye (dpe); chestnut (c); stilted legs (stl);
engraved metasternum (em); dent (dt); warped
elytra (we); split (sp); melanotic stink glands
(mgs); deformed legs (di); fused antennal
segments (fas).

For L. oryzae: I (X): red (r); truncated elytra
(te). II: pearl. Unassigned: brown body color
(bwb).

For G. cornutus: II: pearl; III: light ocular
diaphragm (lod).

14.20. Genetic Correlation and Asymmetry of the
Correlated Response from Selection for In-
creased Body Weight of Triboiium in Two
Environments. A. Earl Bell and H. W. McNary
(Lafayette, U.S.A.).

Genetic parameters needed for predicting
direct and correlated responses from selection
for increased body weight (pupa stage) of
Triboiium castaneum in each of two environments
(70 per cent versus 40 per cent relative humidity)
were estimated in a random mating Base Popu-
lation. Heritabilities of body weight in the two
environments were not significantly different
(0.58 in Wet and 0.55 in Dry), but the phenotypic
variation in Dry was approximately twice that in
the Wet Environment. The genetic correlation
between the two traits was estimated as -f 0.98.
The predicted direct and correlated responses in
both environments were checked in a replicated
selection experiment spanning nine generations.

Good agreement between predicted and
observed direct response was obtained for
each of the four selected populations. Also, the
average observed correlated response including
both environments was accurately predicted
(Predicted/observed = 147. 5/141. 5 ug and 139.5/
138.5 |ag per generation for Replication 1 and 2,
respectively). Yet the observed correlated
response in each replication for the population
selected in Dry was approximately twice that
predicted, while those observed in the popu-
lation selected in Wet were only half of the
predicted values.

When the realized genetic correlation, Tg, for
each population was calculated from the
observed heritabilities, direct and correlated
responses and phenotypic variances t1), it was con-
cluded that the observed asymmetry of correlate
response was due to the effective genetic corre-
lations being different for the two environments
(rg= +0.64 and +0.67 for the two replications
of selection in Wet and +0.93 and +1.12 for
selection in Dry).

Supported by Grant G-15824, National
Science Foundation.
1 . Falconer's Formula 2, J. Heredity 45, 42-4.

14.21. Selection for 13-day Larval Growth in Tribo-
iium under Two Nutritional Levels. Yamada
Yukio and A. E. Bell (Lafayette, U.S.A.).

Selection for large and small 13th-day larval
weight in Triboiium castaneum has been investi-
gated for 16 generations to evaluate the effec-

256

Section 14 — Animal genetics and breeding

tiveness of various selection methods and the
importance of genotype by environment interac-
tions under two levels of nutrition. The principal
difference between the levels consisted of 10
per cent dried brewers yeast in the Good ration
while the Poor ration contained no yeast. All
populations were raised under the conditions
of 33 °C and 70 per cent R.H. In addition to an
unselected control, there were eight experimental
populations as follows: GL = selected large
on performance under the good level each gen-
eration, PL = selected large on performance
under poor [level each generation, gpl = selec-
ted large on average performance under both
levels, gpl = selected large under good or
poor levels in alternating generations, and simi-
lar four populations for small direction. Each
population was represented each generation by
40 single pair matings. Sets of full sibs from each
mating were reared on both good and poor
rations. In addition, each mating was placed in
standard wheat medium for reproduction. Se-
lection was made on the basis of full sibs reared
either under good, poor or average of both levels,
depending on the selection methods. After
choosing the best eight families in each popula-
tion, five males and five females for each selected
family were taken from the sib groups raised
in the standard medium. They were mated at
random with the restriction that full-sib mating
was avoided.

As selection proceeded, the behavior of popu-
lations under two environments showed striking
interactions. The gains per generation in GL
populations under good and poor levels were
65 |ag (direct response) and 60 ug (correlated
response), respectively, and corresponding values
in PL were 55 ug (correlated response) and 98 ug
(direct response). GPL and gpl were inter-
mediate between GL and PL or a little more
similar to those of PL. In other words, regres-
sions of population means on generation under
two environments are nearly parallel for GL but
not in PL, where the absolute difference observ-
ed in early generations lessened or disappeared
in later generations. On the other hand, selec-
tion for small gave a completely reverse picture.
Regressions (gain per generation) for PS under
the two environments were alike (87 ug vs.
73 ug) but were greatly different in GS (106 ug
vs. 53 ug). An interesting response developed in
one of the GS populations. Its average weight
under the poor environment was consistently
heavier than under good for the last 4 genera-
tions. Asymmetrical selection responses observ-
ed were entirely dependent on the environment,
i.e. larger response toward small under good but
reverse under poor level. Realized heritability

seems to be higher for small direction than for
large, although differences between environments
were not appreciable.

Supported by Grant G- 15824, National
Science Foundation.

14.22. Transfer to Descendency of Alterations In-
duced in the White Leghorn by Repeated
Injections of Heterologous Blood. J. Stroun,
L. Stroun-Guttieres, J. Rossi and M.
Stroun (Geneva, Switzerland).

The authors have repeatedly injected blood by
the intra-peritoneal route from the grey guinea
fowl to successive generations of cocks and hens
descending from a white Leghorn strain with
stable characteristics. Concurrently with this
test Leghorn group, they raised a check Leghorn
group treated under identical conditions with
blood from white Leghorn, and also a control
Leghorn group left untreated. In each new genera-
tion obtained through artificial insemination,
fowls remaining up to the standards of the white
Leghorn are selected from both treated groups
and submitted to the blood injections. More-
over, from among the F4 birds in the control
group, the authors set up three new Leghorn
groups treated under identical conditions with
blood from Austhralorp, Rhode Island Red and
white Leghorn respectively.

Results from Fo to F6 (February 1963): No
alterations are noticed in either check or control
Leghorn groups. In the test group, however,
which was submitted to injections of blood from
grey guinea fowl, there are in each generation
from F2 onward a few animals with alterations
in the colour and quality of their feathers and in
the pigmentation of their feet. Such alterations
do not appear in the blood treated Leghorn
fowls, but in birds of the following generation,
independently of any injection whatsoever. They
are maintained in the descendency which is no
longer treated and are transmitted both by the
mother and the father birds. In the groups treat-
ed with blood from Austhralorp and Rhode
Island Red, alterations begin to appear as from
F2.

C.R. Acad. Sciences 255, 781-783, 1030-1032,
1962.
Biol. Med. 1963, to be published.
Arch. Sc, Geneve, 1963, to be published.

257

Section 14 — Animal Genetics and Breeding

14.23. (D). Technic of Repeated Heterologous
Blood Injections to Induce in the White Leg-
horn Alterations Transferred to Descendency .

L. Stroun-Guttieres, J. Stroun and M.
Stroun (Geneva, Switzerland).

To successive generations of cocks and hens
descending from a white Leghorn strain with
stable characteristics, the authors have repeated-
ly injected heterologous blood by the intra-
peritoneal route. The blood is injected every 3
to 5 days as soon as the subjects are 10 to 30
days old and for a period of 6 to 7 months. In
each new generation, fowls remaining up to the
standards of the white Leghorn are again sub-
mitted to the blood injections. In some birds
whose ascendants have been treated, from F2
onward the authors have noticed alterations in
the colour and quality of the feathers and in the
pigmentation of the feet, which are maintained
in the descendency independently of any in-
jection whatsoever. No alteration is noticed
in a check Leghorn group treated under identical
conditions whith blood from white Leghorn.
The authors demonstrate their technic and their
results with diagrams, pictures and preserved
specimens of altered and normal birds.

14.24. Egg White as a Modifying Factor in Heredity.

Luben G. Angeloff (Sofia, Bulgaria).

The present experiments reported were con-
cerned with influence of foreign egg-white
(from 5 to 11 ml) of unfertilized eggs of other
birds, namely, condor (Sarcorhamphus gryphus,
Linn.), wild goose (Anser anser. L.), Chinese
goose (Cygnopsis cygnoides, L.) and Indian
turkey hen (Meleagris galloporo, L.) on the
process of development of chick embryos.
They were designed to determine whether the
transmission of hereditary effects could be
brought about by transforming foreign egg-
white from one strain to another and if the
yolk of the unfertilized egg is a bearer of in-
herited characters with a view to obtaining a
new race of domestic hens possessing more val-
uable characteristics as regards meat, eggs and
resistance to infections.

By injecting eggwhite into eggs of represen-
tatives of different orders of birds the following
conclusions were drawn: transfer of traits by
injection of egg white between representatives
of different orders of birds is possible when the
sexual crossing is impossible. The egg white of
the unfertilized egg was a bearer of hereditary
traits. The experimental birds grew rapidly,
were bigger, and weighed more than the controls.

The hereditary traits appearing in the first gener-
ation were transmitted to the fourth generation.

14.25. Experimental Change in the Hereditary
Character of Gametes in Birds. B. G. Novikov

(Kiev, U.S.S.R.).

This note generalizes the results of the author's
investigations on the change in the hereditary
characters of gametes in birds by cross trans-
plantation of the testicle and injection of
heterogenous desoxyribonucleic acid.

Proceeding from the principle of the formation
of the hereditary characters of gametes in the
process of the entire ontogeny, it may be presum-
ed that during development in the organism of
another breed of animal these characters should
alter in the gametes. The answer to this ques-
tion was found in experiments on cross trans-
plantation of the testicle in fowls and ducks. The
essence of these experiments is that soon after
hatching several testicles from Rhode Island
chicks were transplanted into the body cavity
of castrated Leghorn chicks, while Leghorn
gonads were transplanted to Rhode Island
chicks. In the experiments on ducks interchange
transplantation of testicles was carried out
on fledglings of mallard and peking ducks.
After 1-2 years sperm was taken from the trans-
planted testicle during the breeding season and
used to fertilize artificially females of the breed
to which the transplant belonged.

The investigations showed that in posterity
obtained as the result of such insemination varied
hereditary changes occur which cannot be ob-
tained by ordinary crosses of the initial forms.
The rate of variability was intensified in subse-
quent generations. By selection and closely
related crossing of altered forms it was possible
to stabilize several breed groups of fowls dif-
fering in plumage colour, comb structure, con-
stitution and final body dimensions. A similar
result was obtained in experiments on the cross
transplantation of testicles in ducks.

In later investigations, on analysis of the
mechanisms of the phenomenon under conside-
ration, an attempt was made to connect the
hereditary changes of the gametes that had de-
veloped in the organism of another breed of
ducks with the changes in their desoxyribonucleic
acid. The study of this problem was begun with
experiments on the injection of heterogenous
DNA in ducks. The essence of these experiments
consisted in systematic injections of DNA from
the erythrocytes of a wild duck into peking
ducks during several years, beginning at the
age of eight days; in the converse experiments

258

Section 14 — Animal Genetics and Breeding

DNA of peking ducks was injected into a wild
duck. Under the influence of heterogenous des-
oxyribonucleic acid varied hereditary changes
appeared in subsequent generations in a small
percentage of cases. On this basis two dwarf
forms of snow-white ducks with lighthorn and
yellow beaks were obtained from a wild duck
treated with peking duck DNA. The variability
in ducks due to the effect of DNA injections was
only partially similar to the changes obtained in
cross transplantation of the testicle.

14.26. Abnormal Segregations for the Alleles "single
Comb" and "Rose Comb" in the Domestic
Fowl. Ph. Me rat (Jouy-en-Josas, France).

A study of the segregations of the alleles R
("rose comb") and r ("single comb") in a poultry
population reveals the main following facts:

(a) In the cross Rr x rr (with about 51.000
pedigree progeny) the male chicks show a
highly significant excess of single combs over
the rose combs; the proportion is normal by
the female progeny.

(b) In the cross Rr x Rr (about 8000 pro-
geny), the female chicks show a highly significant
excess of single combs over the expected propor-
tion of 1/4; there is a deviation in the opposite
sense among the male chicks, so that, grouping
the two sexes, the ratio of the comb types is
normal, near 3/1.

For these two crosses, the observed deviation
comes only from the progeny of some males.

(c) For the cross rr x Rr (about 15,000 pro-
geny), on the whole, the ratio of the two comb
types is normal in both sexes.

The hatching percentages, the sex ratio for
each comb type, and the different results ac-
cording to the type of cross, do not support
the hypothesis of a differential embryonic
mortality or of a differential fertility among the
ova.

A progeny-test of single comb males from
"abnormal" families, the compared results in
the 3 crosses studied, and also in numerous
RR x rr crosses, do not lend support to the
hypothesis of modifying genes.

The hypothesis of a selective fertilization
will be discussed.

14.27. Effects of Selection on Reproductive Fitness
in the Fowl. A. W. Nordskog and Margrith
Wehrli (Ames, U.S.A.).

Populations subjected to intensive artificial
selection for specific metric traits are expected
to decline in reproductive fitness. The latter is
defined here as a product of the components:
rate of egg production, fertility, hatchability,
and adult viability. Five lines starting from a
common Leghorn base population have each
been selected for a single trait over six genera-
tions. The breeding population of Line A,
selected for high egg production, consists of
16 sires each mated to 9-16 dams. Lines B and C
are selected for high and low body weight
respectively, and Lines D and E for high and
low egg weight, respectively. These lines are
maintained by 8 single-male pens of 9-16 dams
each. Line A has not improved consistently in
egg production or reproductive fitness. Body
weight has increased 54 per cent in Line B and
fitness has declined steadily by 47 per cent. Line
C has decreased 26 per cent in body weight;
fitness has declined 16 per cent. Egg size has
increased 15 percent in Line D, while fitness de-
creased 39 per cent. In Line E egg size has
decreased 14 per cent and fitness declined 16
per cent, but the latter is not statistically signi-
ficant. A similar selection experiment in the
Fayoumi breed shows good agreement. The
results fit the hypothesis that intermediate values
of body size genes are optimum to fitness. How-
ever, egg size in the original stocks appears to be
above the optimum for fitness such that down-
ward selection has either improved or maintained
reproductive fitness.

14.28. Genetic Variation in Chick Bio-assays for
Gonadotropins. I. Testes Weight and Re-
sponse/1) P. B. Siegel and H. S. Siegel,
(Blacksburg, U.S.A.).

Genetic influences on testes weights at hatch-
ing and testes weight response to anterior pitui-
tary homogenates and purified gonadotropins
were studied in White Rocks. Heritabilities were
estimated from paternal half-sib correlations.
Preparations were subcutaneously injected with
a saline carrier and responses measured as in-
crease in testes weight/100 g of body weight.

Significant differences among sire families
were obtained from injections of anterior pitui-
tary homogenates. Responses among sire fami-
lies to 10 and 20 \xg. of FSH (Armour) were also
significantly different. The rank correlation of
family responses for the two dosages was 0.74
and the mean heritability of response was 0.92.
No significant differences among sire families
were noted for injections of either 100 or 200 \xg

259

Section 14 — Animal Genetics and Breeding

of LH (Armour), however the mean heritability
was 0.24.

Overall response to a combination injection
of LH and FSH (Armour) was significantly
greater than that obtained when either was ad-
ministered singly. An additive effect was noted
within some families while in others the response
was similar to that obtained when either was
administered alone.

When NIH-FSH and NIH-LH were injected,
differences among sire families were not signi-
ficant for the FSH but were for the LH.
Mean heritabilities were 0.03 for FSH and 0.80
for LH.

Significant differences were observed among
sire families for two sources of PMS. The rank
correlation of the familial response to each
source was 0.80.

This experiment demonstrated genetic in-
fluences on the response of target organs to
gonadotropins. Genetic control of assay ani-
mals facilitated excellent precision in bioassays
of gonadotropins.

1. To be published in full in The Virginia Journal
of Science 15.

14.29. Genetic Variation in Chick Bioassays for
Gonadotropins. II. Histological and Histochem-
ical Responses.^1) H. S. Siegel and P. B.
Siegel (Blacksburg, U.S.A.).

Histological and histochemical observations
were made on testes of newly hatched progeny
of White Rock sires after subcutaneous ad-
ministration of purified gonadotropins. Hema-
toxylin-eosin for structural details, methyl
green-pyronin for nucleic acid differentiation
and sudan black for lipids were employed.

Non-significant differences were observed in
the response of seminiferous tubules among
progeny of 5 sires in a dosage range of 5 to 40
Ug/chick of FSH (Armour). However, significant
differences were found when the range was
extended to 625 ug. Similarly, significant sire
effects were observed with NIH-FSH levels rang-
ing from 5 to 125|ig. Differences in tubule res-
ponse among families were not significant when
NIH-LH was used at doses of 1 to 25 ug.
Rankings of families for tubule diameter res-
ponses were not necessarily the same as those
ranked for testes weight response.

Significant linear increases in tubule dia-
meters were found in a range of 5 to 125 |ag per
chick of Armour FSH. When sire differences
were considered, precision (A) for these assays

were 0.35 and 0.11, respectively. A significant
quadratic response between the 125 and 625 pg
levels indicated that this was above effective
assay limits for this method. Precisions with
NIH-FSH and LH were 0.23 and 0.25, respec-
tively.

No noticeable differences were noted in lipid
depletion among families. At 25 ug/chick of
LH, lipoid depletion and increased nuclear
activity were observed. This appeared as an all
or nothing response and could not be effectively
quantitated. Although FSH significantly in-
creased tubule diameters, neither increases in
mitotic figures nor nuclear activity were observed.

1. To be published in full in The Virginia Jour-
nal of Science 15.

14.30. The Effect of Microdose Irradiation of Hen's
Eggs upon Hatchability and Other Characters
of Chickens. H.F. Kushner, I. G. Kostin,

L. A. Zubareva, L. I. Shershunova, N. I.
Kuznetzov, and M. G. Salganick (USSR).

1. The eggs of Russian White and New-
Hampshire hens were used to study the effect
of microdoses of y-rays on economic and repro-
ductive characters of hens.

2. The eggs over the incubation period were
irradiated continuously or intermittently in dif-
ferent trials with uranium and thorium salts.

3. If the eggs during the incubation period
were irradiated intermittently with total doses of
0.003-2.9 r the embryonic mortality was decre-
ased and owing to it the average hatchability
was 3.5 per cent higher than in control.

4. The results of irradiation depended upon
the initial quality of the eggs: the lower the
hatching properties of the control eggs, the higher
was the degree of hatching improvement of
irradiated eggs.

The effect of irradiation on New-Hampshire's
eggs was superior to that on Russian White's
eggs.

A high negative correlation between hatch-
ability of control eggs and the difference in hatch-
ability between the experimental eggs and con-
trols was established (r = —0.585 ± 0.0695).

5. The effect of intermittent irradiation of
0.003 — 0.021 mr doses was equally good.

6. Morphological and physiological studies
on embryos under the intermittent irradiation
showed that the hen's embryos were more sen-
sitive to irradiation at their earlier stages of
development. Treated embryos developed more
rapidly than controls.

260

Section 14 — Animal Genetics and Breeding

7. Respiratory metabolism in irradiated eggs
during 12- 18th days of the incubation period
was more intensive than in the control eggs.
The difference in O2 consumption and CO2
output in favour of treated eggs varied in dif-
ferent trials from 5 to 40 per cent depending on
initial quality of the eggs and consequently de-
pending on the effect of irradiation.

8. The postnatal growth, development and
viability of the chickens hatched from treated
eggs were the same as in the control group.

9. The egg productivity of over 4000 pullets
hatched from treated eggs and maintained at the
range or in the cages exceeded the control by
10 per cent.

10. The quantity, quality and fertilizing ca-
pacity of the sperm of the experimental cockerels
(developed from treated eggs) were the same as
the sperm of the control cockerels.

11. The preliminary observations show that
the progeny of the 1st and 2nd generations from
hens hatched from the irradiated eggs didn't
reveal any abnormalities.

14.31. Selection on X-ray Induced Variation in
Chickens. Hans Abplanalp, Dorothy C.
Lowry and Everett R. Dempster (Davis,
U.S.A.).

weight, at a weeks of age. The coefficient 0.4 is
approximately the mean value of k, the growth
rate of y relative to x (coefficient of heterauxe-
sis) between 2 and 10 weeks. Two further lines,
B (big) and S (small), have been selected up and
down for *io. After 4 generations of selection
H and L differ by 0.067 in i\o (= 17 per cent
difference in shank length at the same body
weight), the realized heritability being 0.57.
After 3 generations B and S differ by 0.143
(=39 per cent) in .no; r.h. = 0.47. Two esti-
mates of the genetic correlation between ;'io
and X10 (- 0.32 from the correlated response in
H-L; + 0.26 from B-S) differ significantly.

Genetic variation in iw arises by (at least)
two distinct developmental pathways; by chan-
ges in post-natal k, and by embryonic changes
without change in post-natal k. The former
pathway accounts for only 20 per cent of the
H-L difference. Measurements of adult skeletons
show far-reaching and anatomically complex
differences in shape between H and L. There
are anatomical and developmental similarities
between H-L and ectomorphy-mesomorphy in
humans. Possible interrelations between different
anatomical and developmental kinds of varia-
tion will be discussed.

Full publication in Genet. Res.4, No. 2, 1963.

Populations of SCWL chickens were given
8000 r X-ray irradiation administered to the
semen of males during seven generations. The
populations used for this study derived from a
flock which had been under selection for high
egg number over 18 years. While irradiated
populations were prepared without artificial se-
lection, a control of equal size was reproduced
under similarly relaxed selection, and the orig-
inal line continued under artificial selection
for egg number. Following the irradiation phase,
all lines were subjected in replicate to four
generations of artificial selection for egg number.
Response to selection for egg number and cor-
related response in egg size and viability are
discussed.

14.32. Quantitative Developmental Genetics of
Body Shape and Size in Domestic Fowl. A. G.

Cock (Edinburgh, Great Britain).

From a population of mixed breed origin,
two lines, H (high) and L (low), have been se-
lected in opposite directions for an index of
relative shank length, /'10 = vio - OAxw, where
ya = logio shank length, and Xa, = logio body

14.33. Response to Selection for Body Size at Two
Ages in the Fowl. E. S. Merritt and S. B.
Slen (Ottawa and Lethbridge, Canada).

In broiler or meat-type chickens rapid early
growth is highly important for efficiency of pro-
duction. Large adult size, on the other hand,
results in increased maintenance and feed re-
quirements of breeding stock. A study was under-
taken to determine whether a selection response
for increased broiler weight could be achieved
simultaneously with a decrease in adult weight.

Three lines were drawn from a random-bred
control strain (Ottawa Meat Control). One was
selected for increased weight at 63 days of age,
one for increased weight at 147 days of age,
and the other for an increase in 63-day weight
but a decrease in 147-day weight. A sample of
the random-bred control population, bred inde-
pendently of these lines, was reared with each
generation of the selected lines.

Heritability estimates for 63- and 147-day
weights calculated on the control or base popu-
lation were approximately 0.55 with a genetic
correlation estimate of 0.75 between them. After

261

Section 14 — Animal Genetics and Breeding

four generations of selection, the response in
lines 1 and 2 was in reasonable agreement with
expectations, being greatest in each case for
the trait under direct selection. In the third line
the males showed both an increase in 63-day
weight and a decrease in 147-day weight. Fe-
males, on the other hand, showed a slightly
greater increase than males in 63-day weight,
but showed no change from the controls in
147-day weight. These results would indicate
that the growth pattern can be changed in the
desired manner by selection.

14.34. Selection for Growth Rate and Correlated
Responses in Chickens. R. George Jaap (Co-
lumbus, U.S.A.).

The paired mating system suggested for
minimum inbreeding in control populations by
Gowe, Robertson and Latter^1) has been used for
two random-bred control lines and five lines
with random-breeding of growth-selected parents.
All lines arose from crossbred ancestry, the
two control and two of the selected lines arising
from the same base populations.

In the growth-selected lines, 20 to 25 per cent
of each generation has been used for reproduc-
tion in 40 paired matings per line. The inbreeding
coefficient (F) in the selected lines increased
approximately 1.0 per cent per generation. In
the random-bred control lines, F has increased
slightly less than 0.5 per cent per generation.
Little change from inbreeding depression or
random gene drift was expected. The present
number of generations of selection is 8, 5, 2, 4
and 3 for lines G, A, AG, RG and GRL, respec-
tively.

Realized heritability of weight at 8 weeks of
age from the first four generations of selection
in line G was about 0.4. Recently, those selected
lines having most rapid growth rate (A, AG and
the last 4 generations of G) have failed to re-
spond as rapidly as RG, GRL and the first four
generations of G. This slackening of the progress
in line G is the first evidence of a plateau in the
rate of progress from selection for growth rate
in chickens.

When week body weight was increased
by about one-half a pound, the following cor-
related responses were observed: Egg produc-
tion decreased 5 per cent, egg weight increased
2.8 g, eggs became relatively broader in shape
and their albumen height increased 0.3 mm.

1. Poultry Sci. 38, 462-471, 1959.

14.35. Influence of Feed on the Heritability of Some
Chicken Traits. J. P. Boyer, X. de Laage
and C. Calet (Jouy-en-Josas, France).

In May-June 1962 3527 chicks were hatched
from 20 sires and 164 dams during 8 weeks —
the hatches 1, 3, 5, and 7 received an all mash
ration with high energy and a coccidiostat; the
hatches 2, 4, 6, and 8 received an usual mash
ration.

At 8 weeks of age, weight, breast angle, keel
length, diameter of shank and weight / shank
length ratio were appreciated. All data were
transformed on a probit scale to avoid the hatch
influence.

Heritabilities of the five characters were esti-
mated by hierarchical analysis of variance.

For males, no great difference was observed
between the two regimes; the percent heritability
was, for "special" and "usual" ration respec-
tively: weight 47.1-48.6, breast angle 38.1-21.3,
keel length 59.2-59.2, diameter of shank 68.4-
56.6; weight / shank length ratio 46.7-39.6.

For females, the differences were spectacular
ones: weight 67.3-41.5, breast angle 42.3-17.2,
keel length 62.1-49.0, diameter of shank 71.4-
59.3, weight / shank length ratio 65.5-42.4.
It is interesting to note that in all cases, the sire
component of variance was greatly increased.

It is concluded that an appropriate feeding
plan permits the manifestation of a genetic
sex-linked variability for meat characters.
Such an environment is able to facilitate selec-
tion.

It is suggested that the absence of response of
males was perhaps caused by a limitation of
available energy in ration.

14.36. Heritability in the Progeny of Hens with
Contrasting and Similar Constitution and
Productiveness. E. E. Penionzhkevich
(U.S.S.R.).

Investigations that are carried out at the
poultry breeding laboratory under the auspices
of the author on heretability of productive,
exterior and interior characters in farm poultry
show hereditary variability of the same characters
depending on methods of reproduction (inter-
breeding, inbreeding, somatic hybridization),
environmental conditions (mainly temperature),
various physiological states of the organism, age
of parents and selection of parental forms among
contrast or similar breeds.

Progeny is affected by parental forms includ-
ing at least two main factors: (a) nutrition during

262

Section 14 — Animal Genetics and Breeding

embryonal development connected with the
maternal organism; (b) hereditary peculiarities
of paternal and maternal organisms.

When crossing birds with contrasting produc-
tiveness and constitution (egg-producing and
double-purpose breeds) the progeny mostly
inherits egg productiveness and physiological
earliness from the paternal organism and live-
weight, hatchability and fattening characters
from maternal organism.

While selecting fathers and mothers with
similar productiveness and constitution (double-
purpose poultry breeds) we observe that in-
breed mating and interbreeding do not result in
the regular paternal or maternal influence on
live- weight.

Only daughters are inclined to inherit live-
weight from mothers and even less so from
fathers.

High meat qualities are inherited by the prog-
eny particularly from the paternal organisms.
The above-mentioned regularities in hereta-
bility of characteristics when selecting poultry
of contrast or similar breeds are efficiently used
by poultry breeders in their work. To produce
highly productive hybrids of double-purpose
breeds, parental forms are selected from egg-
producing breeds or strains, and maternal —
only from double-purpose ones. For the develop-
ment of hybrids, growing for meat, paternal
and maternal forms are selected from special
double-purpose breeds and strains. In this case
the parental form should have high meat quali-
ties which positively correlate with the live-
weight, and the maternal form should have
high egg production and vigorous constitution,
which is characterized by high vitability, hatch-
ability, intensive metabolism and by some
interieur features (development of thumus).

14.37. Diallel Crosses of Inbred Lines of Egg-type
Poultry Repeated over Locations and Years.
I. The Relative Importance of Different Kinds
of Genetic Effects. L. H. Baker and R. B.
Arvidson (Des Moines, U.S.A.).

The now classical concepts of general and
specific combining ability have been extensively
applied in evaluating inbred lines of corn.
However, all published evidence concerning the
relative importance of these genetic effects in the
fowl may be of limited value, since it has all been
based upon results from individual performance
tests.

Because genotype and environment have inter-
acting effects on the phenotypic expression of
quantitative characteristics, the data from a

specific experiment pertain only to the genetic
variance of the population with reference to the
environment (s) actually sampled by the experi-
ment. Therefore, points of major interest in the
design and analysis of a hybrid breeding program
are:

(1) The consistency of estimates of general and
specific combining ability variances from
experiments repeated over a series of
locations and years,

(2) Estimates of variance in genetic and recip-
rocal effects defined as averages for the
totality of environment pertinent to the
destiny of the genetic populations studied,
and

(3) The relative importance of additive and
specific effects in lines selected over years.

The study reported here represents an attempt
to evaluate these points of interest and compare
the results obtained with estimates of genetic and
reciprocal effects available from other studies in
the fowl.

14.38. A Case of Hairlessness in Cattle. V. Derlogea

(Bucarest, Rumania).

Two calves of the Pinzgau breed ($ and ^)
hairless were born in 1962 in the Nasaud county
— Cluj region. Their birth weight was normal
and except the above-mentioned characteristic,
they did not present any other phenotypical
peculiarity. Beginning with their second week of
life, they suffered from repeated indigestions, lost
weight and died, one 52 days old, the other but
16 days old.

As compared to the previously described cases
of semilethal allopecy (Surrarer, Hut and Saun-
ders), these calves presented the particular
characteristic that even their eyelids and tail-
end were bare of hair.

The histologic cross-sections through their
skin showed very rarely distributed hair follicles,
the sheaths of which were much enlarged and
distorted resembling small lodges in which the
hair roots were coiling around several times.
There was a total disturbancy as regards the
orientation of the follicles and an utter impossi-
bility for the hair to push towards the exterior
(Fig. 2).

Breeding history. The mothers of these calves
were daughters of an imported Pinzgau bull
used in the A. I. Centre of the Nasaud county.
These cows were at their first calving. Their mat-
ing had taken place in the conditions of moun-
tainous pasture land where they had been put
together with three bulls of the same herd, two of
which were closely related with the cows, one
even being a half-brother.

263

Section 14 — Animal Genetics and Breeding

It is supposed that the imported bull, the
grandfather of the calves, is the carrier of the
semilethal hairlessness factor, that has been
transmitted to his descendants. Through in-
breeding, this hereditary recessive defect has
finally become obvious.

Observations upon the descendants of this
bull are being continued.

14.39. Gene-Hormone Interactions on Hair Pigmen-
tation in Cattle. L. O. Gilmore and N. S.
Fechheimer ( Wooster, U.S.A.).

Blackish Pattern is a sex-influenced trait with
the responsible gene being found in Ohio Ayr-
shire and Jersey cattle in the respective frequencies
of 0.44 and 0.54. It is assumed to modify Bs
although an insufficient number of test matings
has been made to eliminate the alternate hy-
pothesis of allelism. All eighteen bulls in a
castration experiment lost blackishness in hair
on the body proper but fewer did so in the area
of the crest and head and none on the legs, ears
or switch. The difficulty involved precludes
establishing that the bulls were heterozygotes.
This opens to consideration the possibility of
hormone action overriding that of both genes
present in homozygotes. Testosterone replace-
ment by administration has been found to re-
store the pattern although not with complete
duplication of the original phenotype.

To further test the validity of the premises
underlying the theoretical considerations an
effort was made to superimpose the administra-
tion of testosterone on the heterozygous cow
with intact gonads. Heterozygosity was establish-
ed by inference from observed phenotypes of
dam and/or daughters. Several such cases point
to the validation of the original concept. To
date no attempt has been made to test the re-
sistance of the homozygous genotype in the
female.

It has now been shown that this is a likely
system for studying a simple phenotype resulting
from the combined action of gene and hormone.

14.40. Investigations into the Hereditary Origin of
the Double Cervix Condition in the Meuse-
Rhine-IJssel (MRY) Breed. A. van Loen
(Geleen, The Netherlands).

In a long-term project of research on bovine
fertility within the Meuse-Rhine-IJssel breed
(Netherlands) it was examined whether the dou-
ble cervix condition (DC) is inheritable and,

if so, how it is inherited under normal conditions
of farm-management.

A total of 607 DC-cases recorded as cervix
duplex was encountered among 16,375 cows, i.e.
3.7 per cent.

DC is regarded as a gross genital abnormality
and is characterized on vaginal examination by
two ora uteri externa. The occurrence of this
malformation has to be considered a result of
arrested development of the Miillerian ducts.

The population under study is a random-
mating population, the influence of inbreeding
and assortative mating being negligible. No selec-
tion has taken place according to DC. SC (single
cervix) and DC cows do not differ in reproductive
efficiency.

Heredity is the most obvious cause of the
manifestation of DC in the female offspring of
a relatively large number of sires. The sires
definitely influence the incidence of DC in
their daughters (P<0.0001). Affected dams
produce relatively more affected daughters than
normal ones (/><0.01).

Regarding the mode of inheritance there are
strong indications that the pre-disposition to
DC is due to an autosomal dominant gene (or
gene-complex) (p = 0.17) with incomplete, and
possibly strongly environmental, penetrance
(z = 0.13). For the period in which cattle are
moved from pasture to cowhouse and the period
in which they are inside notably affect the critical
stage of development of the Miillerian ducts
following which normal development is arrested.

14.41. Genetical Study of "Tete de mouton" Cattle.

J. J. Lauvergne and B. Vissac (Jouy-en-Josas,
France).

The abnormality called "Tete de mouton" was
observed for the first time in France in the prog-
eny of a Limousin bull (Charmant) used by
the A.I. Center at SOUAL (Tarn) from Novem-
ber 1957 to January 1960.

The abnormals have a typical appearance:
fronto-nasal trench, convex profiled nose,
brachygnathia of the lower jaw, exorbited eyes
and marked macroglossia. Ossification defects of
skull and abnormalities of the heart (persistent
Botal hole) and of the omasum are equally
established.

Charmant was apparently normal. Both sexes
are equally frequently affected by the abnor-
mality, which had a total incidence of 5.4 per
cent among 81 1 of his progeny, born or aborted.
There was no relationship, either between the
mothers of the abnormals or between the mothers
and Charmant, who was used for beef production

264

Section 14 — Animal genetics and breeding

crosses. In addition, matings between abnormal
half-sibs produced normal and abnormal calves.
Thus, the factor appears to be monofactorial,
autosomal, dominant with incomplete pene-
trance.

The viability of the abnormal heterozygotes is
reduced up to adult age.

(1) Out of 9 "Tete de mouton" calves kept
under special nutritional conditions only
4 lived more than 2\ years.

(2) The perinatal mortality of Charmant's
calves was 32 per cent ± 12.2 among the
"Tete de mouton" calves and only 4 per
cent ±1.4 among the normals.

(3) The embryonic mortality — estimated in-
directly by the proportion of returns for
service among inseminated cows, between
30 and 120 days after insemination — was
higher in the case of Charmant (8.62
per cent ± 0.44) than in the total of all
other Limousin bulls used in the A.I.
Center during the same period (6.02
0.14).

Considering all degrees of expressivity (early
embryonic mortality, perinatal mortality of the
abnormals and viable "Tete de mouton"),
penetrance of the factor can be estimated at 21
per cent for heterozygous fertilized eggs.

Further information on this study will be pub-
lished in Annates de Zootechni.

14.42. Sex Chromosome Mosaicism in Unlike
Sexed Cattle Twins. N. S. Fechheimer, M. S.
Herschler and L. O. Gilmore (Columbus,
U.S.A.).

Female calves born co-twin to males exhibit
maldevelopment of the reproductive apparatus
when there has been vascular anastomosis of
the twins' circulatory systems in utero. Such
twin pairs also exhibit an erythrocyte mosaicism
and a tolerance to reciprocal skin grafts. This
fact has led previous investigators to postulate
that cells other than erythrocyte precursors may
be exchanged.

This hypothesis has been examined using the
leucocyte culture method In spite of the large
diploid chromosome number in cattle (60), the
X and Y chromosomes are morphologically
unique. All autosomes are acrocentric. The X is
large and submediocentric, while the Y is medio-
centric and much smaller than the X. Only the
expected XX or XY sex chromosome constitu-
tion was observed in intact cells from seven
single born females and six single born males
respectively.

Among 13 animals from 11 sets of unlike
sexed multiple births 7 females (presumed free-
martins) and 4 males possessed cells of both
male and female origin. Sufficient numbers of
cells from the remaining two calves were not
examined. Mosaicism of erythrocytes for anti-
genic properties was manifested by the cells of
9 of 10 animals for which a test was made.

These findings raise the possibility that the
intergrade sexuality of females co-twin to males
is caused not by a humoral agent produced by
the male but may be a function of the sex
chromosome mosaicism.

14.43. Heterosis from Crosses among British Breeds
of Beef Cattle. R. C. Carter, W. H. McClure,
J. A. Gaines and D. W. Vogt (Blacksburg,
U.S.A.).

This experiment was designed to estimate
heterosis in beef calves resulting from various
levels of breed crossing, not confounded with
heterosis in maternal traits of their dams. Pure-
bred or high grade Aberdeen-Angus, Hereford
and Shorthorn cows, here designated as
"straightbreds", were mated to purebred bulls of
these three breeds to produce straightbred and
reciprocal two-breed cross calves. Similar
straightbred cows were bred to crossbred bulls
(Fi) to produce three-breed and back-cross calves.
This design permitted comparisons among
straightbred, two-breed, three-breed, and back-
cross calves, born in the same year, and all from
straightbred dams. Five calf crops were produced,
1957 through 1961 , with a total of 504 calves born
alive and 480 weaned.

The most important differences were in fert-
ility and viability. Cows in crossbred matings
had 8 per cent more calves born and 1 1
per cent more calves weaned than those
bred to a bull of the same breed. There was
evidence for heterosis in birth weights, growth
from birth to weaning, and postweaning growth.
Average birth weights, adjusted for sex differ-
ence, were, in pounds: straightbreds, 68.4; two-
breed crosses, 71.4; three-breed crosses, 70.4;
and back-crosses, 69.3. Average weaning weights
for the four groups were respectively: 402, 426,
433 and 413 lb. There were significant deviations
from mid-parental means for birth weight in two
breed and back crosses; and in growth from birth
to weaning and weaning weights, in two-breed
and three-breed crosses. Differences in post-
weaning growth rates were generally smaller
than in growth to weaning, but crossbreds were
heavier at all periods. Differences in feeder

265

Section 14 — Animal Genetics and Breeding

grade at weaning, live slaughter and carcass
grades were small.

Calves sired by crossbred bulls averaged 13
days earlier in date of birth than those sired by
purebred bulls.

14.44. The Relationship between the Performance of
Mothers of Dairy Bulls and the Progeny
Tests of their Sons. L. K. O' Conner (Thames
Witton, Great Britain).

The purpose of this study was to obtain infor-
mation on the relationship between the perform-
ance of mothers of bulls and the progeny tests
of their sons, which would be useful in develop-
ing a practical method of pedigree evaluation
for young dairy bulls.

The data consisted of the progeny tests of 736
Friesian bulls with contemporary comparisons
based on a weighting of 10 or more, and the
305-day lactation records of their 640 dams.
Where possible, the first five lactation records of
the dam were included. The differences between
the dams' lactation records and the appropriate
herd average, and the contemporary comparisons
of their sons, were used in the milk yield anal-
yses. In the case of fat percentage, the dams'
own records and the first lactation daughter
averages of their sons were used.

For milk yield and for fat percentage, regres-
sions of sons' progeny test on dams' rating were
calculated for each lactation of the dams sep-
arately, and for averages of increasing numbers
of dams' records up to five. Estimates of the
repeatability of the dams' milk yield and fat
percentage were also obtained. It was found
that whilst the regressions for fat percentage
were in good agreement with those expected
in theory, the regressions for milk yield for sec-
ond and subsequent lactations of the dam were
less than expected.

A full report of this work will be submitted
for publication in Animal Production.

14.45. A Genetic Study of Feed Efficiency in Holstein-
Friesian Cattle. R. D. Plowman, N. W.
Hooven Jr. and W. R. Harvey (Beltsville,
U.S.A.).

This study was conducted to determine if
genetic differences existed in gross efficiency of
feed conversion among progeny groups and if
feed efficiency could be estimated from a portion
of the complete lactation. Efficiency in this paper
is defined as:

Fat Corrected Milk Yield

Therms of Estimated Net Energy Consumed

The data consisted of 584 lactation records
completed on 332 cows. Eighteen sire groups
were represented and 254 daughter-dam pairs.
The variables considered were: FCM yield,
feed efficiency, body weight, body weight change
and therms of estimated net energy consumed.
Results from the first lactation analyses indicated
significant differences among the sire groups in
feed efficiency and body weight. Significant
positive correlations were found between feed
efficiency and FCM yield. Body weight and
body weight change were negatively associated
with feed efficiency. Heritability and repeata-
bility estimates of feed efficiency are similar
to those of milk production.

Correlation between the third 60-day period
and the total lactation feed efficiency ranged
from 0.88 to 0.94 for different seasons of the
year. Corresponding correlations for the sec-
ond one hundred days as compared to the
total were 0.92-0.96. It seems possible to predict
feed efficiency from part lactation data.

14.46. Effects of Crossbreeding and Crisscrossing on
the Birth Weights and Gestation Periods of
Dairy Cattle. R. W. Touchberry (Urbana,
U.S.A.).

The data for this study consists of the birth
weights and gestation periods of 1205 calves
arising from various crosses and crisscrosses of
the Holstein and Guernsey breeds. The calves
were born over a period of twelve years and are
categorized into five generations. The first
generation consists of purebred Holsteins and
Guernseys and reciprocal crossbreds and was
produced by mating unrelated Holstein and
Guernsey heifers to Holstein and Guernsey
sires. The second generation consists of Hol-
steins, f-Holsteins, f-Guernseys and Guernseys
and was produced by systematically mating the
fiist generation females to purebred Holstein
and Guernsey sires. The third, fourth and fifth
generations were produced in a similar manner
by systematically mating the females of the
second, third and fourth generations to Holstein
and Guernsey sires. For the first generation data,
crossbreds were 2.8 ± 1.6 lb heavier than
purebreds, calves out of Holstein dams were
18.7 ± 1.6 lb heavier than those out of Guern-
sey dams, while those by Holstein sires were only
6.9 ± 1.61b heavier than those by Guernsey sires.
Crossbred calves were carried 0.54 ± 0.78 days
less than purebreds, those out of Guernsey dams
were carried only 3.2 ± 0.76 days more than

266

Section 14 — Animal Genetics and Breeding

those out of Holstein dams, while those by
Guernsey sires were carried 4.8 ± 0.76 days
longer than those by Holstein sires. The results of
analyses of the data for other generations will be
presented. This paper will be submitted to the
Journal of Animal Science for publication.

14.47. Some Observations on Reproduction, Weight
Changes under Lactation Stress and the
Mothering Ability of British and Crossbred
Zebu Cattle in the Tropics. G. H. Lampkin
(Kikuyu, Kenya) and J. F. Kennedy (Rock-
hampton, Australia).

Data collected at Belmont on the performance
of British, British cross Africander and British
cross Brahman females was examined. Details of
2871 matings were available. Of the cows mated
only 56 per cent of the homebred British cows
produced live calves compaied with 77.3 and
73.8 per cent from Africander and Brahman
crossbred cows respectively. Evidence was found
that the low calving percentage for British
females was connected with lactation stress, the
British cows being frequently unable to maintain
weight if two pregnancies occurred in two
successive years. Under similar stress crossbred
zebu cows could thrive and crossbred Brahmans
in particular would normally gain weight.

Although F-2 calves out of crossbred zebu dams
were significantly smaller at birth than contempo-
rary crossbred calves obtained from British
dams, this ability of crossbred zebu mothers to
thrive whilst lactating was not at the expense of
their ability to rear their calves. Calves out of
crossbred Brahman cows, in fact, grew sig-
nificantly faster than crossbred Brahman calves
obtained from British dams. Between birth and
weaning this difference for males and females
was 0.23 and 0.26 lb/day respectively.

14.48. Serum Transferrin Type and Milk and
Butterfat Production in Dairy Cows. G. C. Ash-
ton, G. R. Fallon, and D. N. Sutherland
(Rockhampton and Brisbane, Australia).

The serum transferrin types of 225 Jersey cows
in 13 herds, and 433 Australian Ulawarra
Shorthorn cows in 24 herds, in two regions of
Queensland were determined. The effect of
transferrin type on lactation length, milk yield,
butterfat yield and butterfat percentage was
assessed. The results for the two breeds did not
differ significantly. Combining the data from
both breeds, it was found that on average Tf

D/D cows had lactations 13.9 days longer than
Tf A/ A cows (P < 0.01), and yielded 450 lb
(204 kg) more milk (P < 0.01). The results for
Tf A/D cows were intermediate. No effect on
butterfat percentage was found.

The proportion of the genetic variance in
milk yield due to the transferrin locus was 10.4
per cent in the Jerseys and 6.0 per cent in the
Australian Ulawarra Shorthorns. This implies
that the manipulation of this gene could raise
milk yield by about 5.1 per cent in two gener-
ations in these herds by quite simple selection.

The nature of the effect is now known but is
not due solely to increased length of lactation.

14.49. Blood Groups in Czech Brindled Cattle and
Czech Large White Pigs. K. Hala, J. Hojnv,
J. Matousek and J. Schroffel (Libechoy,
Czechoslovakia).

Besides the formation of antibodies against a
great number of new blood factors following
isoimmunizations of the Czech Brindled cattle
a large number of phenogroups in the B and S
systems is characteristic for this breed.

In 656 bulls and cows of this breed, both
phenogroups having been determined by family
analysis, 144 phenogroups of the B system and
24 of the S system have been proved. In the
Czech Brindled breed all types of transferrins
have been found. From the above results and in
comparison with homogene breeds it may be
assumed that the Czech Brindled breed is
heterogene.

In the Czech Large White pig 28 blood factors
have been found to this day, the majority of
which are classed in 1 1 known genetic systems.
Five new blood factors are being studied as to
their relationship to genetic systems.

By electrophoresis on a starch gel between the
start and the alpha-fractions a protein fraction has
been found, appearing on sexual maturing and
lasting till old age. By means of antisera gained
by isoimmunization of sows by boar sera a close
relationship of this serum protein to the protein
parts of the fluid contained in the seminal
vesicles of boars has been observed.

14.50. The Variability of the Number of Vertebrae
in Domestic Pigs and Their Relation to
Economically Important Criteria. J. K. Hin-
richsen (Giessen, Germany).

The numbers of vertebrae can be of interest
for the producers and the consumer of pork, if

267

Section 14 — Animal Genetics and Breeding

they are in correlation with the content of lean
meat and the content of high prized cuts in the
carcass. The importance of this criterion has
been discussed in numerous publications. The
fact, that longer and leaner pigs are prefered
nearly all over the world, can not be without
influence on the distribution of animals with
different numbers of vertebrae.

The material to be discussed comprised
822 pigs of German improved Landrace (ver-
edeltes Landschwein) in the years 1954 and 1955,
all of them animals of the old type of German
pigs.

The amelioration with boars and sows of
Dutch or, if we look further back, Danish origin,
began later. Therefore the material can be seen
as sufficiently uniform. The distribution of
thoracal and lumbar vertebrae is presented. A
comparison of body length and number of
vertebrae shows that length is in dense corre-
lation with vertebrae numbers.

The figures show that the average length of
vertebrae is nearly the same in all classes of
vertebrae numbers. It is to be stated that pigs
with a higher number of vertebrae are longer in
the average. A selection with the aim of in-
creasing the number of vertebrae must be
successful in increasing the length of the animals
and if length is connected with lean characteris-
tics, also in improvement of quality. Practical
breeding is following this trend.

14.51. Selection for High and Low Fatness in Duroc
and Yorkshire Swine. H. O. Hetzer,
W. R. Harvey and W. H. Peters (Beltsville,
U.S.A.).

Individual selection based on backfat thickness
at a live weight of 175 lb has been carried in
both upward and downward directions through
7 generations in Duroc swine and through 5
generations in Yorkshire swine. By generation
7, b.f.t. in Durocs averaged 2.01 in the high-fat
line, 1.22 in the low-fat line and 1.50 in a
randomly selected control line. The correspond-
ing values for the 5th generation Yorkshire lines
were 1.43, 1.15 and 1.28 inches, respectively.
Realized heritabilities computed as the regression
of generation means on cumulative selection
differentials were 0.64 ± 0.06 and 0.42 ±0.07
for high- and low-fat Durocs and 0.36 ±0.12 and
0.35 ±0.05 for high- and low-fat Yorkshires. The
significantly slower change in the low-fat than
in the high-fat Duroc line (P < 0.05) indicates
that the response in the two lines was asymmetri-
cal. Heritability of b.f.t. in the Duroc and

Yorkshire control lines was estimated at 0.53 ±
0.07 and 0.32 ±0.09 from the intra-group
regression of offspring on mean of parents. These
values agree very well with the corresponding
averages of the realized values and indicate that
Yorkshires are less variable genetically for fatness
than Durocs. Litter size and litter weight
differed little among the Yorkshire lines but
declined in both the high- and low-fat Duroc
lines. Post weaning daily gain tended to be
higher for low-fat than for high-fat Duroc pigs
(1.44 vs. 1.38 lb), while the reverse was true for
high- and low-fat Yorkshires (1.38 vs. 1.29 lb).
Feed requirements per 100 lb gain were sub-
stantially lower for low-fat than for high-fat
lines in both breeds with control lines inter-
mediate. Low-fat Durocs produced substantially
higher yields of lean meat and less fat than
high-fat Durocs. Low- and high-fat Yorkshires
showed similar though somewhat smaller differ-
ences.

14.52. Genetic Change of Backfat Thickness in the
Danish Landrace Breed. Charles Smith
(Edinburgh, Great Britain).

Data from the Danish progeny test reports
were used in an attempt to measure the genetic
change in backfat thickness in the Danish
Landrace breed from 1952 to 1960. Over this
period the average backfat thickness of tested
pigs fell from 34.2 mm to 28.5 mm, a change of
two standard deviation units.

The method used to measure genetic change
depends on the difference in performance in
two or more years of progeny from particular
sires of dams. Environmental differences between
the years are avoided by measuring performance
relative to the year mean. However, allowance
has to be made for selection among parents on
the basis of their first set of progeny records,
through adjusting the initial records by theoreti-
cal regression factors. The genetic change is
then estimated as a function of the difference
between the adjusted first progeny records and
the records of subsequent progeny groups.

Separate estimates of the genetic change in
backfat thickness were calculated in this way
from the progenies of sires and of dams at each
of the three stations. These were in general
agreement and indicated that there was some
genetic improvement in backfat thickness in the
Danish Landrace from 1952 to 1960 but that not
all of the observed change was genetic change.
The overall estimate of the genetic change
was — 0.15 ±0.10 mm per year and this repre-

268

Section 14 — Animal Genetics and Breeding

sented about one-fifth of the observed change.
Other less critical results also lead to the same
general conclusion.

14.53. Longevity in Sardinian Sheep. Pietro Dassat
and Domenico Bernoco (Turin, Italy).

The objective of this research was to study
the longevity of Sardinian sheep and to in-
vestigate the relationship between longevity and
age at first lambing. The analyses were made
using milk records collected in the Sardinian
flock at Barumini (Cagliari) during the period
of 15 years beginning in 1943.

The data comprised 2670 lactations of 678
ewes disposed of; their average age was 4.8
years. This represents an average productive
life of 3.3 years and a replacement rate of about
30 per cent.

Of the 678 ewes of the sample 505 (74 per cent)
first lambed at one year and the rest not until
two years of age. The age at disposal of the
former group was 4.7 years and for the rest
5.1 years, with a average productive life of 3.4
and 3 years respectively.

In estimating the average productive life in
terms of times lambed, results showed 2.48 lac-
tations for the whole sample: 2.55 lactations
for the ewes first lambed at one year compared to
2.27 lactations for the animals first lambed at
two years. The difference is statistically sig-
nificant (P <0.01) indicating that early lambing
increases the length of the productive life even if
it reduces longevity.

The present note describes the histologic
changes of the thyroid gland in five ophthalmic
lambs as compared to one normal lamb.

The current methods of fixing, of inclusion and
of staining of the samples were used in this
experiment:

The prepared samples were examined at the
following magnification.

The form of the follicles in the ophthalmic
lambs was between oval and round, with an
irregular cornered outline.

The mean height of the follicular epithelial
cells was 8.84 u.

The mean frequency of the various epithelial
cells was of 52.08 per cent cylindrical cells,
46.42 per cent cubic cells, 1.25 per cent flat cells.
Resorption vacuols existed in almost all the
follicles of the thyroid gland. In many follicles
the resorption of the acidophil colloid was
almost total.

In the normal lamb the proportion of the
epithelial cells was different: 42.00 per cent
cylindrical cells, 54.00 per cent cubic cells and
4.00 per cent flat cells. The mean height of the
epithelial cells was 8.07 u.

In the ophthalmic lamb, the thyroid gland was
in a state of hyperfunction. The weight of the
lambs decreased with the increase in intensity
of the disorders (symptoms).

14.55. (F). Science and Sheep-breeding. H. N. Tur-
ner (Glebe, Australia).

The data will be pusblished in full on the
Italian magazine La Nuova Veterinaria.

14.54. Contributions to the Histologic Study of the
Thyroid Gland in Tzigaia Lambs with Here-
ditary Ophthalmy. N. Teodoreanu and Lucia
Gurau (Bucarest, Rumania).

Previous studies (Gh. Radu et «/., 1947;
N. Teodoreanu et ai, 1952; and N. Teodoreanu,
1958) have stated the occurrence of hereditary
semilethal ophthalmy for the first time in
lambs of the black-headed Tzigaia breed, at the
Slobozia experimental research station. The
disease appears between the 2nd and the 60th
day of life, ending with the death of the lambs
at the age of 2-3 months.

The film deals briefly with current methods of
sheep selection, wool classing and wool buying
for Merinos in Australia, all of which rely
heavily on hand-and-eye methods of appraisal.
The theory behind the use of measurement, the
estimation of repeatability and heritability and
their application are then demonstrated, and
current C.S.I.R.O. selection experiments are
described and filmed, including methods of
wool-sampling and detailed measurement. In
these experiments, wool weight has been raised
by 1 lb per head; the absence of any important
correlated change is discussed.

The film then moves to a commercial shed and
shows how simply fleece-weighing can be
introduced in practice.

The film was made for general use to demon-
strate the experimental work which lies behind
a widespread extension campaign in Australia
for the introduction of objective methods in sheep
selection.

269

Section 14 — Animal Genetics and Bre edmg

14.56. Current Progress in Three Selection Experi-
ments with Hill Sheep. A. F. Purser (Edinburgh,
Great Britain).

Three separate two-way selection experiments
started by the Animal Breeding Research
Organization in 1953-54 with hill sheep are
described briefly. In one experiment, selection
for cannon bone length at eight weeks of age
has resulted in marked changes in cannon bone
length at all ages from birth onwards. Other
skeletal and body weight changes have also
occurred. In the second experiment selection for
the average degree of fibre medullation in the
lamb fleece has altered adult fleece type and the
timing of the waves of shedding of kemp
fibres. The third experiment involves selection for
two extremes of birthcoat type. Correlated
changes have occurred in birth weight, lamb
mortality and in adult fleece grade and fleece
weight, with the intermediate birthcoat type
being generally better than either extreme type.
The heritabilities of all three characters are 45-55
per cent and response to selection in each line
has been approximately as predicted. However,
evidence of a decline in genetic variance in
each of the selected characters will be presented.

14.57. The Effect of Two Associated Biochemical
Polymorphisms on Red Cell and Production
Traits in Sheep. J. H. Watson, A. G. H. Khat-
tab and R. F. E. Axford (Bangor, Great
Britain).

Two genetic loci governing haemoglobin type
and erythrocyte potassium concentration, show
evidence of association in the College flock of
Welsh Mountain sheep. In 1777 sheep departure
from randomness of association stems from an
excess of haemoglobin B homozygotes among
high potassium animals. This inbalance extends
to the three remaining phenotypes containing the

haemoglobin B gene but not to haemoglobin A
homozygotes.

Differences in potassium concentration be-
tween the three haemoglobin types are accounted
for by their packed cell volumes. At both high
and low potassium levels haemoglobin A
homozygotes have higher blood potassium
concentration and packed cell volume than
haemoglobin B homozygotes. AB heterozygotes
are close to mid-parent values. Red cells con-
taining haemoglobin A, although more numerous
than those containing haemoglobin B, are also
more fragile.

Differences in production records of these
sheep are small, rarely significant, but consistent.
Low potassium types are marginally superior to
high potassium types in all liveweight measure-
ments and first fleece weight. Haemoglobin A
homozygotes are marginally better than BB
homozygotes, with AB heterozygotes inter-
mediate.

14.58. Some Genetic Studies on the Buffaloes in the
U.A.R. A. A. Asker, L. H. Bedeir, A. A. El-
Itriby and I. A. Ahmed (Cairo, Egypt).

Records collected on two herds of buffaloes
maintained by the Ministry of Agriculture were
analysed to study the inheritance of some dairy
traits and to investigate the interrelationships
between these characters. More than 400
buffaloes having 1614 records were included in
this report. The repeatability of 305 day milk
yield, total milk yield, maximum weekly pro-
duction, intensity, persistency and lactation
period were 0.40, 0.41, 0.34, 0.39, 0.18 and 0.27
respectively. The corresponding heritability
estimates were, 0.26, 0.27, 0.43, 0.38, 0.33 and
0.27 respectively. The phenatypic as well as the
genetic correlations between characters studied
were high which indicate that most of these
characters are affected by the same sets of genes.

270

SECTION 15

HUMAN GENETICS

15.1. Biological and Genetical Foundations of Great
Historical Features. Mario Tirelli (Rome,
Italy).

A brief exposition of the phenomena, observa-
tions, criterions which may permit previsions
on the evolution of human groups, and thus on
the evolution of the reciprocal relations between
the groups themselves, from which depend pre-
cisely the development and the succession of the
great features and historic events.

15.2. Phylogenetic Relationships of the Forms of
Erythrocyte Carbonic Anhydrase in Primates.

Richard E. Tashian and Donald C. Shreff-
ler (Ann Arbor, U.S.A.).

Characterization of the non-heme erythrocyte
proteins from a number of representative pri-
mate species of the families Lorisidae, Cebidae,
Cercopithecidae, and Pongidae has revealed the
presence in some species of two distinct mole-
cular forms of carbonic anhydrase (CA). These
forms are designated as CA-I and CA-II. In
addition to carbonic anhydrase activity, these
enyzmes also exhibit esterase activity toward a
and (3-naphthyl acetate, with CA-I possessing
markedly greater esterase activity than CA-II.
The electrophoretic separation patterns and de-
gree of esterase activity for CA-I appear to be
species specific. However, two variants of
carbonic anhydrase-I (CA-Ib and CA-Ic) have
been described from human hemolysates and
demonstrated to be under genetic control;
a variant of CA-I from an orangutan hemolysate
has also been observed.

Additional evidence that the CA-I of different
species are homologous enzymes and that CA-I
is distinct from CA-II has been obtained from
immunodiffusion studies utilizing specific rab-
bit anti-human CA-1 serum.

Often, both electrophoretic and immunolo-
logical characterization were necessary to establish
tentative phylogenetic relationships among spe-
cies. For example, although limited immuno-
gical differences could be detected within the
species of a particular family, they could usually
be readily separated on the basis of their com-

bined electrophoretic patterns and esterase
activity. To a lesser extent, species exhibiting
similar electrophoretic patterns and enzyme
activity were shown to differ immunologically.
The application of these methods to the study
of primate evolution will be discussed.

15.3. Modern Concepts in Clinical Genetics. Robert
L. Tips and G. Smith (Portland, U.S.A.).

In modern practice clinical genetics can be
considered the application of genetic knowledge
in an effort to solve the diagnostic, prognostic,
and management problems of the patients and
their families. Subtle but severe medical emo-
tional, and socio-economic disturbances are
universally expressed by these families following
the introduction into the family environment of
a child with disease of genetic origin. A specifi-
cally trained team, coordinating their efforts,
can isolate and evaluate these problems, so that
the affected family can gain understanding and a
healthy adjustment.

The patient, parents, and siblings are evaluated
in an appropriate clinic setting by the specialists
— the geneticist, who explores the total kindred
history with emphasis on reproductive patterns;
the physician, who evaluates the impact of the
pathological processes on the entire family;
the social worker, who relates to the problem
extended into the socio-economic milieu of the
kindred ; the technicians, who perform the battery
of genetic tests on each member of the family.
The elimination of emotional factors emanates
from conference discussion of the team after
their interviews and tests are complete.

The discussion is amplified by a review of
four families whose problems were resolved by
the clinical genetic counseling team.

15.4. A Genetic Discriminant for Diseases of Hetero-
geneous Origin. Bertram L. Hanna, A
Donald Merritt, Charles Wm. Todd, Jr.
and Terry L. Myers (Indianapolis, U.S.A.).

Many diseases which segregate within families
as expected under a Mendelian model demon-

271

Section 15 — Human Genetics

strate clinical heterogeneity which suggests that
all cases may not be of the same genetic etiology.
Some have been separated with discriminate
function analysis of clinical data or by the de-
monstration of distinct biochemical differences
within diagnoses. These methods have failed
to define homogeneous groupings within other
diseases, including diabetes mellitus and cystic
fibrosis. On examination of the frequencies
of occurrence of a given disease in relatives of an
affected sibship an unbiased estimate of the
allelic frequency in the population may be ob-
tained on the assumption that homozygosity at
a single locus is responsible for all cases. The
population incidence estimated under this as-
sumption will be less than the observed popula-
tion incidence if the same gene locus is not segre-
gating in all observed families.

Differences in the survival of affected indi-
viduals, age of onset and parental genotypes
require that different models be developed for
diabetes mellitus and cystic fibrosis on the as-
sumption that both of these are autosomal re-
cessive traits. The problem of reliable sampling
in cystic fibrosis families may lead to gross bias
in the fitting of the theoretical model. A method
is developed to obtain an approximation to
the population allelic frequency which deviates
by only 0.5 per cent from that value expected
under the theoretical model.

From study of 754 cousins of cystic fibrosis
probands a population incidence of 1/3824 is
obtained. The similarity of this estimate to that
obtained from an epidemiologic study of a
comparable population (1/3700) suggests that
only one gene locus is involved in the etiology
of cystic fibrosis.

15.5. (D.) The Application of Automatic Record
Linking Procedures. J. H. Edwards, I. M.
Leck, T. McKeown, and R.G. Record (Bir-
mingham, Great Britain).

The application of automatic record linking
procedures and other methods dependent on
advanced computational methods to the Birm-
ingham data. These include detailed notifica-
tions of all births, with special reference to mal-
formations, and extensive linking of other
data. Births are identified by both mother and
child and sibships formed automatically as they
arise.

cordant as to a given disease is always very im-
portant for Clinical Genetics, especially when
it is monozygotic. In Clinical Genetics, isolated
pairs occur to the physician, and the diagnosis
must therefore be based on intra-pair compari-
son. The statistical treatment of the case must
consider the fact that a concordant monozygotic
twin pair represents a statistical universe rather
than a statistical unit. This entails taking into
account many traits concerning the time of onset,
the symptoms, development, result of treat-
ment, etc. Suggestions are made for the statis-
tical treatment of such cases.

15.7. First Findings on the Behaviour of Red Blood
Cells from MZ and DZ Twin Pairs in Isonia-
zide-fixation Tests (in Vitro). M. Bolognesi,
U. Fantoli and M. Milani-Comparetti
(Rome, Italy).

An isoniazide-fixation test, described in the
paper, was carried out in vitro on red blood cells
from 30 female twin pairs, in order to investigate
its usefulness in zygosity determination. The
zygosity classification of the material was based
on several criteria of similarity including blood
groups (ABO, NM, Rh-Hr).

The material was divided in two groups, res-
pectively under and above 16 years of age (to
guard against the higher frequency of fast inacti-
vators among individuals under 15, denounced
by Sunhara).

Several tables contain the findings and statisti-
cal analyses; in particular Table V indicates
that only the variability due to zygosity exeeds
Fisher's F, being ascribable to chance only
with one probability out of 1000. The authors
conclude that the factor responsible for individual
behaviour in the test as applied seems to be under
genetic control, thus making it potentially useful
as a further test in zygosity determination. Fur-
ther research tends to:

— determine the correlation between be-
haviour in this test and the fast or slow isoniazide
inactivator trait in vivo.

— establish the limits of individual and in-
tra-individual variability for methodological codi-
fication.

— extend the study to a wider sample, ap-
plying Kallman's twin family method to identify
the mode of transmission of the trait.

15.6. On the Statistical Significance of One Pair of
Monozygotic Twins in Clinical Genetics. Luigi
Gedda (Rome, Italy).

A case of one twin pair concordant or dis-

15.8. Blood-groups and Haptoglobines by 200
Twin-pairs. Hans Niermann (Miinster, Ger-
many).

For the diagnosis of twins it can be important

272

Section 15 — Human Genetics

to use blood-groups and haptoglobines. The
blood-groups and haptoglobines were deter-
mined in 200 twin-pairs. All of 56 monozygotic
twins were concordant, while only three of 81
twins with the same sex were concordant.

somatic cells preserved by freezing. An increase in
abnormalities of progeny has not accompanied
the use of frozen-stored bovine and human sper-
matozoa. The current view is that there is no
such genetic damage.

1. Fertil. & Steril 14, 49-64, 1963.

15.9. Disparity of Colour Vision in Uniovular Twins.

J. Zanen and A. Meunier (Brussels, Belgium).

The authors relate two cases of uniovular
twins. The colour vision normal for one of them,
is wrong for the other and belongs respectively
to protanopia or deuteranomaly group.

They add a third case where the twins are both
deuteranomalous, but the disparity was so slight
that it was not significaat.

15.10. Banks for Frozen-Stored Human Spermato-
zoa. J. K. Sherman (Little Rock, U.S.A.).

Feasible applications of frozen-stored human
spermatozoa include: clinical treatment of in-
fertility with donor or husband's semen, main-
tenance of husband's fertility in absence of
testes, in old age and in death; ready availabi-
lity of spermatozoa bearing desired genetic
characteristics for unusual blood types, etc;
protection from radiation induced genetic alter-
ations on earth and in space; and eugenically
directed population control and selection.
Successful banking of human spermatozoa pre-
served by freezing is dependent upon several
considerations. In addition to simplicity and
efficiency, methods employed must favor mini-
mal loss of spermatozoal motility, retention of
fertilizing capacity, and absence of genetic
alteration. Superiority of the author's combined
freezing and storage method using liquid ni-
trogen-vapor(1) is shown in comparison with
earlier dry-ice method. After an average freeze-
thaw survival of 70 per cent, no further motility
loss occurred during storage at — 196°C for three
years, the longest period observed, while 10-20
per cent was lost at — 75°C. Retention of fertiliz-
ing capacity of human spermatozoa preserved
by nitrogen-vapor method is reported. Possibility
of freeze-thaw induced genetic damage is re-
cognized. Integrity of normal and malignant
cells for research is maintained in frozen-storage
and frozen bovine semen is used routinely to
breed millions of dairy cattle. No alteration in
chromosomal structure or reflected cellular
function has been reported with mammalian

15.11. Selection Relaxation in Man. R. H. Post
(Ann Arbor, U.S.A.).

The relaxation of selection pressures against
deleterious traits resulting from cultural growth
can be demonstrated by the markedly higher
frequencies of red and green "colorblindness"
among populations in Europe and the Far East,
contrasted with the lower frequencies prevalent
among populations living in hunting habitats,
such as Australian aborigines, Eskimos and
certain American Indians. Frequencies are
approximately four times higher in the former
populations than the latter, i.e. some 6 and 1.5
per cent, respectively, for the mutant alleles at
the deuteron locus, and some 2 and 0.5 per
cent respectively, at the protan locus.

The demonstration depends upon certain
assumptions which appear reasonable, although
they are not yet proven. Evidence is presented
for the selective disadvantage of "colorblindness"
in hunting habitats and its relative unimportance
in most walks of life among non-hunters.

The two loci are quite closely linked on the
X-chromosome. Diagnosis is fairly facile and
reliable at early ages, environmental effects and
age changes are negligible, and published data
are sufficient to permit comparisons among
populations which vary in habitat or cultural
history.

Rough estimates of mutation rates can be
made by dividing the difference in gene frequen-
cies between hunters and non-hunters by the
number of generations which have elapsed since
the ancestors of the latter relinquished their
hunting habitats, under the assumption that
relaxation has been complete since that time.

Analysis of the frequencies of "colorblindness"
among geographic areas within several countries
together with their cultural histories gives
further support to the relaxation hypothesis,
viz. Britain and France, Japan and China.

Population differences in vision acuity and
hearing acuity offer less satisfactory opportuni-
ties for demonstrating selection relaxation.
Frequency data for several different populations
are presented, with discussion.

273

Section 15 — Human Genetics

15.12. (D). Consanguinity Studies in Italy. A. Moroni
(Parma, Italy).

An analysis of consanguineous marriages
which have taken place in Italy from 1910 to the
present has been started, using consanguineity
dispensations available in bishopric archives and
at the Vatican.

About 500,000 dispensations have been
examined and are being transferred to magnetic
tape for (a) statistical analysis with a view to
detect what can be learnt on population struc-
ture from the study of the frequency of consan-
guineous marriages, (b) linkage, with other types
of investigations on records (e.g. hospital,
school records, etc.) in order to estimate the
effect of inbreeding on mortality disease,
anthropometric characters, etc.

15.13. Recent Investigations about the Direct
Estimation of the Mutation-Rate of Autosomal
Dominant Genes in Men. Wilhelm Tunte
(Miinster, Germany).

The problem of the frequency of spontaneous
mutations in men is an important field of
research at the Institute of Human Genetics
in Miinster. The starting point for these investi-
gations is the Genetic Register for the adminis-
trative area of Miinster having been established
in 1957. On the basis of experience hitherto ex-
isting, it will be discussed to what extent special
hereditary diseases are suitable for estimating
the mutation-rate. In this connection investi-
gations are reported that are now in operation
or have been closed in the institute mentioned
above. Preliminary findings will be discussed for
syn- and polydactylie. Complete results will be
brought about multiple cartilaginous exostosis
and Osier's disease.

these conditions were fulfilled with only 183
women and 748 men. The material could be
expanded by cases which had been collected by
E. Kruyff in an investigation on the induction of
leukaemia following radiotherapy of ankylosing
spondylites. In addition, progeny was scored
from a group of males having received low
gonad doses of 1-10 rads. Data on the sex ratio,
expressed as the percentage of males in the
progeny, were extracted from birth records in
the municipal registration offices. Following
maternal irradiation with 70-270 rads the sex-
ratio decreased from 0.541 (242 births) before
irradiation to 0.485 (230 births) after irradiation.
The shift is not significant, but the data show no
heterogeneity with those obtained by Turpin,
Lejeune and Rethore (1957) in France. For the
combined sets of data the decrease of the sex-
ratio after exposure to radiation of women is
significant (P= 0.023). Since any of the well-
known factors influencing sex-ratio, such as
birth order, paternal age and annual fluctuations,
including war, cannot account for the observed
decrease in sex ratio, we feel justified in con-
cluding that it was brought about by radiation-in-
duced genetic changes. Non-disjunction would
have resulted in an increase of the sex ratio. The
consistent decrease in this and similar studies
suggests then that the frequency of non-dis-
junction in irradiated women, is negligibly small
relative to the frequency with which recessive
lethals or loss of the X-chromosome are in-
duced. Following paternal irradiation (25-370
rads) the sex-ratio increased significantly (P —
0.035) fiom 0.470 (1258 births) before irradiation
to 0.525 (932 births) after irradiation. Here, how-
ever, the sex-ratio before irradiation was ab-
normally low and differed significantly from the
population average, thus no definite conclusions
can be drawn. In contrast to results of Lejeune,
Turpin and Rethore (1960), no change in the
sex-ratio was observed after paternal exposure
to doses of 1-10 rads.

15.14. Sex Ratio Shifts among Progeny from Patients
having received Therapeutic X-Irradiation.
P. J. L. Scholte and F. H. Sobels (Leyden,
The Netherlands).

The material was collected from four hospitals,
situated in The Hague, Leyden and Rotterdam.
Selection was made for patients who, following
exposure to gonad doses of a few hundred rads,
had progeny and who at the time of irradiation
did not age more than 36 years for women and
41 years for men. Out of a total of 85,000 patients

Subsidized by the Organisation For Health
Research T.N.O.

15.15. Investigations on the Low Sex Ratio in a
Human Population. I. Lester Firschein and
Diane Sank (New York, U.S.A.).

A further statistical analysis of the sex ratio
within families will be made in a population
known as the "Black Caribs" of British Hon-
duras, Central America. Previously it was shown

274

Section 15 — Human Genetics

that in a sample of mothers with sickle-cell
trait (HbA Hbs) the sex ration among their
children is normal whereas in the sample of
HbA HbA mothers, the sex ration was low. The
observed equilibrium values for sex ratio in this
population are very close to the calculated
equilibrium values based on the observed
frequency of the sickle-cell trait in adult males
and females. The relationship between sex ratio
of a population and level of malarial immunity
will be explored.

15.16. Does there Occasionally Exist an Incom-
patibility of Mothers with Their Male Off-
springs? K. O. Renkonen (Helsinki, Finland).

The known phenomena of incompatibility are
enhanced by repeated pregnancies or transplan-
tations. Accordingly, in a study of incompati-
bility of mothers with their male offsprings the
first question should be: how are the sex ratios
(m/f) of live births influenced by repeated
pregnancies. It is well-known that the sex ratio
is steadily decreasing by birth order. As such it is
not a sufficient argument for incompatibility,
because the primary sex ratio could also have
been decreasing by birth order. That would imply
a decrease also in the sex ratio of stillbirths by
birth order, but there is a remarkable increase
instead.

If the decrease of the sex ratio by birth older
is due to incompatibility, it should be manifested
not only by repeated pregnancies but also by
repeated births of boys. The families that have
already got both boys and girls should then have
the highest sex ratio of the next child, if there is
only one boy among the precedent children ;
lower, if two boys were already born, and lowest,
if there were three of them. The observed
statistical data are in agreement with this
prediction.

Let us suppose that some of the mothers in one
type of families are incompatible with their male
offsprings, but all are compatible in the other.
Then the birth interval to the next child should
be lengthened among the families including
incompatible mothers. The observed data
suggest that it is so. In addition, the lengthening
of the birth interval should be caused only by
those intervals terminating with a girl, just as it
is, for, if the birth interval is terminated with a
boy, the mother can hardly be incompatible.
In conclusion I should like to interpret the
statistical data to support the assumption of an
occasional incompatibility of mother with her
male offspring.

15.17. Studies on Colour Blindness among the
Tribals and Non-tribals of Andhra Pradesh,
India. K. R. Dronamraju (Orissa, India)
and P. Meera Khan (Andhra Pradesh,
India).

The Polavaram Agency area of Andhra
Pradesh in southeastern India has, according to
the 1961 census, a population of 22,461 non-
tribals and 23,810 tribals. The non-tribals are
largely Hindus, but there are a few Christians
and Muslims also. The tribals include Koya
Doras, Koya Kammaras, Musaras, Konda
Reddis, Konda Rajus, Sugalis and Pandava
Nayakas. Tests using Ishihara (1960) Plates,
showed that 6.5 per cent of 569 non-tribals and
2.5 per cent of 1155 tribals were colour blind.
1.5 per cent of non-tribals and 0.34 per cent of
tribals were Protans. The tribes in which one or
more colour blind men were discovered are
Koya Daras, Musaras, Konda Rajus and
Sugalis. The low frequency of colour blindness
found among these tribals is in general agreement
with that reported in Navajo Indians, Australian
aborigines, and some African tribes. The differ-
ence between the frequencies among the tribals
and non-tribals in Andhra Pradesh is highly
significant (X2= 16.40) and is explained as due
to the relaxation of selection against colour
blindness in civilized communities.

15.18. The Incidence of Cuna Moon-Children.

Clyde E. Keeler (Milledgeville, U.S.A.).

The incidence of Cuna Indian Moon-child
albinos is more than 60 per 10,000 population,
being reduced from about 69 per 10,000 in 1925.
Stout's 1940 estimate of 47 per 10,000 is probably
in serious error. Our 1962 data suggest infanticide
is uncivilized towns and possibly selective inter-
breeding of heterozygotes in the town of Nar-
gana. Absolute population figures cannot be
obtained, due to census methods and unrecorded
absentees. Our figures show albinos to be less
viable than normals, in keeping with general
Cuna belief. Since albinos seldom reproduce,
the gene frequency in one generation without
mutation should have been reduced to 57 per
10,000, so there is evidence that mutation does
take place. It is estimated that about 398 mu-
tations per generation per 10,000 population are
added to the gene pool in order to provide the
5 new albino homozygotes per generation
necessary to keep the incidence at equilibrium.
Examination of our 1950, 1960 and 1962 data
all show Moon-child albinism to be due to a single
pair of recessive, Mendelian, mutant genes.

275

Section 15 — Human Genetics

15.19. Contribution to the Study of Isolates in
Hight-Normandy (France). P.-Y. Rousseau,
C. Ropartz and R. Kherumian (Rouen,
France).

In this data, the study of the isolates has not
been performed with the use of the bias of the
consanguinity coefficient but by the direct study
of populations' movements.

Investigations among 9700 blood donors living
in Seine- Maritime (France) allow us to study and
precise the migrations of weak amplitude inside
a french department.

The following distances have been analysed
for each subject (from 18 to 60 years old).
Distance between the birth-places of its parents
between the birth-place of the subject
and these of its parents
between its birth-place and that of its
spouse.

Many biological characters, with a well
known genetical determinism, such as the blood
groups A. B. O and Rhesus, the dyschroma-
topsia, and the curling have been gathered up in
view to define the best criterium for an an-
thropological sampling.

It has appeared to us that if the coefficient of
consanguinity has lowered is a very important
rate, the whole population geographically stand
very stable.

The mean estimation of the amplitude of the
movements is about 8 to 10 km. Nevertheless,
notable differences are found following the
geographical position = (sea-side for example or
presence of a river).

These phenomenon when studied in function
of the ages of the subjects allow to sketch the
chronological variations.

15.20. Demography and Genealogy of Different Types
of Isolates. Robert Gessain (Paris, France)

The Anthropology Research Centre (Musee
de l'Homme), the establishment of which was
announced at the Rome Congress, has now
become a laboratory of the third section (biology)
of the Practical School of Higher Studies. Its
research workers are now engaged in the study of
several endogamous human groups from the
anthropological, demographic and sociological
view points. These groups, whether monogamous
or polygamous, patrilineal or matrilineal in-
structive, show different characteristics.

The Angmassalik eskimos are a closed isolate
for whom registry office records are available
since 1900, in addition to which we have census
lists for the entire population for 1 884, the data of

their discovery, and for 1935, the date of our
study.

In Brittany, the two villages under study form
what can be called "open" isolates for which are
available all relevant Registry Office records
since 1800.

In Senegal, we are studying two groups,
without any such records, on the basis of
incomplete lists taken at the official census and
genealogical data collected directly from the
inhabitants. One of these groups is an almost
completely closed isolate whose patrilineal
inhabitants are strongly attached to the soil; the
other is an isolate "open" on neighbouring
groups, matrilineal in structure, with frequent
comings and goings between villages near and
far.

In Angmassalik only one migration, affecting
one third of the population, male, female and
children, has taken place at a precise date, while
in Brittany emigration, which affects 25 per cent
of the inhabitants, is continuous and difficult to
situate at any given time; in Senegal the societies
being studied have only been exposed very
recently to migration.

Thus the varying state of these isolates, some
more open than others, the completeness or
lack of population statistics and the different
state of migrations in each condition the differ-
ence in methods and lead to fruitful comparisons,
although these isolates are far removed from
each other in space and in their degree of
endogamous evolution.

15.21. Human Genetic Studies on an Isolated Popula-
tion in Aland (Finland). A. W. Eriksson and H.
Forsius (Helsinki, Finland).

In Kokar, an isolated island in the Aland
archipelago lying in the Baltic Sea between
Sweden and Finland, investigations on popula-
tion genetics have been made. The well-preserved
church records going back over 300 years have
provided an invaluable source of information
on pedigree, sex ratio at birth, twinning rate,
consanguinity, etc. Medical, and especially
haematological and ophthalmological examina-
tions, blood and serum grouping, serum choles-
terol, serum B12 vitamin, P.T.C. taste tests and
other biochemical and anthropological tests
have been performed on the ca. 550 registered
Swedish-speaking residents.

The investigations are still in progress but
among the results that have already emerged,
the following may be mentioned: endogamous
marriages have been very common; the rate of
multiple confinements has been very high; the

276

Section 15 — Human Genetic*

dizygotic twinning rate has decreased significant-
ly during an observation period of 200 years
(similar results have also recently been observed
for Aland as a whole and in Sweden).

Not only the inhabitants shown by the genea-
logical analysis to be of pure native descent
for at least 4 generations but also the population
as a whole, have some extreme blood groups
frequencies. The A-blood group percentages
67-70 (gene frequencies: pi = 0.27-0.28, P2 =
0.17-0.18) are to our knowledge the highest
hitherto reported for a white population. The
frequency of D-negative individuals is very
high. The frequencies of the following genes
are low: q in the ABO-system, M in the MN-
system, Hp1 of the haptoglobin serum groups,
Gmx of the gamma globulin groups and Gc1 of
the group specific components (Gc1 = 0.615 is
the lowest yet recorded in the literature).

Some hereditary diseases, such as recessive
autosomal tapetoretinal degeneration and the
Aland Islander bleeder syndrome (v. Wille-
brand's disease, Thrombopathia v. Willebrand-
Jurgens) have a noteworthy frequency.

15.22. The Blood Groups of Ashkenazic Jews.

F. Ottensooser, N. Leon, M. Sato and P. H.
Saldanha (Sao Paulo, Brazil).

The genetical diversity of Jewish people and
the persistence of their communities as isolates
within other populations pose interesting evolu-
tionary problems.

From 586 B.C. to modern times there have
been several Jewish dispersal movements which
resulted in four main Jewish groups: Ashkenazic
or Eastern European Jews, Sephardic or Medi-
terranean Jews, Oriental or Asiatic Jews and
Yemenite Jews. Before and during dispersal,
Jewish populations acquired various non-
Jewish components and gene flow into Jewish
communities appears to have been strong during
captivity in Egypt and in the Roman Empire.

Several polymorphic traits have been studied
among Jewish populations of various origins
and blood groups proved to be especially useful
for comparing Jewish populations with their
neighbours.

The ABO, MN and Diego blood groups of
100 Ashkenazic Jews in Sao Paulo, Brazil were
investigated. All subjects were Diego-negative.
The ABO and MN frequencies were similar to
those in previous Askhenazic samples. Rh fre-
quencies presented typical Mediterranean dis-
tributions, Ashkenazic Jews showing higher B
and R° gene frequencies and lower r frequency
than their neighbours. Probably these characteris-

tics were inherited from ancient Hebrews in
Palestine.

The high B values of the Ashkenazim and their
low values of non-tasters and Rh negatives are
not derived from intermarriage with Mongo-
loid people but a Mediterranean heritage as
evidenced by absence of Diego-positives and
high R° values. The incidence of R° gene also
suggests that an African component similar to
that found today in Egyptians was present be-
fore the diaspora. The Jews that preserved a
small but constant Negro component during thou-
sands of years are an example of social isolate
comparable to endogamous groups in India.

15.23. A Comparative Study on Blood Groups Dis-
tribution among Normal and G6PD Deficient
Subjects in Israel. A. Adam, L. Dressler,
C. Sheba and A. SzeInberg (Tel Hashomer,
Israel).

A recent investigation demonstrated signifi-
cantly lowered frequency of the E gene (Rh
system) among glucose-6-phosphate dehydroge-
nase (G6PD) deficient American Negroes, as well
as a tendency toward differences in the B and O
blood groups distribution, between them and the
normal controls (*)

In a similar study, carried out in Israel, about
a thousand normal and 350 G6PD deficient
subjects were tested simultaneously for G6PD
activity and for the distribution of the Rh and
ABO blood groups. The investigated popula-
tion included representative samples of several
communities (Jews from Kurdistan, Iraq, Iran
and Yemen and Israeli Arabs) in which G6PD
deficiency is frequent.

No significant differences were detected be-
tween the distribution of the various Rh genes
among C6PD deficient and normal subjects in
each of the communities investigated.

Similarly no deviations from homogeneity
was found in the ABO distribution except for the
Kurdish community. In this sample (102 nor-
mals and 112 G6PD deficients) a significantly
lower frequency of 0-group and higher frequency
of A group was found among the G6PD defi-
cients (P<02).

A larger population sample is being studied
in order to examine the significance of this
observation.

1. Tarlov, A. R., Brewer, G. J., Carson,
P. E. and Alving, A. S. Primaquine sensitivity,
Arch. Int. Med. 109, 209 (1962).
This investigation was supported by grants

277

Seciion 15 — Human Genetics

A-2740 (Hema) from the U.S.A. Public Health
Service and R/00014 from the World Health
Organization.

15.24. The Problem of the Associations Between
Blood Groups and Disease. Angelo Cresseri
and Angelo Serra (Milan, Italy).

It has been frequently stated that research
on the associations between blood groups and
disease would have ensured an important con-
tribution to the understanding of the workings
of natural selection, and given the chance of
opening up new methods of research on impor-
tant diseases.

Of course both are goals that suppose (1) a
detailed knowledge of all the blood groups of a
given system effectively associated with a partic-
ular disease, and of what is the degree of asso-
ciation; (2) a definite demonstration of the
constancy of the phenomenon of the association
between blood groups and a disease.

With a view to get consistent information on
these to fundamental points, an analysis has
been done — according to classic models and
with the help of an electronic computer — of
most published data since 1953. 1162 samples
in all were analysed; 115 of them ought to be
excluded, because the frequency distribution of
the blood groups in the control subsamples
deviated significantly — the significance level
having been fixed at the probability point
0.01 — from that expected in the hypothesis of
panmixia, thus suggesting non-randomness in
the sampling. The other 1057 samples are un-
evenly assignable to 273 diseases; subdivisions
by age, sex and clinical criteria increase up to 330
the independent classes.

The detailed results will be published else-
where. A few general conclusions seem worthy
to be outlined here.

(1) No clear-cut statement, relevant to the
two fundamental points mentioned, may at
present be done for the majority of the diseases
studied up tonow, because of scarcity of samples
available for the analysis.

(2) The relative risks of morbidity of given
blood groups, estimated from various samples
of the same disease, are often significantly he-
terogeneous, even though either each one or
most of them deviate significantly from unity as
the combined estimate also does. That happens,
for instance, for the risk A : O in the cases of
gastric cancer (58 samples), peptic ulcer (35 sam-
ples), duodenal ulcer (31 samples ) and of the
cancer of uterus (cervix) (16 samples). This
fact would suggest that, when the existence of

a peculiar association may be considered as
statistically proved, one should take in account
also the possibility that systematic factors af-
fect differently the degree of correlation.

(3) Many more researches at the biometric
level are still highly desirable, in order to reach
reliable and meaningful conclusions.

15.25. The Genetics of Insensitivity to Pain: Studies
on Two Families. N. Leon, W. Becak:, M. L.
Becak:, B. J. Schmidt, F. Ottensooser and
P. H. Saldanha (Sao Paulo, Brazil).

Congenital analgesia is a very rare disease.
About sixty cases have been reported in pertinent
literature but its etiology seems rather obscure
mainly because of the variable expressivity of the
disease. Most propositi are sporadic cases which
are identified by their indifference to painful
traumatisms. They frequently have sequels of
bone fractures and tongue cuts. Both sexes seem
to be equally susceptible and occasionally
members of the same sibship present the same
condition. The parents of the affected subjects
are frequently normal and sometimes consan-
guineous.

Two families, including various members with
congenital analgesia have been examined. Both
families live in country towns of the State of
Sao Paulo. In the first family, the propositi are
children of normal and unrelated parents. The
sibship is represented by 8 living and two de-
ceased sibs. Two brothers, 8 and 2 years of age,
are affected, one of them suffering from bilateral
cryptorchidism; a third affected brother died
with six months of age. A girl belonging to the
same sibship has heterochromia iridis and a
cousin of the propositi, on the paternal line, has
club foot.

The second family includes ten living and one
deceased sibs. Two of them, 6 and 1 1 years
old, present analgesia. The parents of the propo-
siti are normal and display multiple consan-
guinity ( / = 0.0703). It is noteworthy that the
father is the only son in a sibship of 1 1 indivi-
duals. Cleft palate occurred in two cousins of the
propositi.

It should be emphasized that in the two fami-
lies examined, the parents are normal, one of
the families presenting a high inbreeding coef-
ficient. The rate of normals to affected members
in both families is 5 : 16. These findings suggest
recessive monogenic inheritance.

The ABO, MN, Rh blood groups and sali-
vary secretion have been analysed in all members
of both families. No close linkage between these
"markers" and analgesia congenita is apparent.

278

Section 15 — Human Generics

Cytologic studies of peripheral blood from
affected as well as normal members were per-
formed. A defect involving an extra chromosome
was found to be present.

15.26. Deviations from the Mendelian Laws in
Human Families. The Action of Natural Selec-
tion on Blood Groups in Twins. L. E. Nijen-

huis (Amsterdam, the Netherlands).

Previous population studies gave evidence for
strong action of natural selection on blood
groups, which varies more together with the
genome composition than with environmental
factors!1). The explanation could be that cer-
tain genomes provide more viability than others.
Other indications for this phenomenon might be
found in family studies, and especially in twin
studies, because the less viable partner may be
superseded by the other. Dizygotic twin partners
are more often double concordant of discordant
for factors belonging to two different blood
group systems than would be expected.

As to the MN and Rh groups an excess of
10 per cent double discordance and concordance
is observed for the single gametes transferred by
the parents, independent of the way of combina-
tion, i.e. MN and Rh alleles from the same parent
or not. This gives rise to the conclusion that
selection takes place after fertilization. If in the
parents combination heterozygosis exists for
both systems, the excess is 22 per cent. This is
only 12 per cent if both parents are homozygous
for MN or Rh, which indicates that the selection
is exercised on genes linked with the blood group
loci. Excess of double discordance and con-
cordance can also be observed, though in a
lower degree, in non-twin family studies.

1. Nijenhuis, 2nd Intern. Conf. Hum. Genet.,
Rome, 1961.

15.27. Genetics and Linkage Relations of the Lp
Serum Type System. Jan Mohr and Kare
Berg (Oslo, Norway).

The discovery of a serum type system con-
cerning human ^-lipoproteins is reported.

From a material of Norwegian unrelated
individuals and families a hypothesis of deter-
mination by a gene pair Lpa and Lpa is proposed,
the estimates of gene frequencies in the Nor-
wegian population being about 0.19 and 0.81
respectively.

Linkage relations with other marker systems
are considered.

15.28. Linkage Studies in Families with Pathological
Dominant Traits. Walter Scholz (Munster,
Germany).

Linkage studies in man are made more pro-
mising by the availability of an increasing num-
ber of well-suited and well-defined marker genes,
especially in serology. Families showing regular
dominance of pathological traits are best suited
for such investigations. The following antisera
have been used in the present investigation,
anti-A, -B, -AB, -Ai, -A2, -M, -N, -S, -s, -P, -D,
-C, -O, -c, -E, -e, -K, -k, -Fya, and Jkb.—
Haptoglobin types and gamma globulin groups
Gm(a) and Gm(x) were used as additional mar-
kers. Linkage data were received for families
with dominant characters as hereditary mul-
tiples exostoses, telangiectasia hereditaria (mor-
bus Osier), Polydactyly, syndactyly, ectrodactyly
and others. The results of these investigations
will be given in particulars and discussed.

Details will be published in the Z. /. menschl.
Vererb.-u. Konst.-Lehre, 1963.

15.29. Scoring for Linkage Between Several Loci.
Cedric A. B. Smith (London, Great Britain).

The detection and estimation of linkage be-
tween two loci is most simply done by using
Morton's z scores. However, when tests are
made on three or more loci on the same chro-
mosome, as already happens on the X chro-
mosome, new problems arise. Families may be
investigated because of the presence of one
sex-linked character, and then used to deter-
mine the strength of linkage between two
others. This will require the use of new types of
score correction.

15.30. Male and Female Recombination Fractions
in Man. J. H. Renwick (Glasgow, Great
Britain).

The linkage data from 27 pedigrees of the
nail-patella syndrome have been analysed by
the computer programme of Renwick and Schul-
ze (1961). A search has been made for incon-
sistencies in the nail-patella: ABO linkage of the

279

Section 15 — Human Genetics

type (1) that might indicate the existence of a
mimic locus and of the type (2) that might in-
dicate differences (a) between recombination
fractions in men and women or (b) between re-
combination fractions in persons of different
ages. The findings will be discussed. It is hoped
that the analysis will be published in the Annals
of Human Genetics.

15.31. Hereditary Factors in Longevity. M. Hauge,
B. Harvald and B. Degnbol (Copenhagen,
Denmark).

The influence of hereditary factors on the life-
span of the individual has been estimated by
comparing the intra-pair difference of age at
death in 239 monozygotic, 414 dizygotic,
same-sexed and 387 dizygotic, different-sexed
twin pairs. The average differences of age at
death in these groups were 14.5 years, 18.7
years and 20.4 years; the difference between
monozygotic and dizygotic pairs was significant
(0.01 yP >0.001), thus proving that heredity
plays a role as a determinant of the life-span.
The relative importance of heredity compared
with that of the sum of environmental factors
turns out to be increasing within modern so-
ciety, where the environment is nearly optimal,
and environmental differences are unimportant.

15.32. Family Patterns of Mortality and Longevity.

Bernice H. Cohen (Baltimore, U.S.A.).

Two studies wil be discussed: (1) a community
based population study and (2) a special group
study.

The first investigation is designed to deter-
mine whether there are family patterns of mor-
tality and, if so, the nature of those patterns
in terms of age at death, cause of death and
cause-age interaction. For this purpose, a sample
of 550 deaths occurring in Baltimore city to
U.S. born white residents has been selected and
is being matched with living individuals (controls)
of the same sex, race and neighborhood and
U.S. born within 5 years prior to the deceased
probands. The mortality experience of relatives
will be compared with that of the corresponding
family members of the matched living controls.
Interfamilial comparisons of probands dead at
different ages and of different causes will be
examined.

The second investigation deals with "Test
Responses, Aging and Family Mortality
Patterns" in a group, comprising 500 living white

males from 24 to 100 years of age who have
volunteered to participate in a longitudinal
study of aging being carried out by the National
Heart Institute, with periodic physiological,
biochemical, psychological tests and anthropo-
metric measurements. In addition, this investi-
gator is obtaining PTC tests, smoking histories
and detailed family and personal data similar
to that being collected in the community study.
The purpose of this study is to determine whether
there is a correlation between family mortality
patterns and differences in test responses of
living individuals, the latter possibly being
precursors of a genetically determined "death
syndrome" to follow.

15.33. Dominant Inheritance of a Metric Trait in
Man. Italo Barrai (Pavia, Italy).

The mode of inheritance of stature and chest
girth in man has been investigated using measure-
ments taken from 3202 males of average age
of twenty years, whose immediate inbreeding
value was known.

Environmental factors were found to play an
important role in the final phenotypic expres-
sion of the trait stature.

Chest girth shows less sensitivity to social and
environmental factors; the data agree quite
satisfactorily with the hypothesis of an average
dominance of factors determining "large" size.

15.34. Familial Correlations in Human Obesity.
R. F. J. Withers (London, Great Britain).

Obesity has been defined as being 20 per cent
overweight. Parent-child and sib-sib correlations
have been calculated for several populations
of individuals. These include a group of adopted
children. This enables the amount of environ-
mental variance to be calculated. A study of the
body build of the people investigated and fami-
lial correlations of the components of their
somatotype, show that overweight by itself
does not appear to be a reliable phenotype for
use in the genetical study of human obesity.

15.35. The Effects of Parental Blood Groups on
Birth Weight of the Offspring. Henry Gersho-
witz, Atmaram H. Soni and Millicent W.
Payne (Ann Arbor, U.S.A.).

From among a sample of 742 married couples

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Section 15 — Human Genetics

with 1820 children, selected for completed re-
production (wife age 40 or over), complete
birth weight information was secured for 1715
children. Tests performed on the parents of these
children included blood type determinations
for the ABO, MN, Rh, P, K, and Fya systems
and secretor status for ABH blood group sub-
stance. Birth weights for all children were as
reported by the parents.

Since the usual weight differential for the
two sexes (892 males, 823 females) was found
(P<;0.001), all analyses were performed on male
and female children separately. The following
significant effects were noted for male children
only, after correcting for the effect of parity:
(1) paternal main effects: (a) ABO (P<0.025)-
type B fathers have heavier sons, type A fathers
have lighter sons ; (b) MN (/><0.05)— type N
fathers have heaviest sons with M and MN
fathers following in that order; and (c) Rh
CP<0.005) — the paternal gene r seems to con-
tribute most to an increase in birth weight,
while the R2 gene seems to be associated with
a below average weight, and (2) maternal main
effects: confined to the Kell system (P<0.05)—
Kell positive mothers have heavier sons than
Kell negative mothers. Probability values of
5 per cent may be subject to some uncertainty
in view of the number of tests of significance
performed.

The data for female children are being analyz-
ed and will also be presented.

Supported in part by U.S.P.H. Grant H-4145
from the National Institutes of Health and in
part by AEC Grant AT (11-1) 405.

15.36. A Study of Seven Blood Group Systems in
Sterility and Child Mortality. T. Edward
Reed, Henry Gershowitz, and Atmaram
Soni (Ann Arbor, U.S.A.).

A sample of 742 married couples, the wife
being 40 or more years of age, from a town in
Michigan, U.S.A., was studied. All couples were
of European ancestry and the only selection was
for willingness to cooperate. For each couple a
personal, social and reproductive history was
obtained and blood group phenotypes in the
ABO, Rh, MN, Kell, P, and Duffy systems, as
well as ABH secretion, were determined. The
results for two reproductive indicators, (1)
proportion of couples (first marriages, married
for 10 or more years) who were sterile, and (2)
proportion of liveborn children born to these

couples who died non-accidentally under 5 years
of age, are reported here.

Two-way analysis of variance (husband's
group one way, wife's group the other) for each
of the 14 blood group-indicator combinations
yielded the following significant items — Sterility
analysis: MN group of the wife (P <0.005, N
women most sterile), Kell group of the wife
(P <0.025, Kell + wives most sterile); Child
mortality analysis: ABO group of father {P <]
0.001, O fathers highest), P group of mother
(P <0.005, P— mothers highest), P group of
father (P <0.05, P— fathers highest), Rh group
of mother (P <^0.05, R1R2 mothers highest), and
MN group of couple (P < 0.05, interaction
item).

This research was supported by a grant from
the U.S. Atomic Energy Commission. A com-
plete report of this study will be submitted to the
American Journal of Human Genetics.

15.37. (F). Teratology. P. H. Saldanha (Sao Paulo,
Brazil).

1st part: Genetical teratology

The first part of the film describes simple
rules of dominant and recessive inheritance.
As examples of these hereditary patterns, pedi-
grees of achondroplasia and the Laurence-
Moon syndrome are presented.

The chromosomal constitution of the normal
human cell and the classification of chromosomal
types are analysed. The well known chromo-
somal aberrations found in some clinical entities
are presented, including mongolism, Turner and
Klinefelter syndromes. As an example of rare
disease believed to be associated to a 17-18
chromosomal trisomy, a case of Klippel-Feil
syndrome is shown.

The most characteristic clinical aspects of the
patients affected with the syndromes mentioned
above are indicated in detail.

2nd part: Descriptive teratology

The second part of the film intends to classify
and characterize the most representative ano-
malies associated with embryonic development.
Malformation patterns found in anomalies due
to retarded, excessive and faulty development are
exhibited. Anencephaly, cleft lip, heart defects,
sirenomielia, cyclops, xiphopagus and other
monster types are shown as examples.

Surgical correction of hare lip is presented as
a clinical recoverv.

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Section 15 — Human Genetics

15.38. The Torus Palatinus. C. Maximilian, Ioana
Popovici and R. Popescu (Bucarest, Rumania).

Investigations concerning the incidence rate
of the torus palatinus in vaiious races have led
to the conclusion that this structure is a racial
character. This would account for the different
figures recorded in the populations studied.
Hereditary transmission is considered to be either
of the autosomal dominant or of the sex-
linked type.

We have investigated the incidence rate of
this character in our country during the course
of history, from the neolithic age up to the pre-
sent. In order to determine the figures prevailing
at present, we investigated a number of 1000
skulls and two additional series, one intensely
exogamic, and another endogamic one formed of
20 families comprising 156 subjects from an
isolate in the Carpathians. The incidence rate
of the torus was found to range from 26 to
46 per cent, in all the series investigated, with the
exception of the isolated group, in which it at-
tained 75 per cent. These figures are similar to
those prevailing in peri-arctic groups. The for-
mation is already apparent during the first years
of life. Its development is genetically determined.
In the isolate it was often found to be of consi-
derable size (3rd degree) and clearly detectable in
7- or 8-year children. It is not yet clear whether
this large size is determined by a homozygotic
condition or by other causes. The type of trans-
mission appears to be autosomal dominant,
the incidence rates of the character being almost
the same in both sexes.

The part played by heredity was further con-
firmed by the investigations of 7 pairs of mono-
zygotic twins who were all found to be concer-
dant, and of 2 pairs of dizygotic twins one of
which was discordant. Similar observations have
already been published by other authors.

15.39. The Epidemiology of Spina Bifida Cystica.

J. Lorber (Sheffield, Great Britain).

In the Sheffield Children's Hospital a large
unit has been established for the care and study
of infants born with spina bifida cystica and over
100 new cases are seen each year. A detailed and
prospective family survey of over 500 unselected
families revealed a very much higher incidence
of multiple cases in sibships than hitherto re-
ported. In completing the family pedigree one
must not rely on a single interview or on the
evidence of non-medical persons not trained in
genetics. Frequently parents did not know the
cause of stillbirths or of neonatal deaths which

occurred prior to or after the birth of the "index
case". Repeated questioning, enquiry from hos-
pitals and family doctors often disclosed such
additional cases. Further, a continuous follow-
up study of our patients allowed me to study
prospectively the condition of children born
after the index case. Finally, a detailed history is
taken of any congenital malformations which
occurred among cousins and in previous gene-
rations. Data are available now for thousands of
children. These indicate a strong, possibly
recessive genetic tendency. Among many inter-
esting features I have seven families in which
more than 2 siblings were affected. In these
families over 50 per cent of infants born after
the second affected child had either spina bifida
cystica, anencephaly or hydrocephalus.

15.40. A Study of Mental Deficiency on Some
Causes. Yasuhiko Kobayashi, K. Kishi-
moto, M. Matsui, H. Tsuboi, Y. Shiraki,
and K. Nakai (Nagoya, Japan).

We presume that mental deficiency is only a
"symptom name" due to its etiology. Mental
deficiency may be preferably considered to be a
collective name given to independent diseases
from the nosological point of view.
The following points may be considered:

(1) Race

(2) Sex

(3) Polygenes (subcultural group)

(4) One gene (pathological group-which is
less frequent in Japan than in Europe)

(5) Chromosome aberration

(6) Incompatibility of normal genes

(7) Germinal injury

(8) Foetal injury

(9) Birth trauma

(10) Injury in childhood

Mental deficiency is not necessarily due to
only one cause. Mental deficiency may be in
consequence of the dynamic combination of
many causes.

Here, we only make reference to consangui-
neous marriage, whose frequently in Japan is
10-20 times higher than in Europe, and in-
compatibility of blood types (ABO-type, MN-
type and Rh-type: Incompatibility of ABO-
blood type in Japan is possibly more frequent
than in Europe, because O-type in Japan is less
frequent than in Europe).

For the same purpose we also studied electro-
encephalogram and amino acid in urine both on
patient and parents to find a few special forms
of amino aciduria, e.g. histidinuria, etc. We de-

282

Section 15 — Human Genetics

monstrate a few points on which consanguineous
marriage and incompatibility of blood types re-
vealed to be the causes of mental deficiency.
Nosology of mental deficiency is discussed with
further investigation from the point of physio-
logical and biochemical genetics.

15.41. Juvenile Amaurotic Idiocy in Sweden.

Store Rayner (Lund, Sweden).

During the years 1950-1959 I have collected
and personally investigated 37 cases of juvenile
amaurotic idiocy together with their relevant
family data.

The incidence rate for the disease was esti-
mated to be 0.023 per cent, i.e. about 1 per
50,000 with an approximate heterozygote
frequency of 1.0 per cent. The incidence of first
cousin marriages among the investigated parents
of juvenile amaurotic idiots was 9.1 per cent.
This, together with the observed incidence of
juvenile amaurotic idiocy among the sibs of the
propositi (p= 0.26 ± 0.05) confirmed the earlier
hypothesis of a simple recessive and autosomal
type of inheritance.

Hematological investigation of this, the
largest material of juvenile amaurotic idiocy
collected so far, showed that lymphocytic
vacuolation is an early and constant sign in this
disease. The average frequency of vacuolated
cells in the patients was 20.6 ± 1.6 per cent.

Of the parents 95 per cent had vacuolated
lymphocytes when 500 cells were counted. The
frequency of vacuole positive individuals among
the sibs of the propositi was 65 per cent. The
mean frequency of vacuolated lymphocytes per
individual was about 1 per cent.

These results, together with other findings in
the examined relatives and in various groups of
control materials, strongly suggest that the
occurrence of lymphocytic vacuolation in
healthy relatives of patients with juvenile
amaurotic idiocy is an expression of the gene
for this disease in heterozygous condition and
that, in the patients (homozygotes), it is a pleio-
tropic effect of this gene when present in "double
dose"'.

15.42. A Test for the Detection of Latent Carriers of
the Duchenne Type of Pseudo-hypertrophic
Muscular Dystrophy. J. van den Bosch
(Amsterdam, The Netherlands).

In 1958 a Dutch, seventeen-year-old sister of
several brothers, affected with the Duchenne

form of muscular dystrophy of the sex-linked,
pseudohypertrophic and progressive type, was
examined by means of electromyography, in
connection with the genetical advice she had
asked about her progeny. The electroneurolo-
gist (Dr van der Most van Spyk, Utrecht) found
signs of myopathic changes in both her E MG and
in that of her mother. This finding resulted in a
wider-scale research into the significance of this
phenomenum: during 1960, 1961 and part of
1962 the author visited and examined "possible"
and "assumed" carriers for this disease in the
United Kingdom and made tape-recordings of
the electromyographic responses obtained by a
specially for this purpose designed portable
EMG apparatus. The recordings were subse-
quently replayed at the laboratory (The Galton
Laboratory, U.C., London) and the output of
the taperecorder was displayed on the screen of
an double-beam oscilloscope and photographs of
these visible patterns were taken. The results of
the statistical analysis of these recordings form
the subject of this communication.

15.43. An Unusual Pedigree of Hurler's Syndrome.

L. P. Chiasson (Antigonish, Canada).

This study is based on a pedigree of Hurler's
syndrome for which the previously published
explanations — sex-linked genes, and recessive
autosomal genes — appear inadequate. The af-
fected individuals are children of normal
brothers whose normal wives are unrelated to
one another or to their husbands. The results of
investigations designed to detect heterozygous
individuals and/or low expressivity will be
presented, and the genetic implications of these
results will be discussed.

15.44. The Zollinger - Ellison Syndrome as a Partial
Phenocopy of Adenomatosis of Endocrine
Glands. Paul Wermer (New York City,
U.S.A.).

The Zollinger- Ellison syndrome consists of the
simultaneous development of severe peptic
ulcers and of solitary tumors of the islets of
Langerhans. In adenomatosis of endocrine
glands peptic ulcers of the same character occur
together with isletcell tumors, which are always
multiple and exhibit a specific microscopic struc-
ture: several other endocrine glands also show
excessive growth. The Zollinger-Ellison syn-
drome occurs only sporadically. Adenomatosis

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Section 15 — Human Generics

is a familial disease with autosomal dominant
inheritance.

The tumors in adenomatosis and in some other
inherited deseases always grow in multiple
fashion in the affected organs. In view of the
great regularity of this finding the presence of
multiple tumors in one organ and/or of symme-
trical tumors in paired organs can be used as a
priori evidence of their genetic origin. It is
assumed that tumor-multiplicity comes about
by necessity: it presumably results from the
action of a growth-promoting gene endowed
with pleiotropism of cell-reaction (Hadorn). The
insulomas in the Zollinger-Ellison syndrome are
not inherited and are therefore solitary like other
not genetically determined tumors. Their micro-
scopic picture likewise sets them apart from the
inherited tumors of adenomatosis, which is true
of solitary isletcell tumors in general.

From the genetic point of view the Zollinger-
Ellison syndrome appears as a partial pheno-
copy of the inherited syndrome of adenomatosis
of endocrine glands.

15.45. Hereditary Hyperparathyroidism.

E. Jackson (Bluffton, U.S.A.).

Charles

The purpose of this paper is to present four
families with hyperparathyroidism seen at our
institution in the past six years — the first with
9 members affected in two generations, the
second with a father and son affected, the third
with a woman and her great nephew affected and
a fourth with 5 members affected in two gener-
ations. These pedigrees and those in the literature
suggest an autosomal dominant type of in-
heritance.

The finding of other endocrine adenomata in
members of 2 of these families has emphasized
that hereditary hyperparathyroidism is most
likely a part of the syndrome termed hereditary
endocrine adenomatosis.

The ascertainment of several asymptomatic
cases in these family studies led to the speculation
that hyperparathyroidism might be more com-
mon than had been realized in the past. To test
this possibility, routine serum calcium deter-
minations were performed on 12,000 consecutive
patients coming to our general medical clinic
with the finding of 10 cases of hyperparathy-
roidism (an incidence of almost 1 in 1000). This
again illustrates how genetic studies help provide
data on the true incidence of certain diseases.

During this 6-year period only 10 other index
cases have had parathyroid adenomas removed
at our institution. Since only 11 other families
have been reported in the literature, the finding

of these 4 families in our small series suggests
that heredity is a more important factor in the
etiology of parathyroid adenomas than has been
recognized previously.

15.46. Albright's Hereditary Poly-osteo-chondro-
dysplasia ( Pseudo-pseudohypoparathyroidism).
Luc Goeminne (Ghent, Belgium).

In 1942 Albright described a syndrome with
biological and clinical findings characteristic of
hypoparathyroidism but refractory to parathy-
roid extract. The syndrome was labelled pseudo-
hypoparathyroidism (PHP) because the target-
organ (renal tubule) fails to respond adequately
to parathyroid hormone.

The patients show a typical morphologic
syndrome, various dyschondroplastic anomalies,
tissue-calcifications ossifications, exostoses and
trophic disturbancies (teeth, eye-lens, skin)
(67 cases reported in 1960, first familial cases
published in 1950).

In 1952 Albright described a syndrome without
symptoms or biological findings of tetany, but
with the same somatic features, named pseudo-
pseudohypoparathyroidism (PPHP) (50 cases
known in 1962, first familial cases reported in
1957).

Symptoms are: short size, obesity, round
face, brachydactyly.
We describe a family with

1. 1 case of PPHP, diabetes, hypertension,
polyarthritis, arteritis (femoral artery
ectopie calcifications).

2. 1 case of PPHP with hypertension.

3. 1 case of PPHP with polyarthritis.

4. At least 4 other cases of PPHP and 2 PPHP
"formes frustes".

This is the second observation in Belgium after
the 3 cases described by Nagant and Hoet, in
1960.

Our pedigree is the first in the medical litera-
ture where more than 5 cases of PPHP are found
in 4 successive generations.

The association of PPHP with diabetes
(25 per cent), hypertension (25 per cent),
hypothyroidism (15 per cent), polyarthritis
(15 per cent) and gonadal dysgenesis (25 per cent)
is probably significant.

Hypertension and diabetes might arise from
diffuse calcifications and clerosis of renal and
pancreatic arteries (?).

McKusick, Uhr and Bezahler (1961), Mann
(1962) and Schwartz (1963) suppose a sex-linked
incomplete dominance. Our extensive pedigree
suggests, (yet with one exception of a clear male-
to-male transmission), this mode of inheritance

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Section 15 — Human Genetics

with a varying expressivity and weak specificity
(polypheny).

15.47. An Apparently Sex-linked Heredo-degenerative
Disease of the Central Nervous System
combined with Leber's Optic Atrophy.

L. N. Went and G. W. Bruyn (Leyden,
the Netherlands).

The index case of the family to be reported here
was suffering since his 7th year from a here-
dodegenerative disorder for which he has been
admitted 6 times and which has been diagnosed
on several occasions as spastic paraplegia.
In addition he experienced on acute bilateral
decrease of vision when 36 years old; pale discs,
central scotomas and a vision of 1/60 ODS were
found.

His family could be studied over 7 generations
and revealed in addition 5 persons with both
abnormalities, 6 with optic atrophy only and
6 with the neurological disorder only. All
patients were males, while their clinical picture
was almost identical. Clinically it seems likely
that a basal ganglia disease is responsible for the
neurological findings. The only female family
member affected (who had an affected brother
and an affected son) was clinically very different
from the others and had actually been diagnosed
as a case multiple sclerosis.

The distribution of the affected patients over
the family is such that it seems justified to
conclude that only one gene, most probably on
the X-chromosome, is responsible for both the
neurological and ophthalmological abnormali-
ties.

Chromosomal and linkage studies and ex-
tensive biochemical investigations have been
undertaken or are under way and will be reported
upon. Our findings will be published in detail
elsewhere.

which some cases belong to Bardet-Biedl
syndrome, congenital tapeto-retinal amaurosis
of Leber, retinitis punctata albescens and fundus
albipunctatus cum hemeralopia. About 90 per
cent of those cases are transmitted by the
recessive mode of inheritance, and 9 per cent are
dominant. One family only presents a recessive
sex-linked transmission of retinitis pigmentosa.
In this family, the female heterozygotes show
the tapeto-retinal reflex which was never ob-
served before in Switzerland. If we keep out the
cases of fundus albipunctatus, because of its
special clinical character, and the cases of
Bardet-Biedl syndrome, we find in 5 cantons
101 living persons suffering from recessive
pigmentary degeneration. This fact allows us to
estimate the frequency of this affection at 1 :7000,
the gene frequency at 1/84 and the frequency of
the heterozygotes at about 1/42. 10 per cent of
these 101 cases are issued from unions between
first cousins.

Among the hereditary affections associated
with tapeto-retinal degeneration, congenital
deafness is known as the most frequent; indeed
we discovered this type of association in about
13 per cent of our material, while psychiatric
disorders as oligophrenia and psychoses do not
seem to be more frequent in the affected people
than in the general population. The Bardet-
Biedl syndrome was observed in four patients out
of a population of 700.000 inhabitants.

The great diversity of clinical forms of the
tapetoretinal degeneration raises the problem
of heterophenia of tapeto-retinal degenerations.
Indeed, the study of an isolate in the canton of
Valais allowed us to demonstrate a phenotypical
alternation between different types of pigmentary
degeneration within several branches of a large
family issued from the same ancestor. This
pedigree comprises 7 cases of peripheral tapeto-
retinal degeneration in 2 sibships, 5 cases of
macular degeneration in 5 sibships and 3 cases
of fundus albipunctatus cum hemeralopia in 2
sibships.

15.48. (D.). Genetic and Epidemiological Investiga-
tions on Pigmentary Degeneration of the Retina
in Switzerland. F. Ammann, D. Klein and
A. Franceschetti (Geneva, Switzerland).

Since 1959 we are dealing with a genetic and
epidemiological investigation on all types of
pigmentary degeneration of the retina in
Switzerland. We relate here the results obtained
until now in about 10 of 22 cantons.

Our material is composed of the following
clinical types of pigmentary degeneration:
typical and atypical retinitis pigmentosa out of

15.49. A Pedigree of Reccessive Deaf Mutism from
Orissa, India. K. R. Dronamraju (Orissa,
India).

A pedigree of Bengali Kayastha settlers in
Orissa, India was investigated for the inheritance
of hereditary deaf mutism. This is a small
endogamous community in which consangui-
neous marriages are scrupulously avoided. The
propositus was a female congenital deaf mute
aged 17 years and was a student at the School
for the Deaf and Dumb in Bhubaneswar,

285

Section 15 — Human Genetics

Orissa. The pedigree contains 182 people in 5
generations and has 5 sibships with at least one
affected member. Two of these sibships are
doubly related, the parents of each, though not
known to be related, being first cousins of the
parents of the other. All the 7 female and 4 male
deaf mutes recorded in this pedigree are the
progeny of non-consanguineous matings between
unaffected people. Three marriages of deaf mute
females to unaffected males produced six
unaffected sons. The evidence suggests that the
deaf mutism represented expression in the homo-
zygote of one of the autosomal recessive genes.

15.50. Studies in the Aetiology of Congenital Deaf-
ness. George R. Fraser (London, Great
Britain).

A series of 2355 children attending special
schools for the deaf in the British Isles was
studied. A clinical examination was performed
and a full family history obtained from the
parents. On the basis of data thus collected
together with a consideration of audiometric
patterns, the children were classified into aetio-
logical categories.

In approximately 33 per cent of cases the
deafness was due to acquired disease in early
life, usually meningitis, and in 17 per cent it was
due to exogenous pre- and perinatal causes, the
most common of which were maternal rubella
and neonatal jaundice.

Analysis of the data revealed that simple
Mendelian inheritance could account for the
deafness of the remaining 50 per cent. Recessive
types were most common but dominant and
sex-linked varieties were also present. Thus, in
12 per cent of this hereditary group the deafness
was inherited in a dominant and in 1 per cent in
a sex-linked manner.

Three recessive syndromes were celarly
different iable clinically. Thus, deafness was
associated with goitre (Pendred's syndrome)
in 10 per cent of this hereditary group, with
retinitis pigmentosa (Usher's syndrome) in
2 per cent and with unique ECG abnormalities
in 1 per cent. In the dominant group, Waarden-
burg's syndrome (white forelock, heterochromia
of the irides, dystopia of the medial canthus of
the eyelid, congenital perceptive deafness) was
frequently seen. In many other families with
dominant deafness, lesser anomalies of pigmen-
tation were encountered.

In 2 per cent of the entire group deafness was
associated with congenital malformations, in
some cases familial. While the Klippel Feil and

Treacher Collins syndromes were the most
common in this group, some of these malfor-
mations were unique.

15.51. Hereditary Hearing Impairment and Related
Anomalies. H. W. Kloepfer, Jeannette
Laguaite, and J. W. McLaurin (New
Orleans, U.S.A.).

This is a preliminary progress report on
genetic, audiological and clinical data collected
over a period of five years from approximately
500 individuals who were selected from 2000
persons distributed in some 250 family units
in which deafness occurred at least in one parent
or offspring. Through a study of 4000 ancestors
of these families specific relationships between
family units and common ancestors can be
traced. Although all individuals have been placed
on 126 major pedigree charts, an IBM computer
program has been developed which will create
the information on a 9-generation pedigree
chart, find and evaluate each ancestor in terms
of association with family units in common, and
group family units according to the most
probable common genotype.

At this stage of the study it would seem that
at least three independent autosomal recessive
genes (one later in life causing in addition retinitis
pigmentosa and cataract) account for the dear-
ness in most instances in a three-parish-area of
Louisiana in which all known cases of deafness
have been ascertained. However, hearing loss
was encountered in one kindred which is caused
by an autosomal dominant gene associated with
ear anomalies, brachial clefts, and ear pits. A
more detailed report will be given on this kindred.

Supported by NIH Grant 22586.

15.52. Deafness Associated with Split Hands and Feet
in Two Siblings. A New Syndrome? L. S. Wil-
dervanck (Groningen, the Netherlands).

Two brothers, pupils of the Royal Institute of
the Deaf, Groningen, Holland, are suffering from
a perceptive deafness with a hearing loss of 60 to
80 db. An older brother and the parents, who are
not consanguineous, are hearing well. There is no
family history of deafness. Moreover the two
boys show split hands and feet, associated with
syndactyly. This anomaly is not known in the
family. Two sisters of the mother and a son of
one of them show a stiff thumb (radiographs

286

Section 15 — Human Genetics

normal). It is highly improbable that these,
apparently fibrous stiffness, is related to the
syndrome. We have to take account of a possible
recessive transmission of a mutated gene. The
most common form of hereditary deafness is a
recessive one. Splithand and foot nearly always
follows a dominant mode of transmission, only
very few (doubtful) recessive cases are known.
The incidence of recessive deafness in Holland is
1 : 5000, splithand and foot occurs in 1 : 100,000.
These figures make it highly improbable that
the association of the two abnormalities is
fortuitous. Being an exogene causation of the
occurring together of the two anomalies in two
brothers highly improbable, I think the most
reasonable explanation is a rare recessive gene
giving rise to the syndrome. Only Birch Jensen
saw one sporadic case and an inquiry to the
other institutes for the deaf in Holland did not
reveal any more cases.

15.53. Inherited Multiple Neoplasia Syndrome.
Eldon J. Gardner (Logan, U.S.A.).

A syndrome including multiple or diffuse in-
testinal polyposis, osteomas, fibromas, epidermal
cysts, and dental anomalies, has been investigated
over a period of 15 years in a Utah family group.
The most serious aspect from the standpoint of
the family under consideration was the polyposis,
known to predispose to carcinoma of the colon
and rectum.

When the study began in 1948, carcinoma
originating in the lower digestive tract had been
the cause of eight deaths among the descendants
of one woman. All 51 living descendants of the
woman were examined. Six were found to have
intestinal polyposis and other manifestations of
the syndrome.

Two cases of fibrosarcoma have been added
to the list of manifestations in people who
evidenced other aspects of the syndrome. While
the family members have been under observation,
two children have developed multiple polyposis
and five others have expressed epidermal
cysts and/or fibromas. These latter types of
lesions appear earlier in life than intestinal
polyps and therefore serve as diagnostic traits for
identifying children who may be expected to ex-
press the more serious aspects of the syndrome
later in life.

Among the children who have lived long
enough to be identified as having or not having
the syndrome and who had one parent who
expressed the syndrome, 20 were positive and
16 were negative. This observation is not

significantly different from the 1 : 1 ratio expected
in cases of single gene dominant inheritance.

15.54. Neoplastic Diseases in Twins. Richard H. Os-
borne and Frances V. Degeorge (New
York, U.S.A.).

This first report of a comprehensive study of
twins in the hospital and clinic populations of the
Memorial Sloan-Kettering Cancer Center, New
York, will introduce the methodological pro-
blems inherent in twin-cancer studies, and
present the variable distribution of twins in
this patient population.

In theory twin studies constitute a simple and
an effective method for investigating the human
cancer problem. In actual practice, however, it
is found that the application of a standard twin
study method results in untenable assumptions
concerning the probable nature of human
cancer. The variable distribution of 166 twin-
born patients in different benign and malignant
diagnostic categories relative to that of single-
born patients demonstrates the unique nature of
the twin-cancer problem and the possibilities
offered by twin study methods for investigating
neoplastic diseases.

15.55. Genetic Factors in Hypophysial Tumours.
H. Hamlin (Boston, U.S.A.).

A statistical relationship between O and A
blood group genes and chromophobe adenoma
of the pituitary has prompted search for genetic
determinants in other types of hypophysial
tumor. There is the chromophil (eosinophil)
adenoma that produces the extraordinary
clinical condition of acromegaly; also the pitui-
tary giant, usually associated with a mixed type
of chromophobe and chromophil hyperplasia.
Such abnormally overgrown specimens of
humanity are counterpoised by the unique
pituitary dwarfs who are usually well-proportion-
ed physically and often superior mentally.
Genetic factors are strongly suggested in the
hypophysial duct tumor (craniopharyngiona)
because of its clearly established patho-embryo-
genesis. Experimental data is cited to suggest that
estrogen-induced pituitary adenomata have
shown genetic linkage in certain Fi-hybrid mice
strains. Examples of human hypophysial neo-
plasia are presented to indicate that constitution-
al factors should always be suspected in these
disorders.

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Section 15 — Human Genetics

15.56. Genetic Variation and Leprosy. S. G. Spickett
(Cambridge, Great Britain).

The causative organism of leprosy is Mycobac-
terium leprae. There is evidence that some human
individuals are resistant to parasitism by this
pathogen. Susceptibility to leprosy would
appear to be determined by a single irregularly
dominant gene. The frequency and penetrance of
this gene varies between populations.

Leprosy may be manifest in several forms, the
relative frequency of these forms shows striking
variation between populations. There is more-
over a significantly higher concordance between
monozygotic than between dizygotic twins in
the form of leprosy suffered. This and other
evidence suggests that the genotype of the human
host is of importance in determining the course
of the disease. Evidence of higher concordance
between unrelated contacts than random expec-
tation and the failure to find any environmental
factor to account for this, is suggestive of an
effect of the genotype of the pathogen on the
course of the disease.

The history of epidemic leprosy is consistent
with there being genetic control of incidence
and form of leprosy by both host and pathogen.

Current studies indicate that drug resistance is
increasing in pathogen populations with the
risk that the control of leprosy may become more
difficult in the future. Moreover, leprosy is
increasing in some populations that have been
free of it for many generations. It is probable
that this is associated with a rise in the gene
frequency for susceptibility (in the absence of
selection pressure against it) coupled with the
greater chance of contact with the disease that
is associated with the greater mobility of indi-
viduals between populations.

15.57. The Role of Genetics in Diabetics. Nancy

E. Simpson (Toronto, Canada).

Analysis of the genetics of diabetics will be
presented from data obtained from a voluntary
register of diabetics in Canada sponsored by the
Canadian Diabetic Association. For the past
two years diabetics have been asked to complete
questionnaires which contain detailed infor-
mation regarding their immediate relatives
(parents, sibs and offspring).

The register will be a continuing one in
which respondents will be asked to keep the
information regarding their families up to date.
Ultimately a history for three generations of
families in which there is at least one diabetic
will be available for genetic analysis. These sort

of data have long been needed to establish
the genetic role in the etiology of this condition
with variable age at onset.

From the register, the proportions of diabetics
among offspring of two diabetic parents and one
diabetic parent (selected through the matings)
are compared with each other and a suitable
population control. The ages at onset and
treatment of the diabetics are taken into con-
sideration. The data suggest that diabetes is not
genetically homogeneous. The results lead to
interesting speculations on the role of genetics
in diabetes and to discussion of future advantages
of the register when complete histories for the
generations become available.

15.58. The Natural History of Hyperuricemia and
Gout. M. T. Rakic, H. A. Valkenburg,
R. T. Davidson, J. P. Engels, W. M. Mikkel-
sen, J. V. Neel and I. F. Duff (Ann Arbor,
U.S.A.).

To determine with more precision the natural
history of hyperuricemia and gout, the families
of 19 proposity (a total of 99 relatives) studied
in 1938-42 or 1946 were reinvestigated in 1961-62.
Nine of the propositi (all still with signs and
symptoms of their disease) and 262 relatives
(69 from the first study, the remainder seen for
the first time) could be examined. In the period
of follow-up gout was present in 14 male
relatives and 6 female relatives. The mean age of
onset gout was in males 39 years and in females
54 years (P < 0.005).

Thirty-five male relatives and 34 female rela-
tives were seen in both studies. Eleven of the 26
male relatives with normal serum uric acid levels
in the previous study developed hyperuricemia
as determined by the colorimetric method and
defined as a level in males of 6.0 mg per cent or
greater and in females of 5.0 mg per cent or
greater. Of these 11 two had developed gout. Of
the 9 hyperuricemic male relatives in the first
study 1 had developed gout. Five of the 9 adult
males with a normal serum uric acid level in the
previous study apparently developed hyperuri-
cemia in the interim. Eight of the 27 female
relatives with normal serum uric acid levels in the
previous study now had hyperuricemia and two
of these had gout. Hence gout developed in equal
numbers in these 35 male and 34 female relatives.
No significant differences existed between the two
sexes regarding the incidence and development
of hyperuricemia in the previous and the present
study. These apparent similar findings could be
explained by a significant difference in mean age
at the time of the first study, the male relatives

288

Section 15 — Human Genetics

being 21.7 years and the female relatives 36.3
years. No differences in mean values and mean
age could be found between the male relatives
who stayed normal during the period of follow-
up and those who became hyperuricemic.

On a NATO Science Fellowship supported by
a grant of the Netherlands Organization for the
Advancement of Pure Research (Z.W.O.).

15.59. Carbohydrate-Induced Hyperlipemia in Child-
hood. S. Jakovcic and David Yi-Yung-Hsia
(Chicago, U.S.A.).

The present paper will describe the biochemical
studies undertaken in a hyperlipemic family,
where 3 of 13 children are affected. The parents
of these children are second cousins.

The children were first placed on a high
carbohydrate diet containing 15 per cent protein,
10 per cent fat and 75 per cent carbohydrate.
During this period the triglycerides increased
from 1670 to 2700 mg per cent and the lipo-
protein lipase remained low with levels of 0.07-
0.13 uEq/ml/min. They were then placed on a
high fat diet containing 15 per cent protein,
40 per cent fat (half corn oil and half cream),
and 45 per cent carbohydrate. During this
period, the triglycerides decreased from 2700 to
605 mg per cent and the lipoprotein lipase reverted
to normal levels of 0.22-0.33 nEq/ml/min. These
data indicate that the children had hyperlipe-
mia of the carbohydrate-induced variety and
not of the fat-induced type. Heparin sensitivity
was also tested and all three siblings showed
more than 25 per cent of clearing of the serum
measured as triglycerides in mgm. per cent.

Plasma from the affected children were se-
parated by ultracentrifugation into the chylo-
microns (< 1.006), the low density lipopro-
teins (< 1.019) and the low density lipoproteins
(1.019-1.063). Each of the fractions were ana-
lyzed for triglycerides, phospholipids and
cholesterol.

Finally, studies were carried out on the parents
and unaffected siblings. While all showed lipo-
protein lipase within the normal range, both
parents showed delayed clearing of triglyce-
rides up to 24-48 hr after a 90 g fat meal.

15.60. A Dual Hereditary Red Cell Defect in One
Family: Hypocatalasemia and Glucose-6- Phos-
phate Dehydrogenase Deficiency. A. Szeinberg,
A. De Vries, J. Pinkhas, M. Djaldetti and
R. Ezra (Tel Aviv, Israel).

Severe hemolytic anemia with sulfhemoglobi-
nemia developed in an Iranian Jew following
contact with a fungicide-zinc ethylene bisdithio-
carbamate. The previous history of the patient
and his family history did not reveal hemolytic
occurrences. Examination of his red blood cells
demonstrated a fully expressed deficiency of
glucose-6-phosphate dehydrogenase (G6PD), as
well as a severe hypocatalasemia (about 8 per
cent of mean normal catalase activity).

Investigation of members of three generations
in this family suggested that the two enzymatic
defects were transmitted independently. The
hereditary pattern of the catalase deficiency was
compatible with transmission by an autosomal
gene of intermediate dominance. In the homozy-
gous state (the propositus) the abnormality
resulted in severe hypocatalasemia, but not in
complete absence of this enzyme. The presence
of slight catalase activity in his red cells was
confirmed by starch gel electrophoresis followed
by specific staining. The heterozygous state
was characterized by intermediate hypocatalase-
mia (50-65 per cent of mean normal catalase
activity). Such intermediate catalase activity was
detected in all the children of the propositus as
well as in several other members of the family
both in those with normal G6PD and with G6PD
deficiency. No difference in the degree of hypo-
catalasemia was observed between those two
groups of subjects heterozygous for catalase
deficiency, suggesting absence of interaction
between these two enzymatic defects.

These investigations were supported in part by
grants from the U.S. National Institutes of
Health, Bethesda (A. 2740), and from the Rocke-
feller Foundation (RF60101).

15.61. Galactosemia and Mongolism in the Same
Family. Brigitte Bahrs and Wolfram
Ostertag (Miinster, Germany).

A family with four children, one of them with
mongolism and the other with galactosemia,
have been investigated. Following modified
manometric methods developed by Kirkman
and Bynum (1959) and by Schwarz, Holzel
et al. (1961) we were able to distinguish the
heterozygous carriers from normal controls by
measuring the activity of the enzyme Gal-l-P
uridyl transferase. All of the children— with the

289

Section 15 — Human Genetics

exception of the galactosemic child — as well as
the parents were heterozygous carriers.

The biochemical as well as the cytological
results of this investigation of linkage relations
will be discussed.

sary, but this is true also of diabetics who re-
quire insulin.

While the "U" series of drugs do not constitute
a cure for mongolism, they do allow mongoloids
to lead a more normal life and point the way to a
genetic solution of the problem.

15.62. Down's Syndrome: A Genetic Abnormality
Improved by Medication. Henry Turkel
(Detroit, U.S.A).

In 1866, both Gregor J. Mendel brought out
his theory of heredity and Dr. Langdon Down
coined the name, mongoloid, for a particular
group of patients, it was not until 1958 that Dr.
Jerome LeJeune pointed out their relationship in
discovering the Trisomy 21.

And in 196J Dr. LeJeune said: "Cytogenics
must not be restricted to genetic counseling.
Even more important than the mapping of
various traits would be the establishment of
solid relationship between cytogenics and bio-
chemistry, leading to the possibility of chemical
correction of inborn errors."

Though various abnormal chemicals have
been found in mongoloids, none have been
pinpointed as the real cause of the various in-
born structural and functional anomalies
characteristic of mongoloids.

Mongolism appears to be a complexity of
problems, but it is like phenylketonuria, galac-
tosemia, diabetes, etc., in that it is a part of the
group of conditions which are inborn. In the
case of, for example, phenylketonuria, the
mother is capable of protecting the fetus, and the
fetus does not begin abnormal development
until after birth. However, in the case of the
mongoloid, the mother is incapable of protecting
the fetus, thus, the mongoloidal fetus is born
with a group of abnormalities which have al-
ready passed into secondary and tertiary stages,
thus, piling abnormalities upon abnormalities.

The treatment for the improvement of the
mongoloid is based upon assisting the mongo-
loid to build through normal means a more
normal manner of growth, removing the se-
condary and tertiary outgrowths of the original
inborn anomalies. This should enable the genetist
to pinpoint the inborn anomalies more easily
with the hope that future findings will dictate a
correction.

Though the use of the "U" series of drugs
does not enable the physician to change the
chromosomal pattern, he can still alter the resul-
tant abnormalities and bring the mongoloid
into the sphere of normal society. Life-long
medication of maintenance doses may be neces-

15.63. Serum Phenylalanine Levels in the Newborn.

David Yi-Yung Hsia, J. D. Berman and
H. M. Slatis (Chicago, U.S.A.).

Phenylketonuria is a hereditary metabolic
disease caused by a deficiency of the enzyme
phenylalanine hydroxylase. The mental retarda-
tion in this condition can be effectively prevented
if treatment with a diet low in phenylalanine
content is started very early in infancy. Two
approaches have been proposed for routine
screening of newborns for phenylketonuria: (1)
testing of urine for phenylpyruvic acid and (2)
determining phenylalanine levels in the serum.
The first has the disadvantage of not being
reliable until 4-6 weeks of age. Methods for the
second have until now only been semi-quantita-
tive based upon the inhibition of Bacillus subtillis
by beta-2-thienyalanine in a minimum culture
medium.

The present paper will describe a survey of
serum phenylalanine levels in 4000 newborn
infants. In each instance, 0.1 ml of capillary
blood was obtained from a single heel puncture
and the serum phenylalanine level determined by
the fiuorimetric method of McCaman and
Robins(1). The data will be analyzed in terms
of sex, age, race, parity, maternal age, and
birthweight, and compared with observations on
known phenylketonuric infants followed in our
own Clinic and from other centers.

Since one laboratory technician can perform
up to 100 determinations of serum phenylalanine
daily, this approach using a quantitative method
should serve as the best means of screening for
phenylketonuria during the newborn period.

1. /. Lab. & Clin. Med. 59, 885, 1962.

15.64. Detection of Carrier State for Phenylketonuria
in Familial Genetic Inquiries. J. Dodinval-
Versie, P. Dodinval, R. Malchair and P.
Moureau (Liege, Belgium).

For a few years, it has been possible to

290

Section 15 — Human Genetics

avoid or improve phenylketonuria or phenyl-
pyruvic idiocy when the children affected with
this recessive hereditary disease are fed with a
phenylalanine-poor diet from the first months of
life. Therefore it is very important to detect this
disease at its very beginning by means of a 10
per cent ferric chloride solution added to the
urine. If there is any phenylpyruvic acid, the
yellow color of the urine becomes rapidly and
intensively green.

Moreover, as soon as the disease is discovered
in a family, it would be desirable, on the one
hand to determine the other diseased persons
and, on the other hand the people who are
carrying the deleterious gene (heterozygotes) and
are likely to pass on the disease to their children
if they marry another heterozygote.

Using the microbiological process, elaborated
by Knox and Messinger, to determine the fasting
phenylalanine level in blood plasma, the authors
were able to find out with sufficient accuracy the
gene carriers in the successive generations of
three families where a phenylpyruvic idiot was
first seen.

The average fasting level of plasma phenyla-
lanine was found to be at 80.33 micromoles per
liter, ± 9.67 with normal individuals where as
the average level with carrier people amounted
to 104.47 micromoles per liter, ± 10.88.

The meaning of the intermediate values be-
tween these two figures can be better estimated by
an oral phenylalanine load test on some in-
dividuals.

groups and the influence of sex, age, body weight,
etc., upon the segregation between the two
groups.

15.65. Phenylketonuria: Further Studies on the
Heterozygous Carrier. William F. Rowley,
Margaret E. O'Flynn, and Paul Wong
(Chicago, U.S.A.).

The present paper will describe a series of
studies carried out using more sensitive and
accurate fluorimetric methods for the assay of
phenylalanine and tyrosine in the serum.
Phenylalanine levels were determined on 0.05 ml
serum using a modification of the fluorimetric
method of McCaman and Robbins.f1) Tyrosine
values were obtained from 0.02 ml serum from a
procedure adapted from that suggested by
Waalkes and Udenfriend.(2)

One hundred parents of phenylketonuric
children and an equal number of controls were
given 0.1 gm per kg L-phenylalanine by mouth.
Blood samples were obtained at 0, 1 , and 2 hours
after the load and analyzed for both phenylala-
nine and tyrosine. The data are analyzed in terms
of the best discriminant for separating the two

1. J. Lab. & Clin. Med. 59, 885, 1962.

2. J. Lab. & Clin. Med. 50, 733, 1957.

15.66. Frequency of Phenylketonuria.

Robert Guthrie (Buffalo, U.S.A.).

The principle upon which the "inhibition as-
say" is based can be applied to the detection of
various "inborn errors of metabolism"^1)
In one application, a microorganism and a
specific inhibitor are incorporated in a layer of
agar culture medium. Upon the surface are
placed rows of paper discs punched from filter
paper containing dried blood spots or urine.
After incubation, the diameters of the growth
zones for the unknowns are compared to dia-
meters of zones surrounding "control" discs.
For example, azaserine and Bacillus subtilis can
be used in this way to detect histidinemia. For
phenylketonuria, B-2-thienylalanine and Bacillus
subtilis spores are used to test dried spots of
blood collected upon filter paper by heel punc-
ture of newborn infants before leaving the hos-
pital nursery. A brief description of this technic
will be presented.

With the demonstration by the Massachusetts
Department of Health that this method is prac-
tical for routine screening for phenylketonuria
in newborn infantsC2) it will now be possible
for the first time to estimate the true frequency
of this disease within the populations screened.
The first 74,000 tests carried out by forty labora-
tories in 29 states (including Massachusetts)
have detected seven cases. The Massachusetts
Department of Health has detected five of these
cases in that State among the first 27,000 new-
born infants tested. At the present time (Febru-
ary 1963), 25,000 total tests are conducted per
month, and the rate of testing is increasing
rapidly. A recent progress report of this program
will be presented. It appears that the frequency
of this disease may be greater in the populations
so far screened than the highest frequency
usually given, 1/20,000. An estimate of the fre-
quency of this disease will be attempted from the
latest results available.

The "inhibition assay" method of testing will
detect PKU cases in which the urine does not
react to ferric chloride, as in young infants.
However, this condition is known to exist
also in some adults ("PKU without PKU"),
but their frequency is unknown. This type of

291

Section 15 — Human Genetics

PKU case may account, at least in part, for
the higher frequency we are encountering with
this method. This possibility will be discussed.

Work supported by grants from the National
Association for Retarded Children, Association
for Aid for Crippled Children, the National
Foundation, United States Public Health Ser-
vice, Grant Number B-1960, and the Children's
Bureau.

1 . Proc . London Conf. Scientific Study of Mental
Deficiency, 1962, Vol. 2, pp. 672-677.

2. New Eng. J. Med. 267, 1208, 1962.

15.67. Pharmacogenetic Aspects of Taste. Roland
Fischer, Frances Griffin (Columbus, U.S.A.)
and A. R. Kaplan (Cleveland, U.S.A.).

parents of children affected with Down's syn-
drome. In the maternal sample the antimode,
dividing the age of younger mothers from that
of older ones, coincides with the threshold anti-
mode dividing tasters from ("non"-)tasters of
PROP. In addition, all but one of the fathers in
the sample can be characterized as markedly
insensitive ("non"-)tasters of PROP.

1. Fischer, R., Griffin, F., and Mead, E.:
Med. exptl. 61, 177 (1962).

2. For taste testing procedure and criteria,
see Fischer, R. and Griffin, F. : Proc. Hlrd

World Congr. Psychiat. June 1961, Montreal,
Vol. 1, p. 542).

3. Nature 195, 362 (1962).

4. Nature 179, 1074(1957).

Very insensitive ("non"-)tasters of HN-c = S
type goitrogens, such as 6-n-propylthiouracil
(PROP), are also insensitive tasters(1) of an
apparently unlimited series of chemically un-
related compounds*2). Differential taste pro-
files for 34 such compounds, mainly drugs,
will be presented and the stereospecificity of
chemoreception described. Sensitive tasters of
PROP can taste 8-times less 1 -quinine (sulfate,
chloride, or free base) — concentrationwise —
than d-quinine, whereas the reverse is true only
for quinine chloride in the case of very insensitive
("non"-)tasters of PROP who constitute about
10-12 percent of a Caucasian population. Taste
testing practice, extending for months, with
mixtures of PROP and quinine — under experi-
mental conditions described by Rubin, Griffin,
and Fischer(3) — can abolish this difference and
make a "learned" taster from a very insensitive
("non"-) taster. This phenomenon can be related
to Beckett and Anderson's model of "footprint-
ing" on stereoselective adsorbents. (4)

If the human oral cavity is regarded as a
pharmacological preparation /'// situ, taste
thresholds for drugs — alone or in mixture —
and drug activity can both be treated as ana-
logous pharmacological responses. By plotting
the taste thresholds of certain molar ratios of
two drugs — in subthreshold concentrations —
isoboles can be obtained which may be inter-
preted as a characteristic systemic response of a
particular subject to a binary mixture of drugs.
Sensitive and insensitive taster (drug responders)
appear to produce different isoboles for the
same pair of drugs.

Another pharmacogenetic aspect of taste is
illustrated by the following observation in 40

15.68. Reexamination of Genetic Aspects of Taste
Thresholds for Thiourea-type Compounds.

Arnold R. Kaplan, Wilma Powell, Ro-
land Fischer and Roger Marsters (Cleve-
land, U.S.A.).

Monozygotic and same-sex dizygotic twins
have been classified according to their taste
thresholds for 6-n-propylthiouracil (PROP) and
various other compounds. Zygosity determina-
tions are based on combinations of morphologic
characteristics and blood types.

A modified Harris-Kalmus testing procedure
has been employed — using distilled water for
the serial dilutions, placebo, and between-
sampling mouth rinses. Other taste-testing me-
thodologies have also been tested, and the
latter yield inconsistencies in the data which
did not occur with the modified Harris-Kalmus
procedure.

Ninety-four monozygotic twins have thus far
manifested 1 00 percent concordance for the 'sensi-
tive taster' vs. 'insensitive ("non"-)taster' dichot-
omy. These data contrast significantly with the
low concordance observed in same-sex di-
zygotic twins. Furthermore, using a series of
fourteen dilutions, all the above monozygotic
twin pairs manifested taste threshold concord-
ance within a single serial dilution. One ex-
ceptional and discordant pair includes one twin
on hormone therapy, who is an insensitive
("non"-)taster while her co-twin is a 'taster.'
Two other variables which evidently affect taste
threshold for PROP are aspirin medication, and
coincidence within the first three days of men-
struation. Threshold discordance coinciding
with either of the above variables disappeared
in each case upon controlled retest.

292

Section 15 — Human Genetics

The data demonstrate the primary genetic
basis of taste thresholds for6-n-propylthiouracil,
when properly tested. They also demonstrate that
medication and menstruation are variables which
may distort the manifest thresholds.

Supported by U.S. Public Health Service,
National Institutes of Health, Grant RG-9885.

15.69. Relation Between the Blood Groups of the
ABO System and the Taste Sensibility to
Phenylthiourea (P.T.C.). Francisco G. Haro
(Caracas, Venezuela).

Relation between the blood groups ABO and
the sensibility of taste to P.T.C. is analysed in
372 students of both sexes between the ages of
16 and 23 years.

The method of Harris and Kalmus (1949)
was applied in the present investigation to deter-
mine the taste sensibility to P.T.C.; and the
slide-test with the whole blood and with an-
tisera A and B to determine the blood group.

The distribution of blood groups was: 50 per
cent O; 32.25 per cent A; 11.3 per cent B, and
6.45 per cent AB.

The threshold distribution of his age group
was 6-7 of the solutions of Harris and Kalmus.

The "non tasters" and "tasters" proportion
was 22.6 per cent and 77.4 per cent respectively.
71 .42 per cent of "non tasters" were of O Group
(50 per cent of the whole group of students was
of O group)

31 per cent of O group were "non-tasters"
(22.6 per cent of the whole group of students was
"non tasters")

To all appearances there are correlations be-
tween the blood group O and the ageusia
("non-tasters").

It is possible to explain these correlations as a
manifestation of linkage of the genes both re-
cessives?

titative aspects of the Ai antigen might be re-
sponsible for the differential behavior, i.e. in-
fants with a strong Ai antigen more likely to be
affected with hemolytic disease of the newborn
than individuals with a weaker Ai antigen.

A method was developed for measuring the
antigenic strength of erythrocytes which depends
on freshly drawn blood for reproducible results.
Density of erythrocyte suspensions, reagent,
complement, temperature and time of incuba-
tion are strictly controlled and the degree of
hemolysis obtained gives a measure of the antigen
strength.

The data obtained thus far reveal considerable
variation in regard to antigen strength between
and among the subtypes Ai and A2. Individuals
whose genotype can be inferred to be AO
show, as a rule, little or no difference within
family units; there is, however, remarkable
variation between families. Thus, the findings
suggest that the alleles conditioning the sub-
types Ai and A2 represent an array of allelic
subunits within each subtype, which differ from
each other in that they are responsible for vari-
ous degrees of antigen strength, but are alike in
their qualitative property. In AB erythrocytes
the A antigen is weaker than in individuals of
AO genotypes, and cells of suspected homozy-
gous A genotype appear antigenically stronger
than when the same allele is in AO combination.

Results will also be presented of a study of
the Ai antigen in families who have had an
infant with hemolytic disease of the newborn
due to A-O incompatibility.

15.71. A Family Pedigree Showing the Transmission
of Rare and "New" Rh-Hr Genes. Lester
J. Unger (New York City, U.S.A.).

This family came from a small village in
Puerto Rico so that the circumstances suggest
inbreeding. The pedigree covers three generations.
The rare gene involved was r° and the "new"
as well as rare gene was rod.

15.70. Quantitative Variation in Antigen Strength
within the A Blood Types of Man and its
Possible Significance. F. J. Grundbacher
(Ann Arbor, U.S.A.).

In hemolytic disease of the newborn due to
ABO incompatibility, the most frequently af-
fected children are of type Ai of O mothers.
Since the majority of ABO incompatible child-
mother combinations cause no obvious fetal
injury, the question arose whether or not quan-

15.72. Lewis Blood Group Haptene in Human
Urine. D. A. P. Evans, R.B. McConnell, and
W. T. A. Donohoe (Liverpool, Great
Britain).

It has previously been found:

(a) that there is a pentasaccharide in pooled

human breast milk which inhibits anti-

Lea0)

293

Section 15 — Human Genetics

(b) that human urine from both salivary
ABH secretors and salivary ABH non-
secretors inhibits anti-Lea(1)

(c) the blood group substance A, B, H and
Lewis (a) purified from human pseudo-
mucinous ovarian cysts have a molecular
weight of about 300,000

This present communication presents data in-
cluding the following:

(a) that the Lea property in human urine is
due to the presence of a freely diffusible oligo-
saccharide. This is found in urine from both
salivary ABH secretors and salivary ABH non-
secretors.

(b) this diffusible Lea haptene is not present in
saliva.

(c) the haptene in the urine of salivary ABH
non-secretors is capable of "coating" Le (a-)
erythrocytes, so changing them to Le (a + )
Some implications of these findings will be dis-
cussed.

1. Watkins, W. M. and Morgan, W. T. J.
(1957) Specific inhibition studies relating to
the Lewis blood-group system. Nature, 180,
1038-1040.

2. McConnell, R. B. (1961) Lewis blood group
substance in body fluids. Proc. 2nd Internat.
Conference of Human Genetics, Rome (in
press).

3. Morgan, W. T. J. (1954). The chemical
basis of blood group specificity in man.
Lectures on the Scientific Basis of Medicine
IV, 92-111.

15.73. Normal Variation in Excreted Amino Acids in
Man, Herman M. Slatis (Argonne, U.S.A.).

Paper chromatography has been used in the
investigation of amino acids and other ninhy-
drin-positive substances in the urine of over
2500 persons. Other characteristics that are
known for these individuals include height,
weight, age, sex, blood type, and ancestry. The
statistical analysis of the chromatographic data
has been based on the ranking of each spot ac-
cording to density, rather than by the usual
practice of expressing the concentration of
each substance in terms of some supposedly
standard constituent of the urine. About 20 sepa-
rate spots were measured on most of the chroma-
tograms.

A number of interrelationships have been
observed between the various chromatogram
spots. There is no correlation of any of these
spots with height or weight but there are correla-

tions with the specific gravity of the urine. The re-
lationships do not seem to shift according to
height, weight, age, sex, ABO blood type, or
Rh blood type.

Most of the persons observed were of Euro-
pean ancestry. The various ancestry groups with-
in Europe do not appear to differ from each
other in significant ways. The non-Europeans
were too few in number to permit an adequate
analysis of possible differences, although some
suggestive trends were observed.

Work supported by U.S. Atomic Energy
Commission.

15.74. Genetic and Biochemical Aspects of Sickle
Cell Dactylitis. W. G. Thurman and
W. Lorraine Watkins (New York, U.S.A.).

Sickle cell dactylitis or the "hand-foot"
syndrome is an infrequent complication of
homozygous S hemoglobin disease. In the popu-
lation groups we have observed over 30 cases
of this syndrome.

Family studies indicate that this syndrome has
a high incidence within the same family group
should more than one sibling have homozygous
S hemoglobin. Four families have been studied
in which three siblings have been affected; one
has been studied in which four children have had
orthopedic difficulties. Possible gene penetrance
as related to male or female "carriers" have been
studied in two families in whom siblings have
had one common parent with subsequent mat-
ing with another parent, also with AS hemo-
globin; comparisons of siblings from the first
mating with siblings of the second mating that
have also developed the syndrome have been
made. The relationship of siblings with this
syndrome to abortions in the mother before or
after the affected pregnancy has also been
studied.

Fragilities, serum iron studies, alkaline and
acid phosphatase determinations, chloride, cal-
cium and phosphorus levels, fetal hemoglobin
studies, relation to infectious processes in other
organ systems and incidence of superimposed
infection in affected joints have all been
tabulated.

15.75. The Chemical Structure of Hemoglobim
Lepore and its Interpretation as the Result of
Non-homologous Crossing-over. C. Baglioni
(Naples, Italy).

Three samples of hemoglobin Lepore, ob-

294

Section 15 — Human Genetics

tained from unrelated individuals, have been
examined. The chemical findings previously re-
ported^) have been confirmed. Hemoglobin
Lepore appears to be made up of two normal a
peptide chains and of two abnormal peptide
chains (designated Lepore chains), resulting
from the fusion of part of the (3 peptide chain
with part of the 5 peptide chain.

A method has been devised to analyze by
fingerprinting the trypsin-resistant "core" of the
Lepore chain. More information on the chemical
structure of the Lepore chain has been obtained
by this method of analysis. The region where
the 5 chain-like portion of the Lepore chain
is joined to the (3 chain-like portion has been
localized in the "core". This region is comprised
between residue 85 and residue 115 of the Le-
pore peptide chain. When digested by trypsin,
this chain yields peptides identical to either (3 or 6
chain peptides. The entire Lepore chain can
now be accounted for; it appears to have the
same length as the (3 and 5 chains. These
chemical findings suggest that a very specific
genetic event is responsible for the formation
of the Lepore chain. The fusion of complemen-
tary regions of these peptide chains is most likely
caused by a non-homologous crossing-over
between corresponding points of the |3 and 5
genes.

1. C. Baglioni, Proc. Nat. Acad. Sc.
48(1962) 1880.

15.76. Serological Recent Data for the Gm Groups.
Their Interest in Sero-anthropology. C. Ro-
partz, L. RiVATand P.-Y. Rousseau (Rouen,
France).

As soon as 1961 we assumed that the Gm(b)
factor had not an elementary structure. It is
but very recently that such a phenomenon has
been highly proved.

Whereas Steinberg found one anti-Gm(b)
specific to the whites, we demonstrated that
what was called Gm(b) in the yellow Races was
in reality a mosaic of numerous unities, specially
among the Japanese : sera, specific for the Gm(b)
factor in the white people, define 3 different
structures among Japanese.

These facts, if complicating the immunolo-
gical study and the analysis of the genetical
determinism, give the gamma-globulins system
"Gm" a capital importance in sero-anthropology
and human genetics.

In fact, specific anti-Gm(b) reagents have

already helped us to review the cross-breeding
percentage of the US Negroes. Moreover deep
qualitative and quantitative differences in the
expression of the mosaic Gm(b) among yellow
races highly contribute to study the problem of
their paleo-migrations and cross-breeding. So,
the Chinese of Macao considerably differ from
the Japanese, and important variations are
found between different tribes of Central and
North American Indians.

Although the genetical determinism of that
mosaic which constitutes the Gm(b) factor is
still unknown, it is beyond doubt that the
facts that we present will help to its study.

Indeed, mind is not satisfied when obliged to
accept the presence of the same gene: Gmab,
which should be omnipresent in the melanoder-
ma populations, only very frequent in the xan-
thoderma ones.

15.77. Studies on the Hereditary Gamma Globulin
Factors: Their Relation to Immune Tolerance
and Studies on the Antibodies to the Hereditary
Gamma Globulin Factors in Man and their Rela-
tion to Immune Tolerance. Arthur G. Stein-
berg and Janet A. Wilson (Cleveland,
U.S.A.).

The hereditary gamma globulin factors (Gm
and Inv) are detected by means of an agglutina-
tion inhibition test. The antibodies which detect
these factors may be formed as a result of trans-
fusions of whole blood, injection of gamma glo-
bulin, or immunization of the infant by the
transplacental "transfusion" of the mother's
gamma globulin. It is of interest that the mother's
gamma globulin does not induce immune toler-
ance even though it is present in high concen-
tration before and after birth.

The antibodies are usually carried by (32 M
molecules but they may also occur on 7sy-
globulin molecules. The latter may cross the
placenta and be found in the infant. The anti-
bodies may be induced in rhesus monkeys,
but it is not yet clear that these are identical
with those formed in humans.

The antigens do not appear to cause trans-
fusion reactions when given to recipients who
have antibodies to them.

Data collected in this and other laboratories
will be presented to substantiate the above
statements.

295

Section 15 — Human Genetics

15.78. New Genetic Variants in the Gc-System of
Human Serum. Hartwig Cleve, R. L. Kirk,
W. Carey Parker, and Alexander G. Bearn
(New York, U.S.A.).

The group-specific components (Gc) of human
serum represent an additional protein poly-
morphism distinguished by differences in relative
electrophoretic mobilities. The common pheno-
types, Gc 1-1, Gc 2-1, and Gc 2-2 are genetically
controlled by a pair of co-dominant, autosomal
alleles, Gc1 and Gc2. The distribution of the
Gc-alleles has been determined in various popu-
lations of different geographical and ethnic
origin. The population surveys have led to the
disclosure of several unusual electrophoretic
variants, which were identified by Immuno-
electrophoresis and starch gel electrophoresis.
Two new variants will be described, which
migrate more rapidly than Gc 1-1. One was
observed in a population of Chippewa Indians
and the second in a sample of Australian Abori-
gines from the Cape York area, and were there-
fore named Gc Chippewa and Gc Aborigine,
respectively. Family data indicated that they
are controlled by additional alleles at the Gc locus
The distribution of the various phenotypes was
in agreement with the assumption of population
equilibrium. The incidence of the variants
was relatively high (GcChiPPewa 0.105 and

Gc Aborigine = 0.046). Gc Chippewa has so far
been found only in Chippewa Indians. The
distribution of Gc Aborigine in a number of
aboriginal populations in Australia will be dis-
cussed and the results related to observations
in populations from South East Asia and Ocea-
nia.

15.79. Contribution to the Genetically Determined
Transmission of the oti-Acid Glycoprotein
Variants. K. Schmid, L. Moroz and
K. Tokita (Boston, U.S.A.).

At the Second International Conference of
Human Genetics in Rome we reported on the
relative incidence of the different oci-acid glyco-
protein patterns in normal white adults. In the
present study, cci-acid glycoprotein was isolated
from plasma of 13 members of a family of
Italian origin and analyzed by starch gel electro-
phoresis at pH 2.9, resulting in most cases in a
pattern with six bands. This binding strongly
supports the concept that the cci-acid glyco-
protein variants are genetically determined.
Further evidence was obtained from the cor-
responding analysis of identical twins.

In continuing this investigation, ati-acid
glycoprotein was prepared from plasma of 44

white adult patients with various diseases and
subjected to the same analytical procedure. The
blood level of this protein was elevated in most
patients, the maximum increase amounting to
300 per cent. The relative incidence of the ox-
acid glycoprotein patterns was found to be
very similar to that of normal white adults and,
as judged from the relatively small number of
analyses, independent of the plasma concen-
tration of this protein. Thus, the genetic trans-
mission of the cci-acid glycoprotein variants
appears to be well established.

Supported by NIH grants GM-10374-01 and
A-3564-C2.

15.80. The Genetically Determined cci-Acid Glyco-
protein Variants. K. Tokita and K. Schmid
(Boston, U.S.A.).

The cci-acid glycoprotein patterns obtained
by starch gel electrophoresis at pH 2.9 appear
too complex for genetic evaluation because of
the large number of bands observed (5, 6, 7
and 8, respectively). In an attempt to arrive at
simpler patterns, ai-acid glycoprotein was in-
cubated with neuraminidase and subjected to
starch gel electrophoresis at pH 4.8. The en-
zymatically modified glycoprotein derived from
pooled normal plasma revealed a pattern with
two main bands of almost equal color intensity
and a minor faster moving band(1).

In the present study cci-acid glycoprotein was
isolated from normal adults, treated with neura-
minidase and analyzed by starch gel electropho-
resis at pH 4.8. Three types of patterns were
noted : the first type exhibited the maximum color
intensity at the slowest moving band and the
second type at the center band. The third type
showed the slow moving and the middle band to
be almost equal in color intensity and very
similar to that of pooled normal cci-acid gly-
coprotein. As judged by the number of analyses
carried out (65) the relative incidence of these
three types were found to be approximately 10,
50 and 40 per cent, respectively. Additional
results obtained from the corresponding study of
identical twins supported the concept of the
genetically determined transmission of the oti-
acid glycoprotein variants.

Supported by NIH grants GM-10374-01 and
A-3564, C2.
1. Nature, 190, 630 (1961).

296

Section 15 — Human Genetics

15.81. Variable Heterozygote Expression in Human
Haptoglobins. H. E. Sutton and G. W. Karp
(Austin, U.S.A.).

Two alleles are commonly recognized at the
human haptoglobin locus, Hpl and Up2. In
most populations, the heterozygous phenotypes
are very similar. However, in Negro populations
a variant, Hp 2-1 modified, has been described.

Our studies of an American Negro population
indicate that a broad range of heterozygous
phenotypes are found, depending on the relative
activity of the two alleles. The limited evidence
suggests that the differences in allelic activity
are not the result of structural differences of the
products. Studies of more than 100 matings of
various types show that in some families there
are factors which influence the activity of the
alleles differentially. Ahaptoglobinemia tends
to occur in the same families.

Analysis of the mating studies will be presented
and the haptoglobin system will be discussed
from the viewpoint of newer ideas on gene regu-
lation.

of the gestation period and of those from the
fathers and from normal controls, are also re-
ported and discussed.

15.82. Possible Influence of Mother's Genotype on
the Foetal Haptoglobin Synthesis. M. Sinis-
calco, G. La Torretta, C. Del Bianco,
S. Marsico and L. Bernini (Naples, Italy).

The incidence of cordal blood plasma with a
detectable haptoglobin pattern has been found to
vary considerably in the different haptoglobin
types of mating, suggesting the possible in-
fluence of the mother's genotype on the foetal
haptoglobin synthesis.

Thus the lowest incidence (4.3 per cent) was
found among the "incompatible matings" i.e.
when the baby must necessarily possess a
molecular haptoglobin species not produced
by the mother, and the maximum incidence
(23.8 per cent) among the full compatible mat-
ings, i.e. those involving a 2.1 mother or parents
with identical haptoglobin genotype.

Passive transfer of haptoglobin from the ma-
ternal to the foetal circulation is excluded on the
ground that the detectability of haptoglobin at
birth is not positively correlated with the
haptoglobin concentration of maternal plasma
and also because of the frequent occurrence of
cord blood plasma with a haptoglobin pattern
unlike that of the mother.

Extensive data on the quantitative hapto-
globin levels of the cordal blood plasma with
or without detectable haptoglobin pattern, of
the blood plasma from the mothers at the end

To be published in full in: Acta Genetica et
Statistica Medica, Karger, Basel.

15.83. Asynchrony in the Synthesis of the Polypep-
tide Chain and Carbohydrate Prosthetic Groups
in Human Transferrin. W. Carey Parker and
Alexander G. Bearn (New York City,
U.S.A.).

Transferrin C, the specific iron-binding com-
ponent of human serum, is a glycoprotein of
molecular weight approximately 90,000 whose
carbohydrate moiety contains glucose, man-
nose, glucosamine, fucose, and sialic acid.
Recent studies on several glycoproteins by
various authors have demonstrated the occurence
of prosthetic groups of multiple carbohydrate
residues attached at various points to the
polypeptide chain of the protein; sugar nu-
cleotides have also been isolated which contain
similar carbohydrate groups. Stepwise removal
of the four sialic acid residues of transferrin can
be accomplished by the enzyme neuraminidase;
transferrin devoid of sialic acid retains the iron-
binding, antigenic, and sedimentation properties
of untreated transferrin. Purification and ana-
lysis of a primate transferrin reveals that it
contains half the human complement of both
sialic acid and glucosamine. Cord serum from
newborn infants contains, in addition to trans-
ferrin C, four additional iron-binding compo-
nents which migrate slightly more rapidly by
starch gel electrophoresis than the correspond-
ing components in the pattern of neuraminidase-
treated adult transferrin. The faint components
disappear within 6-12 weeks of birth. These
observations suggest that the differences in
mobility result from alterations in the molecule
in addition to the absence of sialic acid and are
consistent with the interpretation that a mecha-
nism responsible for the addition of carbohy-
drate prosthetic units (containing glucosamine
and sialic acid) functions imperfectly at birth,
so that not all of the transferrin molecules
receive the full complement of sialic acid-
containing prosthetic groups. A similar pheno-
menon is present in human cerebrospinal
fluid, where two principal populations of trans-
ferrin molecules appear to exist, one with the
full complement of sialic acid-containing pros-
thetic groups, the other devoid of such groups.

297

Section 15 — Human Genetics

15.84. Inherited Structural Variation of Human
Myoglobin. Samuel H. Boyer, David C.
Fainer, and Michael A. Naughton (Balti-
more, U.S.A.).

Although considerable variation occurs a-
mong the hemoglobins of several species, no
such variation has been reported for myoglobin.
We shall describe examples of inherited struc-
tural alteration in myoglobin and indicate simple
methods for their detection.

Myoglobin, obtained from autopsy and diag-
nostic muscle biopsy, was prepared by ultra-
filtration of crude muscle extracts. The ultra-
filtrate was subsequently examined by starch gel
electrophoresis at pH 8.5. These procedures
are simple, highly efficient and well suited for
characterization of large numbers of samples.

Among 159 individuals, two subjects possessed
both normal myoglobin (Mb+) and an electro-
phoretically distinguishable variant. One of
these variants, MbATiercieen, migrates more

slowly than Mb" while the other variant,
Mr>Annapoiisj migrates faster than Mb+.

Mb Annapolis occurred in a mother and her
son both of whom were free of muscular and
cardiac disease. Mb+ and Mb Annapolis are
similar in molecular weight and spectral ab-
sorption but differ in fingerprints of tryptic
digests. Mb Annapolis iacks two normally
present peptides, viz., 7 and 13, and possesses
a new peptide, X. Amino acid analysis of these
peptides and homology with sperm whale my-
oglobin suggest that the peptides are arranged:
NH-2 7 arginine/NH2 13 lysine

The loss of arginine in Mb Annapolis accounts
for the altered fingerprint and the rapid migra-
tion of the whole molecule but has no apparent
influence on function.

Detection and characterization of additional
myoglobin mutants may be helpful in the study
of certain muscular diseases as well as quite
useful in assigning function to various portions
of a molecule whose fine structure is particularly
well delineated.

298

SECTION 16

HUMAN CYTOGENETICS

16.1. (D.) Karyological Evolution in Primates
(Catarrhina Monkeys). B. Chiarelli (Torino,
Italy).

The developments of the in vitro tissue culture
methods are providing considerable insight into
the mechanism of evolution related to chromo-
some variations in mammals.

In this respect some recents works on Primate
chromosomes seem very promising.

During the last four years I had the opportunity
of studing the chromosomes of 112 animals, of
both sex, belonging to nearly all the species of the
Old World Monkeys and Anthropoid Apes.

The chromosome numbers of these groups of
animals show a good deal of variation.

All the species of the genus Macaca, Papio,
Theropithecus and Cercocebus have a diploid
chromosome number of 42. Different species
differ in some morphological characteristics in
their chromosomes.

All the species I have investigated in the genus
Hylobates, Presbytis and Colobus have 44
chromosomes and they differ from the others
for the morphology of some chromosomes.

The species Erythrocebus patas has a diploid
number of chromosomes of 54.

In the genus Ceicopithecus the different
species have a modal number of 54, 60, 66, 72
chromosomes (it is noteworthy that all these
numbers are multiples of 6) the morphological
differences among the different species are
related to the number of acrocentric chromoso-
mes.

All Anthropoid Apes have 48 chromosomes.
Some of their chromosomes are different in
morphology.

Moreover in the demonstration I have pre-
sented some attempts to interpret the variation
in number and morphology of the chromosomes
of the different species with centric fusion, inver-
sion and translocation mechanisms.

meiotic and postmeiotic stages. In Locusta
migratoria (XO), Mus nuisculus, Rattus norvegi-
cus, Mesocricetus auratus, Cricetulus griseus and
Homo sapiens up to pachynema typical sex-
vesicle structures in early meiotic prophase
stages, which include parts of whole sex-chromo-
somes, are visible. The heterologous parts and
the solitairy X-chromosome of Locusta pass
diplonema, diakinesis and metaphase-I in nearly
mitotic shape. Entirely heterologous sex-chromo-
somes are kept together by an unilateral and
achiasmatic end-connection. In Locusta, 54.2
per cent of early spermatids (gynospermatids?)
own a large Feulgenpositive interphase-structure.
which lies in the center of the nucleus. Compa-
rable structures with regard to the X- and Y-
chromosome are found in the spermatids of man.
The sex-vesicle is interpreted as a special
structure of allocyclic sex-chromosomes or
parts of them, corresponding in early meiotic
stages to the chromocenters of mitotic inter-
phase-nuclei. So the heterologous parts are
protected against crossing-over and allowed to
continue in genetic differentiation. As a meiotic
attribute an achiasmatic end-connection is
formed and the division of the centromeres in
metaphase-I prevented. Thus, the meiosis of the
heterologous sex-chromosomes may be thought
as the scheme of a primitive meiosis, developed
secondarily and differentially in the several
species. The formation of the sexvesicle is
independent of the nucleus-orthoploidy and the
DNA-ratio between autosomes and sex-chromo-
somes. Heteropycnotic behaviour of sex-chromo-
somes in spermatids is interpreted as a condition,
capable to block genetic activity, giving equal
chances of fertilization to both types of gametes.

16.3. Studies of Human Pachytene Chromosomes.

J. I. Valencia, N. Cacheiro and C. Sonnen-
schein (Buenos Aires, Argentina).

16.2. Multilateral Aspects of Meiotic and Postmeiotic
Sex-chromosomes in Different Species. Paul
Eberle (Gottingen, Germany).

The X- and Y-chromosomes in the male of
different species have been investigated during

The chromomere pattern of human pachytene
chromosomes is being studied, with the purpose
eventually of constructing a cytological map.
Several bivalents can already by identified
tentatively with relation to the mitotic chromo-
somes, and some gross chromomere counts have

299

Section 16 — Human Cytogenetics

been determined provisionally. For example,
chromosome No. 1 appears to have 57 chromo-
meres and chromosomes 21 and 22 show 9 each.

No chromocentral region has been identified,
even in very well spread pachytenes, but the sex
vesicle has been superimposed on one or more
chromosome pairs, giving the impression of a
chromocenter.

The X and Y chromosome threads have been
observed inside the sex vesicle, apparently
attached by the tips of their short arms. The
long arm of the Y ends in two minute, very
deeply staining appendages which protrude from
the vesicle. The basis of the X-Y pairing is
obscure, but some favorable metaphases reveal
a knot-like structure reminiscent of a chiasma
at the point of association.

At the end of pachytene or the beginning of
diplotene, the sex vesicle begins to disintegrate
and the sex chromosomes become as distinct as
the autosomes, each showing two chromatids.

Pachytene cells show from one to four nucleoli,
none of them associated with the sex pair. Their
size depends upon the number of them present.
In most cases, they are associated sub-terminally
with small or medium-sized acrocentric auto-
somes, but there are some variations which will
be discussed.

16.4. Studies on the Leucocyte Stimulating Factor in
Beans. Kerstin Lindahl-Kiesslins (Uppsala,
Sweden).

Saline extracts from beans (Phaseolus vulgaris)
have been fractionated using different pro-
cedures. The fractions have been tested with
regard to their mitogenic activity, red cell
agglutination, serum precipitation and electro-
phoretic mobility.

16.5. The Non-random Distribution of Specific
Chromosomes in Somatic Metaphase Figures
from Cultured Human Leucocytes and Their
Relation to the Time of Termination of Chromo-
some Duplication. O. J. Miller, W. R. Breg,
A. C. Christakos, B. B. Mukherjee and
A. van N. Gamble (New York, U.S.A.).

are farther from the center of circular metaphase
figures, and more frequently at the periphery of
less selected metaphase figures than most of the
remaining chromosome pairs or groups. Pair
number 3 and group 6-12 + X share this same
rather peripheral location. Chromosome 1 is
closer to the center and less frequently at the
periphery of metaphase figures than any other
chromosome. Pair number 2, group 14-15 and
group 19-20 are almost as near to the center as
pair number 1 .

The location of a chromosome is not related
to its size, but may be related to the time it
terminates DNA synthesis, as shown by auto-
radiographic studies with thymidine-H3. In
general, the chromosomes that terminate their
DNA replication later in the DNA synthetic
period than do the other chromosomes of the
complement tend to have a peripheral location.

16.6. A Possible Mechanism of Dicentric Formation:
"Telomeric Binding" in Cultivated Human
Cells. P. S. Moorhead and Eero Saksela
(Philadelphia, U.S.A.).

Studies of metaphase chromosome rearrange-
ments induced in cultured human cells by the
presence of the simian vacuolating agent
SV-40 have led to the interpretation of certain
abnormal chromosomes as the product of an
end-to-end coupling of morphologically identi-
fiable autosomes into a dicentric which repli-
cates further as an entity. Examples interpreted
as resulting from such "'whole-chromosome-
translocations" have been found within inde-
pendently transformed cultures, including evi-
dence involving diplochromosome homologues
following endoreduplication. Cytologic demon-
strations of spontaneous occurrences of this
process were also found in cells of a mosaic
strain and in the degenerative phase of long
term cultivated diploid strains. Interpretations
of the origin of various other rearrangements and
chromosomes losses resulting from in vitro
SV-40 virus infection will also be considered in
the context of findings from studies employing
human diploid strains and current attempts to
demonstrate similar effects with human viruses
adeno-12 and adeno-18.

The chromosomes are not distributed at
random in flattened metaphase figures from
colchicine, hypotonic sodium citrate treated
human leucocyte cultures. The Y chromosome,
one of the X chromosomes, and pair number 13
tend to occupy a peripheral location. Pair
number 21 and the chromosomes in group 17-18

16.7. Determination of the DNA Content of Cell
Nuclei and Individual Human Chromosomes by
Photographic Colorimetry. E. M. den Tonke-
laar, J. L. G. Gaillard, P. van Duun and
A. Schaberg (Leyden, the Netherlands).

300

Section 16 — Human Cytogenetics

The DNA content of cell nuclei and individual
chromosomes was studied. The cells and chromo-
somes were stained by the Feulgen method and
the DNA measured by a new microspectro-
photometric method based on photographic
colorimetry.

A photomicrograph from the stained object is
taken with monochromatic light. The negative
is enlarged and by a special development a blue
colored print is made. In this print the pictures of
the object to be measured are cut out, the blue
dye is extracted and the extinction of this solution
is determined with a colorimeter.

As is postulated in the constancy hypothesis,
all diploid cells in an organism should have the
same DNA content. When measuring a number
of cells, a certain variation in the results is found.
Analysis of variance was done to separate the
variation in the object from the random measur-
ing error. In this way we established the accuracy
of the method and tested the constancy hypo-
thesis.

We found a high constancy of DNA content.
The measured ratio between the DNA content
of diploid and tetraploid cells also fulfilled the
theoretical requirements.

Photographic colorimetry has the advantage
that objects of very different forms can be
measured by cutting out the pictures from the
photograph. This fact and the results with inter-
phase nuclei led us to use the method for the
determination of the DNA content of individual
human chromosomes. These results will be
discussed.

16.8. Further Characterization of the Chromosomal
Complement of Man Through Autoradiography.

James L. German (New York City, U.S.A.).

In addition to their structural features, the
sequence in which chromosomes complete DNA
replication provides further characterization. This
sequence has been partially elucidated through
autoradiography of metaphase chromosomes
in which the DNA replicated during the terminal
1-3 hours of the S-period had been labeled with
H3-thymidine.

Though variability and homologue asynchrony
are prominent in nucleated blood cells in vitro,
intra-group patterns in Groups 4-5, 13-15, 16-18,
and Y-21-22 serve to distinguish members of the
group. Two in Group 4-5 are among the latest
of the complement to complete replication,

while in the other two there is early completion
in the long arms. In Group 13-15 two are very
late, while two are among the earliest of the
complement to cease the uptake of thymidine.
Pair No. 17 completes replication very early,
pair No. 18 late. Of chromosomes of Group
Y-21-22, in most male cells the Y is last; two of
the group complete replication very early.

The late replicating X of cells derived from the
normal female is clearly distinguished from
others of Group 6-X-12. Its characteristics
include: 51 per mille of the total length of the
haploid complement; arm ratio of 1.75; extreme
peripheral location in 33 per cent of c-metapha-
ses (expected in 32 per cent); secondary con-
striction in the long arm in 3 per cent of cells;
length in relation to the average length of a

average 4-5

Group 4-5 chromosome ( )= 1.25;

inordinate shortening in relation to chromosomes
of Group 4-5 in cells having had exposure to
colcemide for 3 hours.

16.9. Autoradiographic Studies of Normal and
Abnormal Human Karyotypes. Werner Schmid
(Houston, U.S.A.).

Chromosomes replicate segments of their
deoxyribonucleic acid (DNA) strands in non-
random sequences. The use of tritiated thymi-
dine and autoradiography permit the study of
these replication sequences in metaphase chro-
mosomes, a technique which was introduced
into cytogenetics by Taylor, Woods and Hughes
in 1957 (!).

In short-term cultures of human leukocytes
and in embryonic fibroblast cultures, continuous
labeling with tritiated thymidine (specific activity
1.9 C/mmol) at a concentration of 1 uc/ml
medium, was begun 6 hours preceding fixation
of the cells in metaphase. Colcemid was added
for the last two hours, collecting cells in meta-
phase from different terminal DNA replication
stages. The chromosomes were photographed
before and after exposure (4-6 days) to auto-
radiographic film. The often stepwise sequences
in which DNA replication is completed in
individual chromosome pairs will be demon-
strated. Homologous chromosomes go through
the same final replication sequences, usually
synchronously, sometimes slightly out of phase.
Five very marked, late replicating regions in
chromosome pairs Nos. 1,4, 9, 16 and in the Y,
coincide with secondary constrictions which, by
means of special techniques, have been demon-
strated in these chromosomes by Saksela and
Moorhead(2). So far, no significant differences

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Section 16 — Human Cytogenetics

have been found between the terminal replication
patterns of lymphocyte chromosomes versus
chromosomes of embryonic fibroblasts grown in
tissue culture. Results of the application of the
labeling technique to a number of abnormal
human karyotypes, including trisomic and
translocation mongolism and chronic myeloid
leukemia, will also be demonstrated.

1. Proc. Nat. Acad. Sci. U.S. 43, 122.

2. Cytogenetics, in press.

in the fifth, even though breakpoint is <0.2
c.o. -units from +c. (2) Inactivating portions of
the X exert their influence along a gradient.
Thus, the amount of variegation for a given
locus differs for different translocations, one
factor being distance of locus from rearrange-
ment point. Furthermore, we observe a typical
spreading effect for three of the translocations.
The finding that part of the X has no ability
to inactivate translocated autosome suggests
that this part may itself never become inactive
and therefore not fit the single active X hypo-
thesis.

16.10. Structural Variability of Human Chromo-
somes. Thea Luers and Eva Struck (Berlin-
Dahlem, Germany).

Studying the chromosomes in human diseases
sometimes it seems difficult to decide between
structural abnormalities, natural variants, and
artfacts. A collection of karyotypes from
leucocyte cultures is presented and discussed
showing atypical chromosomes such as size
differences between the two homologues, achro-
matic regions, elongated centromere region,
translocation with partial trisomy, ring chromo-
somes, dicentrics, variability of the Y chromo-
some and of chromosomes nos. 21 or 22.

16.11. Evidence from Six X-autosome Translocations
Bearing on the Single-active X Hypothesis.

Liane Brauch Russell (Oak Ridge, U.S.A.).

Recent results confirm our original thesis that
V-type position effects from X-autosome trans-
locations in the mouse (first reported by us in
1959) are found when the translocated X can
act heterochromatically. This occurs only when
there are at least two X's present, one being
required for gene action. Influences from X
heterochromatin suppress near-by autosomal
genes, allowing the recessives on the standard
autosome to express themselves. Lyon later
suggested that one or the other X is entirely
inactive and that the X-translocated autosome
behaves completely like the X to which it is at-
tached. This would imply that, for any given
autosomal locus, variegation characteristics
should be independent of the position of the X
and autosome break points. In work with six
translocations — one T(X;8) and five T(X;l)'s —
we have obtained evidence to the contrary.

(1) Apparently, not the entire X has inactivat-
ing functions. Thus, the r-locus, which shows
typical variegation in four T(X;l)'s, fails to do so

16.12. The Frequency of Sex-chromatin-positive Cells
and the Number of Nucleoli per Cell in Cultures
of Human Tissues. A. J. Therkelsen (Aarhus,
Denmark).

The number of sex-chromatin-positive cells
was counted in tissue cultures of human cells
from bone marrow and from embryonic and
adult human skin. The frequency of sex-chro-
matin-positive cells is low — 40-60 per cent —
in the logarithmic growth phase, but rises to
about 95 per cent in the post-logarithmic phase.
The variation was found in normal females as
well as in sex-chromatin-positive males. The
cause of the variation will be discussed. In the
same type of experiments, cultures from males
and females were grown simultaneously, and the
number of nucleoli per cell was counted in
specimens stained with Feulgen-light-green.
The average number of nucleoli per cell was
shown to be significantly higher in normal
females than in normal males.

This difference between the two sexes may be
explained on the assumption that the female
X-chromosome which forms the sex-chromatin
is nucleolus-organizing, whereas the Y-chromo-
some in males is not.

If this explanation be correct, the average
number of nucleoli per cell in Klinefelter patients
should be significantly higher than in normal
males, whereas the number in Turner patients
should be the same as in normal males. Preli-
minary experiments with Klinefelter and Turner
patients have been performed. The results are
discussed.

16.13. Iso-chromosome X in Man. M. Fraccaro and
J. Lindsten (Pavia, Italy).

In a group of females selected because phe-
notypically similar to XO individuals, we found

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Section 16 — Human Cytogenetics

46 chromosomes instead of the expected 45 and
a structurally abnormal X chromosome. Mea-
surement of this metacentric chromosome
showed that its arms were approximately
equal to the long arm of the X. These individuals
were consistently sex chromatin positive and
measurements of the DNA content of Barr
bodies in their cells (in collaboration with
H. Klinger) revealed that these Barr bodies had
higher DNA content than those from suitable
controls. When labelled with 3H-thymidine (in
collaboration with S. Muldal) late in the syn-
thesizing period the abnormal chromosome was
heavily labelled (i.e. it behaved as one of the
two X chromosomes usually does in this sys-
tem), and symmetrically so along its length.
We tentatively interpret this abnormal chro-
mosome as an iso-chromosome for the long
arm of X.

If the cytological interpretation is correct, it
follows that the carriers of these iso-chromo-
somes are monosomic for the short and triso-
mic for the long arm of X. In one family in our
material there was informative segregation of the
genes for deutan colourblindness and the sex-
linked blood-group Xg. Analysis of this family
(in collaboration with Ruth Sanger and R. R.
Race) gave evidence that both genes are located
on the short arm of the X chromosome. The
origin of the iso-chromosome is probably meiotic
and likely paternal. There was an apparent
deficiency of females among the sibships of
the index cases.

16.14. DNA Replication of X-chromosomes in Cul-
tured Leucocytes from Presumptive XXX,
X XX X Y and XO/ X Iso-chromosome- X Mosaic
Human Subjects. Barid B. Mukherjee,
J. Orlando Miller, W. Roy Breg and Saul
Bader (New York, U.S.A.).

An autoradiographic study of DNA replica-
tion in X-chromosomes was carried out by ex-
posing the cultured leucocytes to tritiated
thymidine for 3-9 hours in the presence of col-
chicine and then harvesting the cells directly
from the isotope-containing medium. Two
chromosomes in group 6-1 2 +X in metaphase
figures from three chromatin two positive
presumptive XXX females and three chromo-
somes of the same group in metaphase figures
from a chromatin three positive presumptive
XXXXY male were consistantly the last chro-
mosomes in the whole complement to complete
DNA synthesis. Two sex chromatin masses were
observed in only two-ten per cent of the inter-
phase nuclei of the three XXX females and three

sex chromatin masses were present in only seven
per cent of the interphase nuclei of the XXXXY
individual. These results support the concept
of a one-to-one correspondence between the
maximum number of sex-chromatin masses and
the number of late-replicating X-chromosomes.
The isochromosome-X was always the last
chromosome to terminate duplication in cultured
leucocytes from the XO/X iso-chromosome-X
female, and the normal X chromosome never
terminated duplication late. According to the
Lyon hypothesis, random differentiation of the
paternally or maternally derived X chromosome
should occur in individual cells during mamma-
lian embryogenesis. In this case, however, the
structurally abnormal X chromosome always
becomes the heteropycnotic, metabolically inert
member of the pair.

16.15. The Incidence of Drumsticks in Normal
Women and in Patients with Chromosomal
Abnormalities. Ursula Mittwoch (London,
Great Britain).

The demonstration of sex chromatin has be-
come an important aspect of human genetics,
both for theoretical and for practical reasons.
Although it is now generally accepted that the
drumstick of the polymorphonuclear leucocyte
bears a direct relationship to the X-chromosome
and is therefore comparable to the Barr body,
the interpretation of findings on drumsticks has
not always been straightforward. For this there
are three main reasons: (1) The low incidence
of drumsticks, which is on the average less than
3 per cent of polymorphonuclear leucocytes;
(2) the wide variation in frequency encountered
in different women; (3) the fact that incidence
of drumsticks is to some extent determined by
the degree of segmentation of the polymorpho-
nuclear nuclei.

An investigation has been made relating the
incidence of drumsticks to the number of nu-
clear lobes in apparently normal women and in
patients with various chromosomal abnormali-
ties. The following individuals were tested: 12
apparently normal women; 27 girls with mon-
golism (Down's syndrome) in association with
27 non-mongol mentally defective girls for con-
trol purposes; 12 patients with Klinefelter's
syndrome (XXY), 3 patients with XXXY chro-
mosome constitution; and 3 patients with XXX
chromosome constitution.

It was found that in the apparently normal
women the correlation coefficient between the
incidence of drumsticks and the average number
of nuclear lobes was +0.44. In all women there

303

Section 16 — Human Cytogenetics

was an increase in the incidence of drumsticks
with an increase in nuclear lobe number from
1 to 5, but the frequency of drumsticks in cells
of any given lobe number showed wide varia-
tions in different women. This difference could
be demonstrated on repeated occasions.

In the patients with chromosomal abnormali-
ties, but not the mentally defective non-mongol
girls, there was a decrease in the average number
of polymorphonuclear lobes and a lower in-
cidence of drumsticks. Moreover, the frequency
of drumsticks per cell of constant lobe number
was less than in the normal women.

It is suggested that chromosomal abnormalities
tend to lower the segmentation of the polymor-
phonuclear nuclei and that this is associated
with an even more marked reduction in the
number of drumsticks.

16.16. (D.) Monozygotic Twins of Different Sex.

T. Dent and J. H. Edwards (Birmingham,
Great Britain).

A case of Turner's syndrome, presenting with
amenorrhoea and dwarfism, was noted to have
a twin brother. The brother appeared to be a
normal male. Blood and serum groups showed
no evidence of dizygosity, and the palm prints
were strikingly similar. Chromosome analysis
from lymphocyte and fibroblast cultures showed
that both twins were preponderately XO in
constitution.

The male twin had marked difference in eye
colour, one eye being blue-green, the colour of
his sister's eyes, and the other brown. This sug-
gests the possibility of a locus on the Y chro-
mosome related to eye colour.

The origin of this chromosomal error was
presumably mitotic non-disjunction at a stage
before the cell lines determining either twin
were defined. The remarkably normal pheno-
type of the boy suggests that, if his tissues are
preponderately XO, then the stigmata of Tur-
ner's syndrome may be related to endocrine
abnormality rather than to defective responses
of structural tissues to directives relating to
growth.

16.17. Lethal Chromosome Constitutions in Man.

K. Patau, S. L. Inhorn, and E. Therman
(Madison, U.S.A.).

The word "lethal" is here restricted to condi-
tions that invariably prevent birth of a live child.
Evidently, polyploidy is lethal. In a study of

placental and fetal tissues from early abortions
a new case of triploidy was found, the first
with XXX and no Y. Among mechanisms that
may cause triploidy, fertilization by a giant
sperm should also be considered. Precise mea-
surements showed these to contain twice the
DNA content of haploid sperm. Nullosomy for
the X is undoubtedly lethal, and so is mono-
somy for any autosome. Viable trisomy is known
only for three autosomes; Di, 18, and the mon-
golism chromosome. Di and 18 trisomies may
have a high pre-natal mortality, but it is at least
as likely that their rarity, compared with
mongolism, reflects much lower non-disjunction
rates. There are reasons for believing that
numerous genes are involved in trisomy effects
and that trisomy for any human autosome inter-
feres in many ways with development. It is con-
cluded that trisomy for all, or almost all, but
the three above mentioned autosomes is lethal.
In the abortion study, no trisomies have turned
up as yet, but at least two cases of monosomy
for a C chromosome were found. In one, there
was an admixture of female diploid cells, but
in both cases the sex chromatin count was so
high that the lacking chromosome must have
been an autosome. In a third case of C mono-
somy no sex chromatin data could be obtained.

16.18. Abnormalities of the Acrocentric Chromo-
somes. Kurt Hirschhorn (New York,
U.S.A.).

A significant number of reports have appeared
during the past few years dealing with various
abnormalities of the acrocentric chromosomes.
These have included conditions recognized as
definitely associated with pathological states
(trisomies, deletions), as potentially pathological
(translocations), and finally, as of purely mor-
phological interest (for example, satellite struc-
ture, distribution, and inheritability). We would
like to discuss several new morphological va-
riants. Three patients and members of their
family were found to possess additional material
in the short arms of either the long acrocentrics,
the short acrocentrics, or both.

The first patient, a 19-year-old unmarried fe-
male with primary amenorrhea, no other clinical
abnormalities, and an excellent response to
exogenous hormonal therapy, had one long acro-
centric chromosome whose short arms were 2-3
times as long as those of the other long acro-
centrics. The same chromosome has been found
in the patient's asymptomatic brother, sister,
mother, and uncle. The second patient, a one-
year-old male child with congenital heart dis-

304

Section 16 — Human Cytogenetics

ease (interventricular septal defect) had two acro-
centric chromosomes involved, one long and
one short. In both chromosomes a prominent
segment was apparent between the short arms
and the satellites, therefore showing two sec-
ondary constrictions in the short arms. The
inserted segments were a bit longer than the
normal short arms which were also present. The
third patient, a 35-year-old female with chro-
matin-positive primary amenorrhea and very
short stature, showed gonosomal mosaicism
(isochromosome-XX/XO). In addition, one small
acrocentric chromosome was noted to have a
very prominent fragment inserted between the
short arms and the satellites, appearing exactly
like the chromosome described for case 2. Only
in the last case was there a possible source for
the fragment ; for example, part of the short
arms of one X. The parents of the other two
cases are being investigated to see if the unusual
chromosomes found in the propositi might be
present in their karyotypes as well as some ob-
vious deletion in another chromosome. Our
working hypothesis is that this additional bit
of chromosomal material may be responsible
for a partial trisomic state in the propositi.

16.19. Chromosomal Break in Autosome No. 2, in a
Patient with Hypogonadism. H. van den
Berghe, H. Verresen, W. de Loecker, O.
Steeno, and P. de Moor (Louvain, Belgium).

A break of the two short arms of one auto-
some no. 2 has been discovered in a 47-year-old
male patient. Numerous chromosomal prepara-
tions were obtained from leucocyte cultures at
different time intervals. The scission of the two
short arms of one autosome no. 2 occurred
in the direct neighbourhood of the centromere,
these two separated arms staying attached to
each other in all the cells.

This chromosomal material, initially belonging
to the autosome no. 2, was found to be transfer-
red and attached either to autosome no. 21 or
22, or even existing freely between the other
chromosomes. This phenomenon was found to
exist in all the examined cells. All the cells
have 46 chromosomes.

The clinical picture of this patient showed
hypogonadism of testicular origin, disturbed
anabolic features, and a series of secondary
abnormalities. The sex-chromosome-comple-
ment is XY in all the cells, and Barr-chromatine
in buccal smear being negative. The karyotype
in one parent and seven sibships was equally
studied.

The incorporation of thymidine-3!! and gly-

cine-2-C14 into the chromosomes of the cells of
this specific case were performed. The effect of
scission of one chromosome and the degree of
incorporation of radioactive precursors into the
different sections of the chromosome involved
as well as in the free existing fragments was ex-
amined.

16.20. Chromosomal Abnormalities in a Familial
Syndrome Involving Labial Fistulae with or
without Hare Lip and Cleft Palate. Adriana
De Capoa, M. Siniscalco, and D. Tarsitani
(Rome, Italy).

Among the types of autosomal trisomies
hitherto described and confirmed, cleft palate
and hare lip are often present though accom-
panied by other congenital malformations.

On the other side, in individuals showing
cleft palate and hare lip unaccompanied by other
congenital anomalies, chromosome studies have
failed to show significant abnormalities.

The authors have recently observed a family
involving a mother and eight children, seven of
which show labial fistulae associated in five
cases with hare lip and cleft palate. Chromosomal
studies performed on peripheral blood of
the mother and three of the affected children
showed the existence of an autosomal mosaicism,
involving an additional chromosome, apparently
belonging to group 4-5. In all the patients,
besides the cells with the extra chromosome, an-
other abnormality was found in all mitoses
observed: i.e. a small centric chromosomal frag-
ment seemed to be constantly present, while
one chromosome of group 18-20 was missing.
This fragment seems to pair with the long arm
of one of the chromosomes of group 18-20, and
it could therefore be interpreted as the missing
chromosome, carrying a deletion on its short
arm. The father was also studied and his chro-
mosome complement was found to be normal.

The interpretation of these findings is, for
the time being, very difficult and indeed hazard-
ous. However, even at this stage, a few conclu-
sions can be tentatively drawn:

(i) On the basis of nuclear sexing, an involv-
ment of the X-chromosome in the abnormal
cells can be excluded ;

(ii) It is very unlikely that the two chromo-
somal abnormalities are independent of each
other, and probably the delegation affecting the
small submetacentric chromosome, being pres-
ent in all the cells, is likely to be the primary
defect.

(iii) The absence of severe malformations and
of mental retardation makes plausible the hy-

305

Section 16 — Hitman Cytogenetic*

pothesis that in the patients referred to there
was no loss or duplication of genetic material,
the primary defect being possibly due to a
(reciprocal?) translocation followed by somatic
non-disjunction in some cell lineage.

(iv) The pattern of inheritance of the present
syndrome (transmission from a parent to all
her children but one) suggests the possibility
of a non random segregation of the aberrant
chromosome pairs during maternal gameto-
genesis.

that some of the genes controlling hemoglobin
synthesis during fetal life are located on the
triplicated chromosome.

To be published in full in Annals of Human
Genetics, London, England.

16.21. Neonatal Hemoglobin Abnormalities in
Trisomy 13-15 Syndrome. Frederick Hecht,
Ernst R. Huehns, and Arno G. Motulsky
(Seattle, U.S.A.).

Linkage of genes to specific chromosomes
was attempted by examining hemoglobin from
individuals with autosomal trisomies by starch
gel electrophoresis. No hemoglobin abnormal-
ities were found in newborns with trisomy 18
or trisomy 21. In two newborns with trisomy
13-15 syndrome an excess of Hb-y4 and a minor
hemoglobin with slow mobility (Hb-XF) were
found. No such abnormalities were found in two
older children (age 20 and 49 months) with
trisomy 13-15.

Hb-"/4 was identified by electrophoresis,
ultraviolet spectrum, and alkali denaturation.
The following studies of Hb-XF have been done.
On starch gel electrophoresis the mobility of
Hb-XF resembled that of Hb-Gower 2. On agar
gel electrophoresis at pH 6.2 Hb-XF migrated
with Hb-A. The alkali denaturation rate of
Hb-XF was between that of Hb-A and that of
Hb-F. The ultraviolet spectrum of Hb-XF was
similar to that of Hb-F. Sedimentation and
recombination experiments suggest that Hb-XF
has the structure of oc gXf,.

The net charge difference of the XF-chain
from the pA-chain makes it likely that there is
more than one amino acid discrepancy between
these two chains. A hemoglobin similar to
Hb-XF has been found in some small embryos
(e.g. the Gower embryo) and rarely in cord blood.
It may therefore represent a normal embryonic
hemoglobin. Hb-XF might thus be a physiologi-
cally and chemically distinct hemoglobin with
its own non-oc-chain: the epsilon chain.

The finding of Hb-XF and excess Hb-y-i in
trisomy 13-15 syndrome raises the possibility

Aided by the U.S. Public Health Service.

16.22. A New Form of Trisomy 13-15. St. M. Milcu,

C. Maximilian, V. Stanescu, I. Florea and
M. Augustin (Bucarest, Rumania).

The case of a child, aged 7, presenting Turner's
syndrome with a male phenotype, numerous
malformations, and a chromosomal trisomy
13-15 with an XY sex karyotype is reported.

The patient had a short stature, a big head with
prominent eminences and metopic suture,,
hyperteloriam, an antimongolian slit, a trilobated
nose, ears with a low implantation and asym-
metric auricles, retrognathism, a short and pal-
mated neck with a low insertion of the hair in
the back, Lyipogenesis of the breast papilla,
right umbilical and inguinal hernia, hyperlaxity
of the teguments and ligaments, hypoplasia of the
genitalia, shortening of the metacarpal and meta-
tarsal bones, clinodactyly. The patient also
presented hepatomegaly and the air mediasti-
nography rendered evident an enlarged thymus.
The right hypoplastic testis presented a dissocia-
ted spermatic cord, and a dysgenetic histologic
structure; the right testis could not be palpated.
Sex chromatin was negative and the culture of
bone marrow tissue showed the trisomy of one
of the 13-15 pairs with an XY karyotype.

The reported case differs from the other cases
of 13-15 trisomy by the clinical aspect of Turner's
syndrome with a male phenotype, emphasizing
thus the diversity of the clinical aspects brought
about by this trisomy. Nevertheless, in this case
an association of the trisomy with submicro-
scopic alterations of the Y chromosome might
exist, but dysgenesis of the gonads might also be
an effect of trisomy.

16.23. A B/G (4-5/21-22) chromosomal Translocation
associated with Multiple Congenital Anoma-
lies. K. H. Gustavson, S. C. FlNLEY, W. H.
Finley and B. Jalling (Stockholm, Sweden).

A case of a male infant with multiple con-
genital anomalies is reported. The anomalies
include scaphocephaly with premature synos-
tosis of sagittal suture, marked hydrocephalus
with dilatation of the ventricular and cisternal

306

Section 16 — Human Cytogenetics

systems, low set malformed ears, slight hyper-
telorism, bilateral blepharophimosis and colo-
boma of the iris, micrognathia and macroglossia.
There were also malrotation of the small
intestine, omphalocele, hiatus hernia, dilatation
of the renal pelves, absence of one umbilical
artery and anal atresia. The finger and toes were
long, and there was a distinct gap between the
first and second toes bilaterally. There was
marked muscular hypotonia and the deep tendon
reflexes were weak. The heart and genitalia were
normal. The child died at the age of six months.
An EEG was normal. Numerous vacuolated
mononuclear cells were found in the peripheral
blood and in the bone marrow.

The mother had had five pregnancies. The
first child was a male who died shortly after
birth. The second pregnancy resulted in a normal
male child. The next two pregnancies terminated
in early abortions. The present case was the
fifth pregnancy.

Chromosome studies of cultured skin and bone
marrow cells of the propositus showed a chromo-
some number of 46 and an XY sex chromosome
constitution. There were only three chromosomes
in group G (21-22), and there was an odd
chromosome which could not be paired. The
mother, who was phenotypically normal, had a
chromosome complement of 46 in cultured skin
cells. ?n addition to the same abnormalities
present in the karyotype of her son, one of the
chromosomes in group B (4-5) lacked the short
arms. The karyotype of the father was normal.

The most likely interpretation of the chromo-
some abnormality is a translocation involving a
chromosome of group 4-5 and a member of
group 21-22.

16.24. Multiple Congenital Anomalies with Autosomal
Trisomy amongst the Chromosomes of Group
16-18 and Their Possible Relations to the "Dys-
cranio-Dysphalangic-Syndromes." M. Tolks-
dorf, H. G. Hansen, H. R. Wiedemann and
W. Lehmann (Kiel, Germany).

We discuss the autosomal trisomy amongst
the chromosomes of group 16-18 associated
with multiple congenital abnormalities.

In view of the similarity in the clinical pattern
of the anomalies in cases so far published we
regard some distinctive clinical findings and other
features occurring facultatively.

In this connection we give a report of a prema-
ture female child with multiple anomalies and a
trisomy 17 demonstrated in blood and bone
marrow cells.

Moreover, we shall try and find possible

relations between the cases with trisomy amongst
the chromosomes of group 16-18 and the various
types of the "Dyscranio-Dysphalangies" (Gi'in-
ther).

16.25. (D.) Trisomy 17 associated with Multiple
Congenital Anomalies. H. G. Hansen,
M. Tolksdorf, W. Lehmann and H. R. Wie-
demann (Kiel, Germany).

We demonstrate figures of a premature female
infant with a relatively paraphased combination
of congenital abnormalities. Cytological studies
on her blood and bone marrow cells were
carried out on short term cultures. The results
show a chromosome number of 47 with a trisomy
amongst the group 16-18 in the great majority
of cells of both cultures.

Some mitoses and karyotypes are demon-
strated.

16.26. Discordant Mongolism in Monozygotic
Twins? J. W. Bruins, J. van Bolhuis,
J. B. Bijlsma and L. E. Nijenhuis (The Ne-
therlands).

In 1 956 van Beukering and Vervoorn published
a case of negro-twins, one of which suffered
from Down's syndrome (mongolism). Investi-
gation of the placenta (plate) proved the twins
to be monozygotic. So this would be a case in
point of discordant mongolism in uni-ovular
twins. It was not possible to examine the blood-
groups or the chromosomes of these negro-
twins.

If the general karyotype of the child with
Down's syndrome presented 47 chromosomes,
this would be a case of non-disjunction which
occurred in the fertilized human egg. Instead of a
derangement in the meiosis, it would be here
non-disjunction in the mitosis.

On October 4, 1962, at Deventer (Nether-
lands), a pair of twins was born that at first
was taken to be dizygotic on account of anthro-
pometrical differences. One of the twins was
normal, the other showed nearly completely
Down's syndrome. Unfortunately the placenta
was not examined but examination of the
bloodgroups indicated that the twins were
monozygotic. To test this, a small reciprocal
skin-transplantation will be made.

The number of chromosomes of the cultivated
white blood cells was 47 for the diseased child,
and 46 for the normal one. So they are "mosaic-
twins", at least if there has been one cell in the

307

Section 16 — Human Cytogenetics

beginning. More investigations will be done
with other tissues of the mongoloid child to
test the number of chromosomes. It may very
well be possible that these tissues will have a
mosaic character as a result of the mitotic
non-disjunction.

The examination offers possibilities as to the
localization of particular bloodgroups on
particular chromosomes. The examination is not
yet finished and will be extended — if possible.

If congenital dysplasias, whatever they may
be, be sometimes discordant in human mono-
zygotic twins and if there be moreover a dis-
congruency in the chromosome-pattern, one
may conclude that their origin lies in a very early
developmental phase.

families of Group A and six families of Group
B. The parents of the mongoloid children had
a normal karyotype in 13 families. Some normal
siblings were found to have a normal karyotype
as well. All but one mongoloid of these thirteen
families has 47 chromosomes with a trisomy Gm.
Translocation of a Gm chromosome as cause of
familial mongolism was found in one family
only, where the mother and one mongoloid
child had 45 and 46 chromosomes respectively
with a 15/21 translocation. The father has a
normal karyotype. The other mongoloid child
died before the chromosomes were studied.
Evidence will be presented that several causes
may lead to familial mongolism and that
familial translocation is not its main cause.

16.27. Chromosomal Mosaicism in a Mongol Child
with Average Intelligence. P. Giraud, R. Ber-
nard, A. Stahl, F. Giraud, M. Lebeuf,
and M. Hartung (Aix-Marseille, France).

The authors are reporting the case of a
sixteen-year-old girl, with the typical features of
Mongol disease (Down's Syndrome).

Her mental level is sufficient for a quite normal
(on the medium range) scholar activity.

The chromosomic study (including a skin
biopsy) shows a mosaicim: Fifty (50) cells have
been counted and analyzed. Among these, eight
(8) cells were found as being 47 chromosomes,
trisomic for the 21 chromosome; the others
being normal, with 46 chromosomes.

The authors are supputing about the con-
nections of a such mosaicism with the different
features of Mongolism, with particular attention
to the intellectual development.

16.28. Familial Mongolism.

(Iowa City, U.S.A.).

Hans Zellweger

Thirty-two families with more than one case of
mongolism are under investigation:

A. In fifteen families more than one mongoloid
has been found in one and the same sibship
(4 times both twins, 9 times two siblings, 2
times three siblings were affected).

B. Seventeen families were found with two to
six cases of mongolism in a wider kindred with
no single sibship (except one) having more than
one mongoloid.

The average maternal age at the time of birth
of the affected children was 29.1 years in Group
A and 34.4 years in Group B. So far chromo-
somal analyses have been performed in eight

16.29. An Extra Chromosome in Patients with
Pelger-Huet's Abnormality of Leucocytes.

H. Siebner, F. Heni and D. Klaus (Tubingen,
Germany).

In several patients of different sibships with a
Pelger-Huet's abnormality of the polymorphonu-
clear leucocytes a small extra chromosome with
subterminal centromere was found in cultures
from peripheral blood and in a higher percentage
in cultures from bone-marrow. Classified by its
size it belongs between the pair of autosomes 20
and 21 following the Denver-classification.

A trisomy 21 appears to be unlikely, because
the leucocyte alkaline-phosphatase activity was
not increased in the studied patients, although
there is a well-known defective lobulation of
polymorphonuclear leucocytes in mongols.
Relations to the Ch^-chromosome and the
Philadelphia-chromosome are discussed.

The extra chromosome may be regarded as
the result of a deletion with nondisjunction of a
larger chromosome. There is also the possibility
of the same mechanism which is assumed by
Patau about the marker chromosome of
Waldenstrom's Macroglobulinaemia. Chromo-
somes of one culture were labelled with tritiated
thymidine.

16.30. Pyknotic and Binucleate Cells in Irradiated
Peripheral Blood Cultures. J. Weuer, Dorothy
L. Weuer and H. E. Duggan (Edmonton,
Canada).

Irradiated peripheral blood when cultured
yields aberrant nuclear forms. Apart from
chromosome aberrations the binucleate cells
and the pyknotic leukocytes are also important.

308

Section 16 — Human Cytogenetics

In order to estimate damage from various
dosages, frequency curves have been compiled
for some of these nuclear aberrations (binucleate
cells and pyknotic leukocytes) using normal
leukocyte cultures irradiated with different
dose levels of X-rays up to 100 r. In addition,
other cultures were supplemented with radio-
sulphur (S35) and radiophosphorus(P32)(togive
accumulated doses up to 200 rad). These experi-
ments were repeated for different culture times.
A correlation was found to exist between the
number of these aberrant forms, increasing
radiation dosage and increasing culture time.
The results with X-ray and S35 were comparable
but P32 gave an enhancing effect due to its
incorporation and subsequent transmutation in
vital cellular components.

16.31. Chromosome Aberration Rates in Human
Somatic Cells Irradiated in Vivo. M. A. Bender
and P. C. Gooch (Oak Ridge, U.S.A.).

Although a fairly large body of data has
accumulated on the induction of somatic
chromosomal aberrations in human cells by
ionizing radiation, the work has necessarily
been done by irradiation of cells /'// vitro. The
recent development of a technique for the initi-
ation of mitosis in human peripheral leukocytes,
however, permits the examination of the chromo-
somes of irradiated humans in the first division
following irradiation. We have examined such
material from a number of normal persons who
received whole-body irradiation. One group of
three men received doses of mixed gamma and
fission neutron radiation estimated at 19, 43,
and 110 rem (estimating the RBE for the
neutrons as 2). A second group of three men
received doses of y-rays estimated at 17, 22, and
57 rad. The frequencies of aberrations observed
in samples obtained immediately after irradiation
were consistent with the coefficients of aberration
production obtained previously for irradiation
of leukocytes in vitroS1) Aberration frequencies
remained at essentially the same frequency until
about 4 weeks, and the aberrations seen provided
evidence that the cells had not divided in vivo
after irradiation. In later samples, however,
aberration frequencies fell, and many of the cells
sampled had evidently undergone division in
vivo. Dicentric chromosomes, symmetrical trans-
locations, and deleted chromosomes continue
to be seen many months after irradiation.

1. Bender and Gooch, Proc. Natl. Acad. Sci.
U.S. 48, 522, 1962.

16.32. Action of X-rays on Transformation of Normal
Cells into Malignant in vitro. Ju. B. Vakhtin
(Leningrad, U.S.S.R.).

It is known that attempts to accelerate the
process of malignant transformation of mono-
layer cell cultures by means of chemical cancer-
ogens were unsuccessful. The cancerogenic
action of ionizing radiation on cell cultures
inducing the high percent of mutations and
chromosome aberrations has not yet been
investigated.

Fibroblasts isolated by tripsinization from
subcutaneous connective tissue of newborn rats
were cultured in the synthetic medium 199 or
0.5 per cent lactalbumin hydrolysate with 20
per cent of horse or bovine serum. Just after
explantation cells of each culture have been
divided into five variants. Four of them were
treated with 10 r 10, 100 r, 100 r 5 and
500 r of X-rays and control variant was not
irradiated. Cultures were maintained for 2-15
months (6-26 passages) and implanted regularly
into newborn rats and adults of the same strain
(1-5 x 106 cells in each inoculum, subcutaneous-
ly and interperitoneally).

No malignization took place in variants of
cultures maintained for 2-8 months. All of these
cultures consisted mainly of cells with diploid
and near-diploid chromosome complements.

Malignant transformation has been found
within the only culture in the control variant and
the variant irradiated with the dose of lOOr
after 12 months of the cultivation. As implanted
into rats these variant cells give rise to transplant-
able sarcomas.

Results obtained are compared with our data
of caryological studies of cell cultures and tu-
mours developed. The importance of genetic and
epigenetic variations for evolution of cell popu-
lations in vitro is discussed.

16.33. The Changes in Somatic (Tumour) Cells
Induced by Specific DNA and RNA. J. M.

Olenov (Leningrad, U.S.S.R.).

Tumor cells are suitable objects for study of the
action of nucleic acids for a selective back-
ground can be applied in experiments in vivo.

The treatment of the sarcolysine-sensitive
variant of the rat sarcoma 45 with DNA from
the sarcolysine-resistant variant of the same
sarcoma causes the transformation of the for-
mer cells (D. Podgajeckaja, V. Bresler, J. Olenov).
DNA-ase inactivates the activity of the prepara-
tion. The transformants retain their properties
in subsequent passages.

309

Section 16 — Human Cytogenetics

Rat liver RNA inhibits the tumor growth in
transplantable rat liver mucous cancer obtained
by Maljugina (N. N. Aksenova, V. M. Bresler,
V. I. Vorobjev, J. M. Olenov). RNA-ase totally
inactivates the preparation. The action of RNA
from other tissues is less pronounced than the
action of liver RNA.

The possibility to transform the nuclear cells
indicates the universality of this phenomenon.
Simultaneously, the result obtained proves the
transgenic origin of sarcolysine-resistant vari-
ant. The transformation frequency cannot be
determined exactly by studying of populations,
but it is likely to be small. The transformability
of non-haploid cells indicates the dominant or
semidominant nature of the marker. Unlike the
transforming activity of DNA, the antitumor
activity of RNA affects all or majority of cells
in the population — the change of some cells
would be ineffective. The paper deals with the
nature of normal tissue RNA antitumor action
(the templates controlling the protein synthe-
sis or repression and derepression of genes).

There was no change in the normal diploid
chromosome count after 4 months of cultivation
(93 per cent diploid cells). The cells were then
exposed to 56C for one hour in an atmosphere
of 4.5 per cent CO2. Most of the cultures dis-
continued their growth, but a few surviving cells
were able to form new colonies. The chromo-
some number of these colonies was determined.
Shortly after recovery there were 52 per cent
polyploids, some octoploids included. After
6 months cultivation the chromosome number
changed again and there were now only 15 per
cent polyploids, 9 per cent subtetraploids and
76 per cent diploid cells.

This change in chromosome number from
almost all diploid to more than 50 per cent
polyploid after heat shock and the slow decrease
of tetraploids, that is increase of diploid cells
and the almost absent fraction of subtetraploid
cells after 12 months of cultivation in Puck's
medium will be discussed.

16.34. The Dependence of Molecular Weight of
DNA and RNA and Its Uptake by Mammalian
Cells. T. Wilczok (Gliwice, Poland).

Ehrlich ascites carcinoma cells were used as
recipient cells for uptake studies of homologous
labeled DNA and RNA. The rate of incorpora-
tion was proportional to the molecular weight
of tracer nucleic acids; the lower molecular
weight, the lower adsorption and incorporation.
The highly polymerized polyanions such as
polyethylene sulphate compete with highly poly-
merized DNA and RNA in the process of its
uptake by recipient cells. DNA and RNA ad-
sorption on the surface of recipient cells pro-
ceeds instantly and this process is not time and
temperature dependent. The incorporation of
DNA into DNase resistant fraction of host
cell is higher at 0° than at 37° and reaches its
maximal level after 5 hours. The amount of
tracer DNA in DNase resistant fraction does
not increase when the exposure is performed
at 37°.

16.35. The Change in Chromosome Number of
Human Cells in Culture after Heat Shock. W.

Ostertag and H. Kruger (Munster, Ger-
many).

The skin (epidermis) of a normal human male
was cultivated in modified Puck's medium.

16.36. Alterations in Karyotype of Mammalian Cell
Populations Exposed to Antimetabolites. J. L.
Biedler (Rye, U.S.A.).

Cytogenetic studies of transplantable mouse
neoplasms treated with several cancer chemo-
therapeutic agents were undertaken in a search
for possible specific relationships between anti-
metabolite, chromosomal alteration, and drug
resistance. Karyotype analyses were carried out
on sublines of sarcoma 180 and ascitic leukemia
L1210 continually exposed to amethopterin,
azaserine, 6-mercaptopurine, or 5-fluoruracil,
singly or in combination, over periods ranging
from a few weeks to several years.

The modal chromosome numbers (70-71) and
4 bi-armed marker chromosomes of the untreated
sarcoma remained unaltered for 3 years. In
contrast, the modal number was reduced in 7 of
8 treated sublines. In all sublines there were
changes in or losses of one or more markers.
The disappearance of a small subtelocentric
chromosome was prevalent, and thus unrelated
to a specific antimetabolite.

The modal chromosome number of the un-
treated leukemia remained 40-41 for 5 years.
Five of 12 variously treated sublines showed a
definite increase in number of chromosomes. In
early transplant generations of 6 of 13 amethop-
terin-treated sublines, a small proportion of cells
contained 2 characteristic subtelocentric marker
chromosomes instead of the usual 1.

These results suggest that the antimetabolites
provoked mitotic non-disjunction, resulting in
decreases in numbers of chromosomes in the

310

Section 16 — Human Cytogenetics

hypotetraploid populations and increases in the
"diploid" populations. The abnormal, bi-armed
chromosomes were apparently preferentially
involved.

Development Award (PCI 1-63) from The Na-
tional Institutes of Health.

Support by CCNSC Contract SA-43-ph-2445.

16.37. (D.). Some Relationships of Viruses and
Chromosomes. Warren W. Nichols, Albert
Levan, Rei Kato, and C. G. Ahlstrom
(Lund, Sweden).

In a long-term study of the relationships of
viruses chromosomes and carcinogenesis, two
model systems, Rous sarcoma in the rat and
measles, have been utilized. The Rous sarcoma
system has been studied from the aspects of
zero passage and serially transplanted tumors,
both in vivo and in vitro, and by the addition of
the virus to normal cells in tissue culture. Both
in vivo and in vitro, the tumor has shown a pro-
gression of chromosomal changes with initially
normal karyotypes, or karyotypes with changes
in the number of chromosomes around the di-
ploid level, followed by changes in chromosome
type, and finally changes in ploidy. The addition
of Rous sarcoma virus to normal rat cells in
tissue culture produced an increased incidence
of chromosome breakage and abnormalities
over the control level.

In the measles system, clinical measles was
found to be associated with chromosome break-
age in the white blood cells. When patients were
studied who received live attenuated measles
vaccine, breakage was again found but to a
lesser extent than in the disease, and there were
indications that natural immunity or injection of
gamma globulin at the time of immunization
offered some protection against the breakage
phenomenon.

Chromosome breakage fits well into a somatic
mutation theory of carcinogenesis and it is in-
teresting that chromosome breakage is a com-
mon denomination in the three classes of material
that are known to be carcinogens; namely, irra-
diation, chemicals and viruses.

This work has been supported in part by
grants-in-aid from The National Institute of
Health (C4953 and C43845 and CA06415) and
The American Cancer Society (ElllB), The Swed-
ish Cancer Society (62:80), The Swedish Med-
ical and Natural Sciences Research Councils
(U212 and T432) and by a Research Career

16.38. Chromosomal Aberrations Associated with
Virus Infections in Man. Ulla Gripenberg
(Helsinki, Finland).

Leucocyte cultures have been raised at the
acute stage of several different infections (1-20
days after getting sick). About 100 karyotypes of
each culture have been examined in order to
determine the frequency and localization of
chromosome breakages. Chromosome prepara-
tions have been made from 1-5 patients with each
of the following infections: morbilli, exanthema
subitum, epidemic parotitis, varicella, infectious
mononucleosis, mesenteric lymphadenitis.

An increased incidence of various chromoso-
mal aberrations was found in the different
cases. (The final percentage of cells with aberra-
tions, as well as the nature and localization of
these will be reported later.)

16.39. Approach to the Cytogenetics of Mammalian
Cells cultured in Vitro. L. De Carli, J. J.
Maio and F. Nuzzo (Pavia, Italy).

Mammalian cells in tissue culture may yet
offer the best approach toward initiating true
cytogenetic studies which could correlate the
data obtained from chromosomal analyses with
variations in cellular phenotypes. The possibility
would then be offered of locating cytologically
the genetic control of some cellular characters
recognizable in vitro. Although variants may be
easily isolated from the heteroploid cell strains,
this material is poorly adapted or even useless
for many experiments of genetic analysis. How-
ever, these same strains could be even preferable
to euploid cultures in some instances if one
would study the correlation between chromoso-
mal variations and the phenotypic changes in a
given character. In fact, the chromosomal varia-
bility of heteroploid strains can be usefully
exploited since it may give rise to a series of
polysomic states for some chromosomes which
may correspond to a gradient of expression in
the cellular phenotype. Furthermore, particular
chromosomal patterns may be stabilized for a
sufficient number of cell generations by means
of clonal procedures.

As an example of this approach to the cyto-
genetics of mammalian cells cultured in vitro,
data are presented concerning the karyotypic
analyses of several clonal lines derived from a

311

Section 16 — Human Cytogenetics

human heteroploid cell strain which exhibit
marked variations in alkaline phosphatase
activity. The karyotypes of the deficient lines
showed a loss of a number of chromosomes.
The detailed morphological analyses of chromo-
somes, made it possible to individuate the
missing chromosomes, thus indicating the possible
site of genetic control of the character.

occur, but in acute myeloid leukaemogenesis
permanent derepression occurs, with subsequent
mutation affecting the reactive site and occa-
sionally mutation affecting other points in the
enzyme protein molecule. These phenomena
have important bearing on gene regulation in
mammalian cells, and on aberrations of gene
control in carcinogenesis.

16.40. Genetic and Biochemical Studies on Mutant
Cell Lines in Culture. Robert S. Krooth
(Ann Arbor, U.S.A.).

Galactosemia in man is a rare recessive disease.
Cell lines developed from the tissue of patients
with galactosemia and from the tissues of normal
controls have been studied after prolonged
growth in vitro. When a suitably constituted me-
dium is employed, the galactosemic cells appear
to be unable to grow in galactose, whereas the
normal cells can. The cultured galactosemic cells
do not oxidize galactose-1-C14 to C1402 in
short term experiments while cultured cells
from control patients perform the reaction readi-
ly. These two properties of galactosemic cells
have thus far proven to be a stable phenotype of
the line. Analogous experiments have also been
performed on cell lines derived from patients
with another recessve abnormality — acatalasia.
Here too the characteristic biochemical abnor-
mality appears to persist in the cultured cells.
Preliminary genetic experiments on these lines
will be described, and the results of a further
search for biochemical markers in cell cultures
will be summarized.

16.42. Mitotic and Immune Response of Human
Peripheral Lymphocytes in vitro. N. Hashem
(New York, U.S.A.).

During the investigation of the mechanism of
the mitogenic action of phytohemagglutinin on
human peripheral blood lymphocytes, it was
noted that mitotic activity is associated with
leucocyte agglutination, gammaglobulin pro-
duction by the lymphocytes and morphological
changes of the cells during culture. This im-
mune-like response prompted investigation of
the action of specific antigens on lymphocytes
from individuals sensitized to the antigens.
With the use of tuberculin purified protein deriv-
ative and pertussis vaccine it was demonstrated
that mitotic activity resulted in cultures of
peripheral lymphocytes from sensitized indi-
viduals. It was also noted that morphological
alterations and gammaglobulin production was
limited to only a group of the cells in culture.
These experiments have demonstrated the po-
tential ability of "clones" of peripheral lym-
phocytes to respond to specific antigens with
gammaglobulin production and mitotic activity.

16.41. Leucocyte Enzyme Induction and Repression
and Human Leukaemogenesis. Chev. Kidson
(London, Great Britain).

Under certain conditions two catalases, a
and b, may occur in human leucocytes of the
myeloid type. Catalase a is always present in
all leucocytes; catalase b, having a different
pH optimum, occurs intermittently in normal
myeloid cells, never in lymphoid or erythroid
cells. In acute myeloid leukaemia catalase b is
always present, but with altered reactive site, and
occasionally altered electrophoretic mobility.
Catalase b is not induced in normal myeloid
cells during m vitro phagocytosis, nor by
anacrobiosis. It appears that catalase b induc-
tion in normal human myeloid leucocytes is
due to reversible derepression: in the normal
cells repression of catalase b synthesis can readily

16.43. Variation of In Vitro Gamma Globulin Pro-
duction by Human Lymphocytes. Fritz Bach
and Kurt Hirschhorn (New York. U.S.A.).

Human lymphocytes, obtained from heparini-
zed peripheral blood by sedimentation and sub-
sequent removal of polymorphonuclear leuko-
cytes, were grown in tissue culture. In the pres-
ence of phytohaemagglutinin and other sub-
stances, all lymphocytes have the potential to
produce gamma globulin. This problem was
studied by both radioactive and fluorescent
antibody techniques. Further studies were done
on the lymphocytes of patients with dyspro-
teinemias including agammaglobulenemia, mul-
tiple myeloma, and macroglobulenemia. In
cases of hereditary agammaglobulenemia both
heterozygotes and homozygotes were examined.
Data about the quantitative production of gam-

312

Section 16 — Human Cytogenetics

ma globulin in the whole population of cells as
well as production of individual cells will be
presented.

16.44. Peroxidase Isozymes in Human Leucocytes.

Barbara Z. Siegel and Kurt Hirschhorn
(New York, U.S.A.).

Isozymes, altered but active forms of a
given enzyme, have been shown to exist in
human tissue and that their presence and in-
tensity may be both genetically and ontogeneti-
cally determined. A preliminary report on the
technique of preparation and identification of the
peroxidase isozymic series from human leuco-
cytes will be presented. Differences between cells
from normal and leukemic peripheral blood,
bone marrow and tissue cultures will be demon-
strated, as well as data on the qualitative and
quantitative variations.

16.45. Influence of Steroid Hormones on Mammalian
Cells and Culture. David Stone (Shrewsbury,
U.S.A.).

Studies on the influence of steroid hormones
on Hela cell cultures have indicated that it is
possible to select cell sub-lines which are in-
hibited in growth by particular steroids, but
not by others. Chromosome studies of these
cultures have indicated that the cell strain which
is inhibited in growth by both desoxycorticos-
terone and testosterone has a chromosome stem
line of 68, whereas the sub-lines resistant to
desoxycorticosterone or testosterone, respective-
ly, each have a stem line of 74 chromosomes.
In order to study the relationship of steroid
sensitivity and chromosomal characteristics
further, a cell strain having more classic diploid
characteristics] has been studied. Using cell strains
of the Chinese Hamster cultured for varying
periods in the presence of certain steroids, it has
been shown that the actions of the hormones
change with the length of culture over a period
of months. Cells which previously grew (at the
normal rate of growth) in a particular steroid
may become inhibited by the presence of that
steroid and later may be stimulated in growth
by the same steroid. Chromosome studies have
indicated that, compared to control cultures
run in parallel, the steroid supplemented cells

exhibit altered numbers and morphologies of
their chromosomes. Even after one to two days
of steroid treatment small but significant changes
can be detected. The effects of steroid hormones
appear to be related to the age of the culture
and the particular batch of serum used in the
medium. These results will be discussed.

16.46. Inherited Variation of Somatic Cells to Polio
Virus Infection. Lawrence N. Chessin (New
York, U.S.A.).

Studies using primary human amnion cell
cultures have indicated that virus resistant
clonal variants can be selected from the parental
population. These variants are morphologically,
karyotypically, and immunologically indistin-
guishable from the sensitive population

Studies on these resistant and sensitive clonal
types haves shown that cellular resistance and
susceptibility to poliovirus infection was clo-
nally distributed in a primary human amnion
population. Data from virus absorption, penetra-
tion and replication experiments will be dis-
cussed from the standpoint that somatic cell
variation to poliovirus infection may be due to
inherent differences in the mechanism for the
control of viral replication.

16.47. An X-ring Chromosome in a Turner Patient
Without a Specific Peripheral Localization.
Th.W. J. HusTiNx(Nijmegen, the Netherlands).

Chromosomal analyses in a Turner patient re-
vealed a ring chromosome in both blood- and
skin cultures. Barr-chromatine was detected in
part of the diploid skin nuclei.

The ring chromosome is presumed to be a
mutated X chromosome. The X-ring seemed to
have preferentially a peripheral localization in
the metaphaseplates; in 42.5 per cent the ring
proved to be totally peripheral. The percentage
of metaphase-chromosomes, which are also pe-
ripherally localized was 41.1 per cent of all
karyograms had a peripheral localization in
the metaphaseplates in 45.3 per cent.

The data for these different chromosome clas-
ses differed not significantly; the hypothesis of a
specific peripheral localization of the X-ring is
discarded.

313

SECTION 17

DERMATOGLYPHICS

17.1. Dermal Configurations: A Study of the Hallucai
Area of the Sole in Mongoloids, Non-Mongoloid
Mental Defectives and a Control Series.

G. F. Smith and G. M. Turral (London,
Great Britain).

In the diagnosis of Mongoloids some of the
most useful of all dermal configurations are the
patterns in the hallucai area of the sole (Ford
Walker, 1958). The usefulness of this type of
information has been somewhat limited by the
lack of information of the distribution of the
variety of patterns in different populations.
In order to compensate for this deficiency the
present study was done on both English and
American populations. The dermal ridge con-
figurations and ridge counts in the hallucai area
were statistically compared in a group of Mon-
goloid patients of both sexes. In addition a
comparison of pattern frequencies and ridge
count distributions were studied in Mongoloids,
Non-Mongoloid Mental Defectives and Normal
individuals.

17.2. Dermatoglyphics in the Diagnosis of Mongolism
and in Family Relations. M. Cerny, M. Barvic
and B. Sekla (Prague, Czechoslovakia).

An aid for the diagnosis of mongolism is
beeing based on the comparison of dermato-
glyphic patterns of mongolic patients and of
normal controls. An analysis of extensive
samples of Czechoslovak population and of
patients with Down's syndrome has shown basic
similarities with the Canadian material (N. Wal-
ker). At the same time the necessity came out
for some corrections; the use of this aid in
different demographic regions must be based
on standards derived from the populations
concerned.

An attempt has also been made of analysing
dermatoglyphic patterns of mongolic patients
as well as of those with other syndromes connected
with karyological abnormalities, in connection
of family relations.

17.3. Characteristics of Finger Prints, Palms and
Soles in Phenylketonurics. Walter Hirsch
(Chicago, U.S.A.).

Since the discovery of chromosomal aberra-
tions, various abnormal patterns have been
described in finger and palm prints. According
to present knowledge, no specific pattern or
combination of patterns should be expected. All
known patterns are occurring in normal popu-
lations too: arch, loop whorl, their borderlines,
combinations, accidentals, and defects on
fingers, palms and soles, the transversal course
of palm main lines, multiplication of achsial
triradii, the simian crease and changes in the
formation of secondary creases and white lines
and dissociations in primary and secondary
creases. But the distribution of these patterns
is different in pathological conditions and very
often this difference is highly significant.

For every one of the possible patterns the
percentage frequency in patients and controls
have been estimated and the logarithm of these
figures has been noted (N. F. Walker). The sum
of these logarithms gives a fairly reliable index
of the deviation from normal averages.

The results of finger and hand printings in
certain groups of mental retardation (Hirsch and
Geipel) and in chromosomal aberrations sug-
gested that similar deviations from the normal
might also be present in "inborn errors of
metabolism". It was the idea of D. Y. Hsia to
examine the group of about 50 phenylketonurics
under his observation. To these the author could
add about the same number of phenylketonurics.

Such a detailed study is also likely to reveal
correlations between the dermatoglyphics and
creases and some biochemical and enzyme
conditions. Preliminary studies of the author
have shown such correlations to be present and
to be significant. The results will be presented and
discussed in detail and a number of slides will
demonstrate the essential deviations from the
normal patterns.

17.4. Dermatoglyphic Anomalies Associated with
Abnormal Sex Chromosomes. Sarah B. Holt
(London, Great Britain).
Striking anomalies have been described in the

315

Section 1 7 — Dermatoglyphics

dermal ridge arrangements of persons trisomic
for autosomes. Anomalies associated with
abnormal sex chromosomes are not, on the
whole, so marked. Nevertheless, dermatoglyphic
peculiarities are discernable. Dermal prints of
cases with various abnormalities of the sex
chromosomes have been analysed. These include
the palm and finger prints of a series of cytologi-
cally diagnosed Turners, collected by Dr. J. Lind-
sten.

In Turner's syndrome, where only one X-chro-
mosome is present in a cell, three genetically
controlled quantitative characters, total finger
ridge-count, a-b count and maximal atd angle
have been studied. The means of the three
distributions differ significantly from those of a
control sample. In each case the values are higher
than in the normal males and females. Similar
differences in means occur in a small samples of
Turners with mosaicism (Xo/XX) and in other
related conditions.

It is intended to publish a detailed account of
the findings in Annals of Human Genetics, as
part of a more comprehensive survey of the
dermatoglyphics of sex chromosome abnormali-
ties.

17.5. Aberrant Dermatoglyphic Patterns and Undiffer-
entiated Mental Deficiency. Dick Hoefnagel
(Hanover, U.S.A.), Jamshed Mavalwala
(Cambridge, U.S.A.).

17.6. Dermatoglyphic Studies at the Anthropology
Research Centre, Musee de l'Homme, Paris.

Monique Gessain (Paris, France).

The Anthropology Research Centre now
possesses several thousand finger prints collected

in France, in the Department of Finistere
during the Pont-Croix surveys;
from numerous families;
from the Criminal Records Office;

/"/; Senegal from the Tendankes in the Kedou-
gou region (East Senegal).

The material collected during the Pont-Croix
surveys relates to more than a thousand subjects
from among the 3800 inhabitants of a highly
endogamous community, which, for two years,
has been the subject of a multi-survey in the
demographic, genealogical, ethnological and
anthropobiological fields.

The material collected from families includes
several hundreds of subjects belonging to 2, 3 or
4 generations.

The material from the Criminal Records
Office relates to 8000 offenders charged in
Paris, classified according to their ethnic origin.

The material from Senegal covers the larger
part of a small isolate of some 900 inhabitants.

The material is now being studied and some of
the results will be presented to the Congress by
Miss de Lestrange, Miss Pee-Laborde and Mr.
Luu. Other studies are being conducted to
evaluate genetical, statistical and anthropological
evidence from these fingerprints.

Patients of the State Schools of Brandon,
Vermont, U.S.A., and Laconia, New Hampshire,
U.S.A., numbering approximately 1500 were
examined. Several patients have been found to
display unusual dermatoglyphic patterns in
that they possess an abnormally low ridge count,
as a result of very vestigial patterns on the
fingers. This finding was associated with chromo-
somal abnormalities, not all of which could be
definitely established in a specific group or
number. All of these patients upon detailed
cytogenetic and clinical examination turned out
to belong to the group classified as "Undifferen-
tiated Mental Deficiency".

The authors believe that the dermatoglyphic
trait of an extremely low total ridge count may
be used as a screening device for ascertaining
undifferentiated mental deficiency. This tech-
nique does not pick up other conditions.

Detailed studies will be reported upon all
patients of undifferentiated mental deficiency.
Patients with other conditions such as Turner's
Syndrome, Klinefelter's Syndrome, Phenylketo-
nuria, etc., are also being investigated.

17.7. Study of the Palmar Flexion Creases.

Marie-Therese de Lestrange (Paris, France).

Genealogical study carried out in French
families, certain members of which have par-
ticular configurations of creases.

17.8. Multivariate Analysis in the Study of Dermato-
glyphics and its Application to the Study of the
Fingerprints of 7000 French Offenders (Symme-
try, Correlations and Structure). Luu Mau

Thanh (Paris, France).

The study of finger prints according to form
gives rise, generally speaking, to particular
difficulties common to all qualitative characteris-
tics.

Various methods provide estimations, for the
most part unprecise, of correlations between the
different fingers. From canonical analysis,
quantitative values can be given to different

316

Section 1 7 — Dermatoglyphics

types of finger prints (according to form) which
until now could only be accorded qualitative
values. These qualitative values enable us to
establish the matrix of correlations between
fingers by the classical method.

The fingers have a particular structure which
can be shown by giving that each finger x expresses
itself by linear combinations of some orthogonal

factors

p

Xi=zl ajeFe

e = i

By appropriate rotations, the different group-
ings of fingers can be located in the space of the
factors.

This work has been done from a sample of
some 7000 French offenders whose finger prints
are analysed according to four types of patterns:
Arc, Radial Loop, Cubital Loop, Whorl.

17.9. Some Remarks on the Study of Finger Ridge
Count. Lucienne Pee-Laborde and Luu
Mac Thanh (Paris, France).

Owing to its quantitative value, the finger
ridge count of patterns lends itself readily to
statistical analysis.

The utilization of finger ridge count, a some-
what too simple index, can give rise to certain
difficulties when an overall study is made relating
to fingers, patterns and individuals as a whole.
There are, in fact, several different populations
following each type of pattern or each finger . . .
These superpositions are discernible on the
frequency distribution curves.

A sample of finger prints of some hundred
French subjects was analysed by counting the
ridges and distinguishing between the different
forms of patterns, which has enabled us to make
a critical study of the two methods.

17.10. R L Symmetry and Asymmetry of Angles atd,
at'd, at'd and atpd and the Corresponding R
minus L difference in the Burman Males (400) —
A Probable Explanation of Serations. Abhi-
manyu Sharma (New Delhi, India).

The study reveals "assumed" symmetry
(0° or — 2° difference between R and L) that
shows the gradation as tp (230/400 = 57.50 per
cent)— 1(116/229 = 50.66 per cent)— 1'( 89/240 =
37.08 per cent)— 1"( 12/71= 16.90 per cent)
whereas total asymmetry (ranging from — 31" to
—3° and +3° to +20°) shows it as tp (159/400 =
39.75 per cent)— 1"( 19/71 =26.76 per cent)— t'

(56/240= 23.33 per cent)— 1(3 1/229= 13.54 per
cent). These figures are understandable for
t, t', t" but tp ranking number one appears
problematic only initially. Its clue lies in the
incidence of "indeterminate" cases (N or ?)
that show the gradation as t" (40/71 = 56.34 per
cent)— 1'(95/240= 39.58 per cent)— 1(82/229 =
35.81 per cent)— tp(l 1/400= 2.75 per cent) which
is indicative of fairly high incidence at t, t', t"
(from 35.81 per cent to 56.34 per cent) relative to
only 2.75 per cent at tp! High incidence of
symmetry at tp may be explained by taking
example of a person whose right shows only one
axial triradius t but located very close to t'-
segment (Sharma, 1961a: E 111; 1962a: in the
press) while his left shows only one as t' but
located fairly close to t-segment implying thereby
that both are near borderline cases between
t and t' -segments. It is indeterminate for two
categories, angles atd as well as at'd, but not if the
distalmost axial triradius tp (Penrose, 1949, vide
Penrose 1954: 10-11) is considered ignoring
threefold distinction between t' and t". Differ-
ences of 0 to : 2 (up to 2° in excess either in
rights or lefts) will add to the incidence of
"assumed" symmetry while 3° to that of
asymmetry. Angle atd shows the least extent of
morphologically-evidenced fluctuation relative to
that shown by Angles at'd and at" d that can be
explained in geometrical terms.

17.11. The Palmar Dermatoglyphics of a Group of
People of Lazio (Italy), and Some Remarks on
Methodology. G. Alciati (Rome, Italy).

The results of the examination of the palmar
dermatoglyphics of 320 individuals, all born at
Pofi, district of Frosinone (South Lazio), are
here exposed. The study was performed in
compliance with the "revised methods" (Cum-
mins et al., 1929) and is demonstrative of the
fact that the palmar epidermal ridges of the
above-mentioned group show a behaviour
similar to that of other European groups which
are already known in literature, both for their
mainly transverse direction and for their per-
centage frequency of patterns in the five palmar
areas.

The palmar prints were moreover examined
following application of the new method which
was introduced by the author during the Ilnd
International Conference of Human Genetics
(Rome, 1961); the results thus obtained were
compared with those derived from the above
mentioned "revised methods": a close relation-
ship was noticed, wherefrom the author draws
the cue for some practical considerations.

317

Section 1 7 — Dennatoglyphics

17.12. Eurasians Digital and Palmar Dermatoglyphs.

George Olivier (Paris, France).

The fingerprints of 160 French-Vietnamese
crosses (100 male and 60 female) are studied.
They are compared with two new series of 500
Frenchmen and 250 Vietnamese. The charac-
teristics of the female group have been estimated
approximately.

In general, the frequencies of the different
patterns fall between those of the Vietnamese
and those of the French. In males, they get
nearer to the Vietnamese frequencies; in females
they get nearer to the French frequencies; trans-
mission thus seems to vary somewhat according
to sex. The dominance of digital loops (Euro-
pean type) found by Abel, Cummins, Kimura
and Mikami, have not been found. The Viet-
namese realize at a maximum the dermato-
glyphic patterns typical of the xanthodermic
populations.

The transverse palmar fold (simian fold) is
three times more frequent in Vietnamese than in
French populations. Its frequency in Eurasians
is intermediate.

The only positive results are:

1. The radial loops of the hypothenarian
eminence seem to be recessive (this contradicts
Kimura).

2. The thenarian patterns seem to be recessive.

17.13. On the Optimal Extraction of Genetical
Information from Fingerprints. Friedrich
Keiter (Hamburg, Germany).

Fingerprints (and toeprints) are highly patter-
ned, tenfold, in a complicated manner intercor-
related morphological traits. The tenfinger-
ridgecount is a very incomplete indicator of
genetical information contained in them. Direct
finger-to-finger-comparison on ridgecounts and
on degree of similarity in controlled estimation
yields 20 (with toeprints 40) single data, whose
score can be combined into a simple discrimi-
nant system (method of "Trennlogarithmus",
separating logarithms). In a sample of Mato
grosso-indians (Chavantes) by this procedure
the "critical values" (ratio of the frequency of
the same finding in related and random pairs)
have been found 8.40:1 for pattern similarity,
2.85:1 for finger-to-finger-ridgecount against
1.22 only for ten finger-ridgecounts alone. The
same multiplied discrimination effect of parents-
children or sibs compared with randomly
chosen pairs of nonrelated individuals is well
established for Northern Germany since 1955.

Agreement with or deviation from multifac-
torial-additive inheritance can be checked using
the same principles.

17.14. Genetics of the a-b Ridge Count on the Human
Palm Prints. Jose Pons (Oviedo, Spain).

The present study deals with the distribution
of the number of ridges between the triradii a
and b of the human palms. As an individual
trait the sum of the a-b counts of the two hands
is used and, as a measure of bimanual asymmetry
we analyze the differences between hands (right
minus left). After some brief remarks on racial
differences, we consider the heritability of this
trait by calculating the intraclass correlation
among sibs, as well as the parent-child correla-
tions.

For the study in the general population a
series of 412 Spaniards (200 males and 212
females) is used, comprising unrelated persons
only. For analysing the heritability we have a
series of 307 sibs falling into 123 sibships and of
57 families composed of both parents and at
least one child.

17.15. Inheritance of Dermatoglyphic Formulae. — I.
Ulnar Loops in Complete or Partial Symmetri-
cal Formulae.C. Lazaro (Montevideo, Urugay).

In a previous work we have found that the
observed frequency of the ten most common
dermatoglyphic formulae for the population of
Montevideo departs significantly from the cal-
culated one. Based on this conclusion, we pre-
sent in this paper the results of the study of
ulnar loops in complete or partial symmetrical
formulae in all possible combinations in 200
families taken at random in our population.

The results of our data do not show apparently
any simple mendelian mechanism of inheritance;
they rather suggest the action of a polygenic
system, governing the genetics of dermato-
glyphic formulae.

17.16. The Hereditary Pattern of the Quantitative
Value of Fingerprints from a Critical Aspect.

Margarete Weninger (Vienna, Austria).

Among the characteristics of the digital pat-
terns the importance of the quantitative value
(QV) has long ago been stressed. In spite of the
great difficulties resulting from the sexual dif-

318

Section 1 7 — Dermatoglyphics

ference of the QV, from its bimanual asym- proaches Bonnevie's first hypothesis in that it

metry and, above all, from the differences be- explains the individual QV, i.e. the value of the

tween the finger categories, researches have been 10 fingers combined as the effect of additive

made to detect the genetic background of the genes.

QV. The respective paper is designed to the prob-

K. Bonnevie (1924) developed a hypothesis of lem from a critical point of view.
5 pairs of polymeric genes in order to explain
the hereditary pattern of the so-called individual

QV. This hypothesis was rejected by herself a The paper will be probably published in full

few years later in favour of another one. in the Mitt.d.Anthropol. Ges. Wien or in the

The modern English biometrical school ap- Zeitschr. f. Morphol. u. Anthrop.

319

SECTION 18

CITATION INDEXING

18.1. Dissemination and Retrieval of Genetics In-
formation Through Interdisciplinary Citation
Indexing. Eugene Garfield and Irving H.
Sher (Philadelphia, U.S.A.).

The difficulties in handling the highly inter-
disciplinary genetics literature by conventional
methods led to a study of citation indexing, a
method used in legal literature. Citation indexing
overcomes the terminological barriers of lan-
guage-oriented systems and does not require
scarce scientifically trained indexers. It uniquely
discloses the bibliographic descendants of ear-
lier papers rather than their antecedants — as in
conventional systems. A citation index iden-
tifies the subsequent papers citing earlier target
papers.

For three years methodologies for compiling
citation indexes by computer have been studied.
Several experimental indexes were compiled
and compared to other systems. Over 2,000,000
citations from the published literature have
been processed! Invaluable bibliographical,
sociological, and historical statistical data have
been obtained as by-products and various
author, journal, and chronological utilization
factors determined.

Citation indexing is based upon a posteriori
indexing by citing authors, making the litera-
ture a dynamic tool for creative research. New
or previously unknown correlations and in-
sights are obtained since the citation index
integrates the "old" literature with the new,
often in dramatic ways. The enormous prolif-
eration of the literature, anathema and disabling
in conventional a priori systems is highly
beneficial in citation indexing. Its self-organizing
properties automatically increase specificity as
the literature grows. As citation input is
increased one approaches critical mass — the
coverage necessary to insure a known degree
of reliability for a negative search result, an
important indicator that new research is needed
or justified.

18.2. (D) The Genetics Citation Index. Irving H.
Sher and Eugene Garfield (Philadelphia,
U.S.A.).

A citation index is a directory of cited litera-
ture references, each of which is accompanied
by a list of citing source documents. Typically,
the user specifies a target paper in which he is
interested. In the citation index he locates the
author and specific article first. From there, the
index displays the more recent papers that have
cited the target paper, thus bringing him for-
ward in time through subsequent interdis-
ciplinary developments. We have prepared sev-
eral types of genetics citation indexes for com-
parative evaluation including:

1. Comprehensive interdisciplinary citation
indexes compiled from 2,000,000 references.

2. A Genetics Citation Index selectively ex-
tracted from a file of 1.4 million references
appearing in 550 life science journals
published in 1961.

3. Genetics Citation Indexes derived from
long runs of "hard core" genetics journals.

Microfilms of the indexes will be displayed and
a sample search of the files will illustrate the
unique speed and power of this new tool for
information retrieval.

In contrast to conventional systems, valuable
information is obtained from a Citation Index,
even when no citations are disclosed, since this
implies that the target paper has not been ex-
tended. A demonstration of "cycling" will
show how a search can be expanded by selecting
new targets from the bibliographies of papers
which cite the target paper, continuing the
process until an optimum list of papers has
been retrieved. After the pedagogical demonstra-
tion, spontaneous literature searches will be con-
ducted for each visiting geneticist and copies of
the search bibliography provided.

321

INDEX OF AUTHORS

Aastveit, K. 13.16.
Abbadessa, R. 2.3.
Abdel-Azim, O. T. 9.72.
Abeelen, J. H. F. v. 14.4.
Abel, W. O. 2.13.
Abplanalp, H. 14.31.
Adam, A. 15.23.
Ahlstrom, C. G. 16.37.
Ahmed, I. A. 14.58.
Ahuja, M. R. 7.4.
Aigaes, N. S. 13.23.
Akira, Y. 6.7.
Alciati, G. 17.11.
Alderson, T. 5.31.
Alexander, M. L. 5.45.
Alikhanian, S. I. 5.5, 5.10.
Allard, R. W. 9.17.
Allderdice, P. W. 8.16.
Altenburg, E. 1.4., 1.7.
Altwerger, L. 4.48.
Ames, B. N. 4.8.
Ammann, F. 15.48.
Amos, D. B. 11.19.
Anagnostopoulos, C. 4.51.
Anders, A. 10.27.
Anders, F. 10.27.
Anderson, L. E. 6.1.
Andor, B. 13.104.
Andresen, E. 11.8.
Angeloff, L. G. 14.24.
Angulo, D. 5.90, 6.38.
Appella, E. 4.46.
Ardhendu, M. 10.7.
Arkel, G. A. van, 3.19.
Arnason, T. J. 5.106.
Arnold, P. 14.1.
Arsenieva, M. A. 5.79.
Arvidson, R. B. 14.37.
Ascenso, J.C. 13.57.
Ash, W. J. 10.48.
Ashton, G. C. 14.48.
Ashri, A. 12.9.
Asker, A. A. 14.58.
Astaurov, B. L. 14.18.
Atwood, K. C. 4.22.
Aubertin, M. 12.15.
Augustin, M. 16.22.
Aust, J. B. 11.25.
Avanzi, S. 5.91.
Axford, R. F. E. 14.57.

Bach, F. 16.43.
Backstrom, L. 8.19.
Bader. S. 16.14.
Badr. F. M. 14.9.
Badtke, G. 10.40, 10.41.
Baglioni, C. 15.75.
Bahrs, B. 15.61.
Bailey, D. W. 11.26.
Baker, L. H. 14.37.
Baker, L. N. 11.8
Baker, S. 5.55.
Baker, W. K. 4.12.

Bakhteyev, F. K. L. 8.12.
Bakulina, E. D. 5.79.
Balaban, G. 6.48.
Band, H. T. 9.38.
Bankowska, H. 13.36.
Barak, E. 5.49.
Barat. M. 4.52.
Barbacki, S. 13.97.
Barbour, S. D. 10.4.
Barbour Bendbow, E. 2.3.
Barigozzi, C. 12.19.
Barrai, 1. 15.33.
Barthelmess, A. 5.99.
Barvic, M. 17.2.
Basak, S. L. 7.30.
Bass, I. A. 3.5.
B axeman, A. J. 5.41, 5.42.
Bateman, N. 14.11.
Battaglia, B. 9.26.
Bauchinger, M. 5.99.
Baumiller, R. C. 5.54.
Bayubay, S. J. 13.90.
Beard, B. H. 13.92.
Beardmore, J. A. 9.39.
Bearn, A. G. 15.78., 15.83.
Beatty, R. A. 14.12.
Becak, M. L. 8.17., 15.25.
Becak, W. 8.17, 15.25.
Beck, S. L. 10.42.
Becker, W. A. 9.32.
Beckwith, J. R. 4.9.
Bedeir, L. H. 14.58.
Belajev, D. K. 14.5.
Belitz, H. J. 5.50.
Bell, A. E. 14.20, 14.21.
Bell, S. 5.81.

Benazzi Lentati, G. 14.13.
Benazzi, M. 14.13.
Bender, H. A. 10.12.
Bender, M. A. 16.31.
Bennett, D. 1.10.
Ben-Gurion, R. 5.21.
Ben-Zeev, N. 5.55.
Berendes, H. D. 6.36.
Berg, K. 15.27.
Berg, R. L. 9.40.
Bergann, F. 10.29.
Bergen, P. 13.45.
Berghe, H. van den, 16.19.
Berman, J. D. 15.63.
Bernard, R. 16.27.
Bernini, L. 15.82.
Bernoco, D. 14.53.
Bernstein, N. 5.49.
Bernstein, S. E. 10.52.
Bertucci de Lozzio, C. 5.87.
Bevan, E. A. 5.13, 12.6. 12.7.
Bhaskaran, S. 5.109.
Bhattacharjee, J. K. 2.15.
Bianchi, A. 5.110.
Biedler, J. L. 16.36.

BlELAWSKA, H. 2.7.

Binet, F. E. 9.12.
Binnard, R. 6.47, 6.48.

323

Index

Biola, M. T. 6.37.

BlRNAURE, I. 13.34.

Birsan, M. 13.34.
Blomback, B. 3.8.
Bochnig, V. 5.59.
Bodmer, W. F. 3.34.
Bogyo, T. P. 9.32.
Bohidar, N. R. 9.14.
Bohme, H. 3.44.
Bolthuis, J. VAN, 16.26.
Bolognesi, M. 15.7.
Bolsunov, I. 13.6.

BORAKER, D. 11.3.

Borek, E. 3.4.

Borojevic, K. 5.114.

Borojevic, S. 13.44.

Borstel, R. C. von, 7.23., 9.27.

Borthwick, M. 3.14.

Bosch, J. van den, 15.42.

Bosiger, E. 9.41.

Boskovic, E. M. 13.46.

Bouharmont, J., 13.18.

Bouw, J. 11.12.

Bowler, K. 9.42.

Boyd, W. J. R. 13.49.

Boyer, J. P. 14.35.

Boyer, S. H. 15.84.

Boyes, J. W. 8.22.

Bozzini, A. 5.115.

Bradley, S. G. 2.16.

Bradshaw, A. D. 9.2.

Braend, M. 11.11.

BRAGD0, M. 13.19.

Brawner, T. G. 4.38.

Bray, M. 11.2.

Breg, W. R. 16.5., 16.14.

Bremer, G. 6.40.

Bremer-Reinders, D. E. 13.24.

Bresler, S. E. 5.3.

Brewbaker, J. L. 13.9.

Brick, Z. 13.63.

Bridges, B. A. 5.120.

Brieger, F. G. 9.25.

Briganti, G. M. 13.68.

Briggs, B. G. 8.1, 13.7.

Brink, J. M. van, 8.22.

Brito da Cunha, A. 9.45.

Brix, K. 7.10.

Brncic, D. 9.43.

Brown, K. S. 11.30.

Brown, M. S. 8.3.

Browning, L. S. 1.4, 1.7.

Bruins, J. W. 16.26.

Bruyn, G. W. 15.47.

Bryan, J. H. D. 6.39.

Bryssine, P. 5.119.

Buiatti, M. E. 5.93.

Bull, A. L. 10.11.

Bunzo, S. 8.29.

Burdick, A. B. 2.3.

Burk, R. R. 4.41, 4.42.

Burnet, B. 10.26.

Burnett, J. B. 4.33.

Burnham, C. R. 6.30.

Burns, J. A. 4.26.

Burt, B. E. 6.42.

Burton, G. W. 13.61.

Bush, D. J. 1.11.

Butler, L. 13.72.

Butterfass, T. 10.35.

Buxton, E. W. 3.45.

Cacheiro, N. 16.3.
Calef, E. 3.21, 4.15.

Calet, C. 14.35.

Cameron, D. R. 12.10.

Campos, F. F. 5.118.

Carey-Parker, W. 15.78, 15.83.

Carlson, E. A. 5.30.

Carsiotis, M. 4.46.

Carson, H. L. 5.57.

Carter, R. C. 14.43.

Casciano 3.48.

Case, M. 1.17.

Caspari, E. W. 5.88.

Caulton, J. 11.13.

Centeno, A. J. 5.62.

Cerny, M. 17.2.

Cessain, M. 17.6.

Cetrulo, S. D. 5.9.

Chai Hyun, Y. 3.1.

Chandley, A. C. 5.41., 5.42.

Chandra, M. 13.69.

Chang, S. T. 13.52.

Chase, S. S. 13.60.

Chen, C. C. 6.32.

Chen, C. H. 7.11.

Chessin, L. N. 16.46.

Chiarelli, B. 16.1.

Chiasson, L. P. 15.43.

Childs, J. D. 4.5.

Chiyoko, T. 10.6.

Chouinard, L. A. 6.41.

Chovnick, A. 1.1, 1.2, 4.28.

Chozo, O. 9.60.

Christakos, A. C. 16.5.

Chu, E. H. Y. 5.84.

Chung-Suk Kim, 13.32.

Ciofu, A. 13.34.

Clarke, C. A. 11.32.

Clarke, C.H. 5.16.

Cavilier, L. 4.10.

Cleve, H. 15.78.

Cock, A. G. 14.32.

Cockerham, C. C. 9.23.

Cockrem, F. 9.33.

Coe, E. H. (Jr.) 4.23.

Cohen, B. H. 15.32.

Cohen, J. A., 3.16, 3.29.

Cohn, N. S. 5.82.

Colberg, J. E. 1 1.17.

Connolly, K. 14.1.

Constantin, M. J. 5.103.

Cooper, T. P. 10.33.

Cordeiro, A. R. 5.60, 5.61, 5.62, 9.59.

Costello, W. P. 5.13.

Coston, J. 5.2.

Coutinho, P. 13.88.

Cove, D. J. 4.36, 4.37.

Cox, B. S. 2.21.

Cox, D. F. 5.68.

Coy, D. O. 3.47.

Craciun, T. 13.34.

Craig, G. B. (Jr.) 10.9.

Cresseri, A. 15.24.

Cribbs, R. 4.6.

Crippa, M. 8.20.

Crosby, J. L. 9.15.

CSUKAS-SZATLOCZKY, I. 10.19.

Cunha, A. B. da 9.45., 9.46.
Cushing, J. E. 11.3.
Cuzin, F. 4.16.

Da Cunha, A. B. 9.46.
Dahl, B. 3.39.
Dahlberg, J. 3.6.
Daigoro, M. 9.27.
D'Amato, F. 5.115.

324

Index

Dassat, P. 14.53.

David, J. 10.53.

Davidson, R. T. 15.58.

Davies, W. I. C. 8.15.

Davis, G. 7.31.

Davis, R. H. 4.43.

Dawson, G. W. P. 4.20.

De Capoa, A. 16.20.

De Carli, L. 16.39.

Dee, J 10.55.

De Fries, J. C. 5.64.

Degenhardt, K. H. 10.40, 10.41.

De George, F. V. 15.54.

Degnbol, B. 15.31.

de Groot, B. 3.22.

de Laage, X. 14.35.

Del Bianco, C. 15.82.

Delden, W. van, 9.39.

De Lestrange, M. T. 17.7.

De Loecker, W. 16.19.

De Lozzio, C. B. 5.87.

Del Vecchio, V. 4.35.

de Magalhaes, L. E. 9.45, 9.46.

Dempster, E. R. 14.31.

De Moor, P. 16.19.

Dent, T. 16.16.

Den Tonkelaar, E. M. 16.7.

Dfol, M. S. 10.44, 10.45.

Derlogea, V. 14.38.

de Robichon-szulmajster, H. 3.53.

de Sa, J. 3.33.

De Serres, F. J. 1.16., 2.10.

de Toledo, J. S. 9.45, 9.46.

de Toledo, S. A. 9.45.

De Vries, A. 15.60.

De Zorrilla, N. B. 8.30.

Dhillon, T. S. 6.44.

Dickie, M. M. 10.15.

Dietrich, H. F. 13.106.

Dionne, L. A. 13.14.

Di Pasquale, A. 10.25.

Djaldetti, M. 15.60.

Dodinval, P. 15.64.

DODINVAL-VORSIE. J. 15.64.

Donohoe, W. T. A. 15.72.
Doolittle, R. F. 3.8.
Doschek, E. 7.29.
Douglas, L. T. 6.21.
Downs, W. G. 14.8.
Dozortseva, R. L. 5. 113.
Drawert, F. 10.27.
Dray, S. 11.17.
Dressler, L. 15.23.
Driscoll, C. J. 7.19.
Dronamraju, K. R. 15.17, 15.49.
Dryagina, I. V. 13.79.
Dubinin, N. P. 5.36, 5.73.
Duff, I. F. 15.58.
Duggaun, H. E. 16.30.
Dunn, L. C. 1.10.
Duijn, P. van, 16.7.

Eberle, P. 16.2.
Edward Quinn, C. 11.5.
Edwards, J. H. 15.5., 16.16.
Ehling, U. H. 5.76.
Ehrman, L. 9.47.
Em, S. E. 6.46., 8.10.
Eigsti, O. J. 13.59.
Eisenstark, A. 5.15.
Ekfr, R. 10.49.
El-Itriby, A. A. 14.58.
Ellis, J. R. 13.3.
Eloff, G. 9.48.

El-Sadek, L. M. 5.106.
Emerson, S. 10.22.
Engels, J. P. 15.58.
Englesberg, E. 4.6.
Enikeyev, Kl. K. 13.13.
Epstein, C. J. 3.7.
Eriksson, A. W. 15.21.
Ernhaft, J. 10.39.
Esser, K. 4.45.
Evans, D. A. P. 15.72.
Evans, L. E. 8.14.
Ezra, R. 15.60.

Fabian, G. 10.39.
Fahmy, M. J. 5.32.
Fahmy, O. G. 5.32.
Fainer, D. 15.84.
Falk, R. 5.49., 5.55.
Fallon, G. R. 14.48.
Faludi, B. 13.87.
Faludi-Daniel, A. 13.96.
Fantoli, U. 15.7.
Fasculas, A. 13.107.
Fechheimer, N. S. 14.39., 14.42.
Fedorcsak, I. 3.41.
Fejer, S. O. 9.3.
Feldman, M. 13.63.
Fernandez, G. 6.3.
Ferrando, M. 13.34.
Filippov, V. D. 3.43.
Fincham, J. R. S. 4.42.
Finley, S. C. 16.23.
Finley, W. H. 16.23.
Finn, R. 11.32.

FlRSCHEIN, I. L. 15.15.

Fischer, R. 15.67., 15.68.

Fischer-Fantuzzi, L. 3.21, 4.15.

Fleissner, E. 3.4.

Fling, M. 4.34.

Florea, I. 16.22.

Forrest, H. S. 1.9.

Forsius, H. 15.21.

FORSTHOEFEL, P. F. 10.47.

FORTSON, J. C. 13.61.
Fraccaro, M. 16.13.
Frahm-Leliveld, J. A. 8.4.
Franceschetti, A. 15.48.
Francisco, H. G. 15.69.
Frankel, F. R. 3.11.
Frankel, O. H. 10.31.
Frankel, R. 12.14.
Fraser, G. R. 15.50.
Fredericq, P. 4.19.
Fregin, A. 11.13.
Fried, V. 3.6.
Friedman, J. 11.13.
Fries, J. C. De, 5.64.
Frus, J. 1.18.

Froese-Gertzen, E. E. 5.105.
Fuerst, R. 4.39.
Fuller, J. L. 14.2.
Fuscaldo, K. E. 4.35.

Gaillard, J. L. G. 16.7.
Gaines, J. A. 14.43.
Gajewski, W. 2.7.
Gamble, A. N. van, 16.5.
Ganesan, A. T. 3.34.
Gans, M. 3.46.
Garber, E. D. 7.5.
Gardner, E. J. 15.53.
Garfield, E. 18.1, 18.2.
Gartlery, S. M. 6.42.

325

Index

Gay, H. 6.8.
Gedda, L. 15.6.
Geissler, E. 3.26.
Gell, P. G. H. 11.18.
Generoso, W. M. 5.118.
Gerald, L. 11.17.
German, J. L. 16.8.
Gershenson, S. 3.36.
Gershon, D. 14.10.
Gershowitz, H. 15.35., 15.36.
Gerstel, D. U. 4.26.
Gessain, R. 15.20.
Geyer-Duszynska, I. 7.34.
Gibson, I. 12.1.
Gibson. J. B. 9.49.

GlLDENHUYS, P. 7.10.

Giles, J. A. 13.39.

GlLLHAM, N. W. 12.3.

Gilmore, L. O. 14.39., 14.42.

Gimenez-Martin, G. 6.3., 8.18.

Giraud, F. 16.27.

Giraud, P. 16.27.

Glassman, E. 4.29.

Glavinich, R. 13.102.

Glavinich, R. D. 13.55.

Glazer, V. M. 3.43.

Glenn-Wolfe, H. 14.7.

Gloushcenko, I. E. 13.26, 13.27, 13.28.

Glover, S. W. 3.23, 3.24.

Goeminne, L. 15.46.

Goldberger, R. F. 3.7.

Goldin, E. 12.9.

Goldschmidt, E. 5.49.

Gollub, E. G. 4.50.

Gompachiro, Y. 6.11.

Gonzalez-Fernandez, A. 6.3.

Gooch. P. C. 16.31.

Goodale, H. D. 9.35.

Gorla, M. S. 12.19.

Gorlenko, G. M. 3.5.

Gots, J. S. 4.50.

Gottlieb, F. J. 10.16.

Gough, F. J. 13.48.

Gowen, J. W. 5.69, 5.71, 5.72, 9.52.

Graham, K. M. 13.14.

Grant, C. J. 5.95.

Grant, W. F. 8.5.

Green, E. L. 5.70.

Green, M. M. 10.12.

Greenblatt, G. M. 6.2.

Gregg, T. G. 7.32.

Grell, E. H. 2.2.

Grell, R. F. 2.1.

Grenson, M. 12.5.

Griffin, F. 15.67.

Gripenberg, U. 16.38.

Grobman, A. 13.58.

Grogan, C. O. 12.12.

Groot. B. de, 3.22.

Grosch, D. S. 5.53.

Gross, J. D. 4.17.

Gross, S. R. 1.15.

Grun, P. 12.15.

Grundbacaer, F. J. 15.70.

Gugler, H. D. 5.33.

Gurau, L. 14.54.

Gustavson, K. H. 16.23.

Guthrie, R. 15.66.

Guttes, E. 3.9.

Guttes, S. 3.9.

Guttman, R. D. 11.25.

Gutz, H. 1.19.

Gyorffy, B. 3.28.

Gyurjan, I. 13.95.

Hackel, E. 11.33.
Haefner, K. 5.27.
Haga, T. 9.22.
Hagemann, R. 2.9.
Hagman, M. 13.8.
Hala, K. 14.49.
Halfer, C. 11.6.
Halkka, O. 6.10.
Halle, E. S. Von, 1.3.
Hamlin, H. 15.55.
Hanna, B. L. 15.4.
Hannah-Alava, A. 5.38.
Hansen, H. G. 16.24, 16.25.
Hansen-Melander, E. 6.14.
Hanson, W. D. 2.5.
Harle, J. R 5.108.
Haro, F. G. 15.69.
Harrington, F. E. 9.36.
Harris, D. L. 9.16.
Harris, J. M. 11.29.
Hartman, P. E. 4.8.
Hartshorne, J. N. 13.70.
Hartung, M. 16.27.
Harvald, B. 15.31.
Harvey, W. R. 14.45, 14.51.
Haselkorn, R. 3.6.
Hashem, N. 16.42.
Haskell, G. 13.42.
Hassanein, S. H. 13.109.
Hauge, M. 15.31.
Hauschteck, E. 8.28.
Hawthorne, D. C. 3.49.
Hecht, A. 13.10.
Hecht, F. 16.21.
Heed, W. B. 8.25.
Hefner, R. E. 5.105.
Heldenmuth, L. H. 3.35.
Helling, R. B. 4.7.
Henderson, S. A. 6.28.
Heni, P. 16.29.
Henricson, B. 8.19.
Herschler, M. S. 14.42.
Herzenberg, Leonore A. 11.22.
Hetzer, H. O. 14.51.
Hess, O. 6.26.
Heuts, M. J. 9.75.
Hexter, W. M. 1.6.
Heyne, E. G. 13.47.
Hildreth, P. E. 10.8.
Hill, D. L. 9.36.

HlLLMAN, R. 10.4.

Hinrichsen, J. K. 14.50.
Hinako, S. 13.99.
Hirono, Y. 2.20.
Hirsch, W. 17.3.

HlRSCHHORN, E. 13.101.

Hirschhorn, K. 16.18., 16.43., 16.44.
Hitoshi, K. 13.65.

HOCHMAN, B. 1.5.
Hoecker, G. 11.20.
HOEFNAGEL, D. 17.5.
HOF, J. VAN 'T, 5.98.

Hojnv, J. 14.49.
Holden, J. H. W. 8.11.
HOLLIDAY, R. 2.14.

Hollingsworth, M. J. 9.42.
Holm, G. 1.12.
Holt, S. B. 17.4.
Hooven, N. R. (Jr.) 14.45.
Horowitz, N. H. 4.34.
Hottinguer-de-Margerie, H. 5.26.
Hsia, D. Y. Y. 15.59., 15.63.
Hubacek, J. 3.42.
Huchns, E. R. 16.21.
Hull, P. 9.34.

326

Index

Humihiko, O. 8.29.
Hunziker, J. H. 8.13.
Hurst, R. L. 9.14.
Hustinx, W. J. 16.47.
Hutchison, D. J. 13.89.
Hwang, Y. L. 2.15.
Hyun, S. K. 13.32.

Ingraham, L. 3.11.
Inhorn, S. L. 16.17.
Ionescu, M. 13.34.
Irgens-Moller, H. 9.4.
Issaev, S. I. 13.94.
Ivanascu, M. 13.34.

Jaap, R. G. 14.34.
Jackson, C. E. 15.45.
Jacob, F. 4.16.
Jain, H. K. 7.30.
Jain, S. K. 9.17.
Jakob, H. 3.50.
Jakovcic, S. 15.59.
Jalling, B. 16.23.
James, A. P. 5.28.
James, S. M. 11.4.
Janick, J. 13.41.
Jansz, H. S. 3.16, 3.29.
Jaranowski, J. 8.8.
Jaynes, R. A. 12.8.
Jenkins, J. A. 13.73.
Johnson, F. N. 14.6.
Johnson, L. A. S. 8.1.
Johnston, C. O. 13.47.
Johnston, T. D. 13.81.
Jones, K. 7.9.
Joset, F. 5.24.
Judd, B. H. 1.8.
Jurasits, P. 8.16.

Kaczmarczyk, W. 4.35.

Kada, T. 5.22.

Kafer, E. 2.18.

Kalmus, H. 11.29.

Kamra, O. P. 10.28.

Kaplan, A. R. 15.67., 15.68.

Kaplan, W. D. 5.33.

Karp, G. W. 15.81.

Kato, R. 16.37.

Kaudewitz, F. 3.18.

Kazimierski, T. 13.1.

Kcesin, R. B. 3.5.

Keeler, C. E. 15.18.

Keep, E. 13.5.

Keiter, F. 17.13.

Keller, E. C. (Jr.) 9.50.

Kelton, D. E. 10.46.

Kelus, A. S. 11.18.

Kenichi, K. 9.51.

Kennedy, J. F. 14.47.

Kerkis, J. J. 5.74.

Kernaghan, R. P. 1.1, 1.2, 4.28.

Khan, P. M. 15.17.

Khan, S. U. T. 7.18.

Khattab, A. G. H. 14.57.

Kherumian, R. 15.19.

Khitrinsky, V. F. 13.86.

Khush, G. S. 7.2.

Khvostova, V. V. 5.100., 13.23.

Kidd, K. K. 5.33.

Kidson, C. 16.41.

Kidwell, J. F. 9.52.

Kihara, H. 13.65.

Kihlman, B. A. 5.83.
Kikkawa, H. 4.27.
Kilbey, B. J. 5.18., 5.25.
Kim, Ch. S. 13.32.
King, R. C. 10.1.
Kirk, R. L. 15.78.
Kishimoto, K. 15.40.
Kitagawa, O. 9.57.
Klaus, D. 16.29.
Klein, D. 15.48.
Klingmuller, W. 4.3.
Kloepfer, H. W. 15.51.
Knight, R. L. 13.5.
Knolle, P. 3.18.
Kobayashi, Y. 15.40.
Kobozieff, N. 10.50.
Koetn, H. I. 11.26.
Kohoutova, M. 3.31.
Koizumi, S. 11.2.
Kojima, K. I. 9.51.
Kolmark, H. G. 5.18.
Kondo, S. 5.18.
Konicek, J. 3.31.
Konzak, C. F. 5.105, 5.108.
Koo, F. K. S. 5.85.
Koopmans, A. 6.6.
Kopecka, H. 3.31.
Koref-Santibanez, S. 9.43, 9.44.
Korn, R. W. 10.20.
Kossikov, K. V. 3.52.
Kostin, I. G. 14.30.
Kozinski, A. W. 3.12.
Kravina, A. M. 12.19.
Kriebel, H. B. 13.33.
Krimbas, C. B. 9.5.
Krivisky, A. S. 5.4.
Krooth, R. S. 16.40.
Kruger, H. 16.35.
Kruszewska, a. 2.7.
Kruzhilin, A. S. 13.27.
Kubitschek, H. E. 5.8.

KUHLMANN, W. 5.80.

Kunkel, H. A. 5.14., 5.35.
Kupzow, A. J. 9.24.
Kushner, H. F. 14.30.
Kuzmin, V. P. 13.85.
Kuznetzov, N. I. 14.30.
Kyle, W. H. 9.35.

Laage, X. de, 14.35.
Lacour, L. F. 6.18.
Lacroute, F. 4.1 1.
Lacy, A. M. 1.14.
Laguaite, J. 15.51.
Lakovaara, S. T. 10.18.
Lampkin, G. H. 14.47.
Langer, B. 9.70.
Lanna, M. T. 14.16.
Laskowski, W. 5.27.
La Torretta, G. 15.82.
Laughnan, J. R. 7 13.
Lauvergne, J. J. 14.41.
Law, G. R. J. 11.15.
Lazarescu, C. 13.31.
Lazaro, C. 17.15.
Lebfuf, M. 16.27.
Leck, J. M. 15.5.
Ledingham, G. F. 8.6.
Lee, N. 4.6.
Lee, W. J. 7.7.
Legault, R. R. 5.108.
Lehmann, W. 16.24, 16.25.
Leigh, B. 5.39.
Lengerova, A. 5.65, 11.23.

327

Index

Leon, N. 15.22, 15.25.

Leonard, A. 5.77.

Leslie, I. 4.1.

Lestrange, M. Th. de, 17.7.

Leupold, U. 1.18.

Levan, A. 16.37.

Levene, H. 9.18.

Levine, L. 14.3.

Levine, M. 3.14.

Levins, R. 9.19.

Li, H. W. 6.32.

Li-Chun-Li, L. 4.39.

Lieb, M. 3.27.

Lilly, L. J. 5.17.

LlNDAHL-KlESSLlNS, K. 16.4.

Lindegren, C. C. 2.15.

LlNDEGREN, G. 2.15.

Linden, D. B. 5.111.

LlNDSTEN, J. 16.13.
LlNNERT, G. 5.117.

Lints, F. A. 9.53.

LOECKER, W. DE, 16.19.

Loen, A. van, 14.40.
Lopez-Saez, J. F. 6.3, 8.18.
Lorber, T. 15.39.
Lorenz, H. 8.16.
Lorenzetti, F. 13.68.
Lowry, D. C. 14.31.
Lu, K. C. L. 6.32.
Lucchesi, J. C. 10.3, 10.8.
LiiERS, H. 5.29.
Luers, T. 16.10.
Lukyanenko, P. P. 13.91.
Lunden, A. O. 5.103.
Lush, 1. E. 9.31.
Luu, M. T. 17.8., 17.9.
Luzzati, M. 4.10.
Lyon, M. F. 7.33.

Mc Clure, W. H. 14.43.

Mc Connell, R. B. 15.72.

Mc Donald, D. J. 9.30.

Mc Fall, C. 4.39.

Mc Farland, E. C. 10.51.

Mc Ginnis, R. C. 13.49.

Mach, B. 4.52.

Maciel, C. P. 5.63.

Mc Keown, T. 15.5.

Mc Laurin, J. W. 15.51.

Mc Leish, J. 6.27.

Mc Leod, C. M. 3.32.

Mc Leod, H. 4.34.

Mc Nary, H. W. 14.20.

Magalhaes, L. E. de9.45., 9.46.

Magni, G. E. 2.8.

Mahoney, D. L. 13.41.

Maillis, M. 5.2.

Mainx, F. 7.29.

Maio, J. J. 16.39.

Maisin, J. R. 5.77.

Makarewicz, A. 2.7.

Makower, M. 12.6., 12.7.

Malchair, R. 15.64.

Maling, B. 4.48.

Malogolowkin, C. 9.54.

Mann, T. J. 9.23.

Marcovich, H. 5.22, 5.24, 5.26.

Mariani, A. 13.68.

Marquardt, H. 5.11, 12.13.

Marques, E. K. 5.63.

Marshak, A. 3.2.

Marsico, S. 15.82.

Marsters, R. 15.68.

Martin, F. W. 13.11.

Martin, T. G. 9.36.
Mather, W. B. 9.55.
Mathon, C. C. 13.29.
Matousek, J. 14.49.
Matousek, V. 5.65, 11.23.
Matsui, M. 15.40.
Mattoni, R. H. T. 9.8.
Matzinger, D. F. 9.23.
Maumus, L. 8.13.
Mavalwala, J. 17.5.
Maximilian, C. 15.38., 16.22.
Mazoti, L. B. 12.11., 13.67.
Melander, Y. 6.15
Merat, P. H. 14.26.
Mergen, F. 5.112.
Merrell, D. J. 9.6.
Merritt, A. D. 15.4.
Merritt, E. S. 14.33.
Meunier, A. 15.9.
Meyer, G. F. 6.26.
Mickey, G. H. 5.51.
Mikio, M. 13.62.
Mikkelsen, W. M. 15.58.
Milani, R. 14.17.
Milani-Comporetti, M. 15.7.
Milcu, St. M. 16.22.
Miller, J. O. 16.14.
Miller, J. R. 10.43.
Miller, M. W. 5.13.
Miller, O. J. 16.5.
Milohnic, J. 13.20.
Minocha, J. L. 5.106.
Mirjuta, J. P. 13.93.
Mishell, R. I. 11.22.
Misic, P. D. 13.25.

MlTTWOCH, U. 16.15.

Mkrtumian, N. M. 5.5.
Moens, P. B. 7.3.
Moes, A. 5.104.
Mohler, J. D. 7.28.
Mohr, J. 15.26.
Mojaieva, V. S. 13.23.
Mole-Bajer, J. 6.45.
Moll, R. H. 13.37.
Momordica, C. L. 5.118.

MONTALENTI, G. 10.54.

Moor, P. de, 16.19.

Moor-Jankowski, J. 11.30., 11.31.

Moorhead, P. S. 16.6.

Moray, N. 14.1.

Moree, R. 9.56.

Morgan, D. H. 2.24.

Moriwaki, D. 9.57.

Moroni, A. 15.12.

Moroz, L. 15.79.

Morpurgo, G. 5.19.

MOSEMAN, J. G. 1.13.

Mosig, G. 3.15.
Mossige, J. 10.49.
Motulsky, A. G. 16.21.
Mouli, C. 13.69.
Moureau, P. 15.64.
Mousseau, J. 3.55.
Moustacchi, E. 5.26.
Moutschen, J. 5.89.
Moutschen-Dahmen, J. M. 5.89.
Mueller, H. W. 13.84.
Mugnozza, G. T. S. 5.115.
Mukai, T. 9.58.
Mukherjee, A. 10.7.
Mukheriee, B. B. 16.5., 16.14.
Muller, I. 5.121.
Munday, A. 10.31.
Munson, R. J. 5.120.
Muramatsu, M. 13.62.

328

Index

Murata, N. 13.100.
Murray, M. J. 8.9.
Murray, N. E. 2.6.
Muth, F. W. 5. 88.
Myers, T. L. 15.4.

Nakai, K. 15.40.
Napp, M. 5.63, 9.59.
Nash, D. 10.17.
Nash, D. J. 5.69.
Natarajan, A. I. 5.34.
Naughton, M. A. 15.84.
Nazareth, H. R. 8.17.
Neagu, M. 13.71.
Necasek, J. 5.12.
Neel, J. V. 15.58.
Neeley, J. C. 7.28.
Negovsky, N. 13.78.
Neuffer, M. G. 4.25.
Newcomer, E. H. 6.9.
Nichols, W. W. 16.37.

NlERMANN, H. 15.8.

Nilan, R. A. 5.105., 5.108.
Nirula, S. 5.34.

NlSONOFF, A. 11.17.

Niznik, G. V. 5.75.
Nobuo, M. 13.100.
Noda, S. 9.22.
Nordenskjold, H. 6.33.
Nordskog, A. W. 11.16., 14.27.
Notani, N. K. 13.69.
Nothel, H. 5.52.
Novikov, B. G. 14.25.
Nowacki, E. 13.105.
Nuzhdin, N. I. 5.113.
Nuzhdin, N. J. 5.75.
Nuzzo, F. 16.39.
Nijenhuis, L. E .15.26. 16,26.

Oakberg, E. F. 5.78.
O'Connor, L. K. 14.44.
Odintsova, N. 13.35.
O'Flynn, M. E. 15.65.
Oftedal, P. 5.43.
Oganesian, M. 5.10.
Ogita, Z. 4.27.
Ohanessian, A. 12.21.
Oksala, T. 7.26.
Olah, L. V. 6.4, 6.5, 6.43.
Olenov, J. M. 16.33.
Olivier, G. 17.12.
Omar, A. A. 13.109.
Ondrej, M. 2.12.
Ono, H. 8.29.

OOSTERBAAN, R. A. 3.29.

Oppenoorth, W. F. F. 3.54.
Orlova, N. N. 5.79.
Osamu, K. 9.27.
Osborne, R. H. 15.54.
Osborne, T. S. 5.103.
Oshima, C. 9.60.
Oster, I. I. 5.37, 6.47, 6.48.

OSTERGREN, G. 6.45.

Ostertag, W. 15.61., 16.35.
Ottensooser, F. 15.22, 15.25.
Ottolenghi, E. 3.32.

Pair, Y. K. 9.61.
Paleckova, F. 5.23.
Palenzona, D. L. 13.38.
Palm, J. 11.28.
Panella, A. 13.68.

Parker, W. C. 15.83.
Pasquale, A. di, 10.25.
Paszewski, A. 2.7.
Patau, K. 16.17.
Patel, D. G. 9.14.
Pateman, J. A. 4.41., 4.42.
Pa van, C. 9.45., 9.46.
Payne, M. W. 15.35.
Peat, W. E. 13.82.
Pee-Laborde, L. 17.9.
Pelling, C. 6.24.
Penionzhkevich, E. E. 14.36.
Fere, G. 4.10.
Peters, W. 9.62.
Peters, W. H. 14.51.
Peterson, P. A. 4.24.
Phillips, R. J. S. 5.67.
Pinkhas, J. 15.60.
Pipkin, S. B. 9.63.

PlTTENGER, T. H. 4.38.
PlZARRO, O. 11.20.

Platonova, R. 5.101.
Plaut, W. 6.23.
Plowman, R. D. 14.45.
Plus, N. 12.20.
Poehlman, J. M. 13.51.
Pol, J. H. van de, 3.19.
Politis, J. 4.53.
Polivanov, S. 9.64.
Polsinelli, M. 3.37.
Polyakov, I. M. 13.76.
Pomerantzeva, M. D. 5.66.
Pomriaskinsky-Kobozieff, N
Pons, J. 17.14.
Pooley, E. 5.37.
Popescu, R. 15.38.
Popovic, A. 13.54.
Popovici, !. 12.17., 15.38.
Post, R. H. 15.11.
Poulson, D. F. 12.18.
Pouwels, P. H. 3.29.

15.68.

R. 11.2.

4.4.

9.65.

Prokofieva-Belgovskaya, A. 6.20
Prout, T. 9.66.
Prozorow, A. A. 3.5.
Prud' Homme, N. 2.23.
Przybylska, J. 13.98.
Puro, J. 5.40.
Purser, A. F. 14.56.
Pusa, K. 6.29.
Putrament, A. 2.17.

PUTTE, P. VAN DER, 5.20.

Pynacker, L. P. 10.10.

Qualset, C. O. 13.43.
Quinn, C. E. 11.5.

A. 10.50.

Powell, W.
Preer, Jr. J.
Prevost, G.
Prevosti, A.

Rachmeler, M. 4.48.
Radding, C. M. 3.10.
Rai, K. S. 10.9.
Raicu, P. 12.17.
Raievskaia, O. G. 3.52.
Rakic, M. T. 15.58.
Ramage, R. T. 7.16.
Ramirez, C. 1.18.
Rao, S. H. K. 5.116.
Rapacz, J. 11.7.
Rasmusen, B. A. 11.9.
Rastunkova, L. 13.102.
Rauch, H. 10.36.

329

Index

Ravin, A. W. 3.33, 3.38.

Rayner, S. 15.41.

Record, R. G. 15.5.

Redei, G. P. 2.20, 4.14.

Reed, T. E. 15.36.

Rees, H. 8.15.

Reguly, M. L. 5.60, 5.61, 5.62, 9.59.

Reich, E. 4.40.

Reilly, B. 3.39.

Renkonen, K. O. 15.16.

Renwick, J. H. 15.30.

Resch, K. 10.5.

Resende, F. 6.35.

Resnick, B. 10.37.

Rever, B. M. 4.36., 4.37.

Rhode, E. A. 11.10.

Richardson, D. L. 13.89.

Richardson, F. 11.24.

Rick, C. M. 7.2., 13.74.

Rieger, R. 5.92.

Riscutia, C. 8.27.

Ritte, U. 7.24.

Rivat, L. 15.76.

ROBBELEN, G. 12.16.

Roberts, D. B. 11.1.

Robertson, A. 9.20.

Robertson, F. W. 9.67.

Robichon-Szulmajster, H. De, 3.53.

Robinson, H. F. 13.37.

Roderick, T. H. 9.37.

Rohrborn, G. 5.29.

Rommel, M. 13.15.

Ronchi, V. N. 5.93.

Ropartz, C. 15.19, 15.76.

Rorsh, A. 5.20.

Rosen, G. Von, 5.107.

Rosener, A. 5.14, 5.35.

Ross, J. G. 7.11.

Rossi, J. 14.22.

Rotheim, M. B. 3.38.

Rotterdam, C. van, 3.16.

Rousi, A. 8.7.

Rousseau, R. Y. 15.19, 15.76.

Rowlands, D. G. 13.22.

Roy, S. K. 9.7.

Rubenstein, I. 3.13.

Rubini-Francs, M. G. 14.15.

Rubinstein, P. 11.20.

Russell, E. S. 10.14, 10.51.

Russell, J. S. 8.25.

Russell, L. B. 16.11.

Rutishauser, A. 7.6.

Ryan, F. J. 5.9.

Ryuhei, T. 13.50.

Sa, I. de, 3.33.

Sachs, L. 14.10.

Saez, F. A. 8.30.

Sacharov, V. V. 5.101.

Sadaaki, K. 11.2.

Sakaguchi, B. 12.18.

Sakai, B. 8.29.

Saksela, E. 16.6.

Saldanha, P. H. 15.22, 15.25, 15.37.

Salganick, M. G. 14.30.

Salganik, R. I. 5.1.

Samokhvalova, N. S. 5.113.

Sanduleac, E. 5.122, 13.31.

Sang, J. H. 10.26.

Sank, D. 15.15.

Sankaranarayanan, K. 5.58.

Sansome, E. 6.34.

Sari Gorla, M. 12.19.

Sarkissian, I. V. 10.34.

Sarvella, P. 12.12.
Sato, M. 15.22.
Saul, G. B. 4.30.
Savitsky, H. 13.2.
Savitsky, V. F. 13.21.
Scaife, J. 4.17.

SCANDLYN, B. J. 1.3.

Scarascia Mugnozza, G. T. 5.115.
Schaberg, A. 16.7.

SCHALET, A. 1.1., 1.2.

Schaller, C. W. 13.43.
Scharloo, W. 9.68.
Schell, J. 3.24.
Schierman, L. W. 11.16.
Schmid, K. 15.79, 15.80.
Schmid, W. 16.9.
Schmidt, B. J. 15.25.
Scholte, P. J. L. 15.14.
Scholz, F. 13.80.
Scholz, W. 15.28.
Schroffel, J. 14.49.

SCHULZ-SCHAEFFER, J. 8.16.
SCHWAIER, R. 5.11.

Schwartz, D. 4.32.
Schwartz, N. 5.2.
Schwarz, R. 6.47.
Schwinck, I. 10.2.
Scossiroli, R. E. 9.27, 13.38.
Searle, A. G. 5.67.
Sears, E. R. 7.19, 7.21.
Sedlarova, L. 3.51.
Seiger, M. S. B. 9.8.
Sekla, B. 11.14, 17.2.
Selim, A. K. A. 13.109.
Sels, A. 3.50.
Sengun, A. 6.19.
Serban, M. 8.27.
Sermonti, G. 3.48.
Serra, A. 15.24.
Serres, F. J. de, 1.16., 2.10.
Seshachar, B. R. 6.17.
Shackleford, R. M. 11.7.
Shama Rao, H. K. 5.116.
Shams-Ul-Islam, K. 7.18.
Shankel, D. M. 5.7.
Sharma, A. 17.10.
Sharma, R. P. 5.34.
Shaver, D. L. 7.12.
Sheba, C. 15.23.
Shemjakin, M. F. 3.5.
Sher, I. H. 18.1., 18.2.
Sherman, F. 4.49.
Sherman, J. K. 15.10.
Shershunova, L. J. 14.30.
Shestacov, S. V. 3.43.
Shinkovitz, M. 13.35.
Shiomi, T. 5.46.
Shiraki, Y. 15.40.
Shleser. R. A. 2.3.
Shozo, N. 9.22.
Shreffler, D. C. 11.21., 15.2.
Shu, T. C. 13.52.
Shvedskaja, Z. M. 13.27.
Sickle, R. van, 5.15.
Siebner, H. 16.29.
Siegel, A. 3.20.
Siegel, B. Z. 16.44.
Siegel, H. S. 14.28, 14.29.
Siegel, P. B. 14.28, 14.29.
Siegel, R. W. 10.21.
Silagi, S. 4.40, 12.4.
Silio, F. 5.90, 6.38.

SlMANTEL, G. M. 7.11.

Simmons, A. S. 9.54.
Simpson, N. E. 15.57.

330

Index

Singleton, J. R. 7.8.

Siniscalco, M. 15.82., 16.20.

Sin-Kyu Hyun, 13.32.

Six, E. W. 3.25.

Skiebe, K. 13.17.

Skleranik, R. 11.4.

Slatis, H. M. 15.63., 15.73.

Slen, S. B. 14.33.

Slizynski, B. M. 6.22.

Sly, W. 3.53.

Smith, C. 14.52.

Smith, C. A. B. 15.29.

Smith, D. A. 4.5.

Smith, G. 15.3.

Smith, G. F. 17.1.

Smith, H. H. 5.86.

Smith-Keary, P. F. 4.21.

Snaydon, R. W. 9.1.

Snoad, B. 7.1.

Sobels, F. H. 15.14.

Sokal, R. R. 9.9.

Sokoloff, A. 14.19.

Sokolova, L. K. 13.27.

Solima Simmons, A. 9.54.

Somers, C. E. 6.12.

Sondhi, K. C. 10.38.

Soni, A. H. 15.35, 15.36.

Sonnenschein, C. 16.3.

Southin, J. L. 5.30.

Spada-Sermonti, 3.48.

Sparrow, A. H. 5.98.

Sperlich, D. 9.69.

Spickett, S. G. 14.9, 15.56.

Spiess, E. B. 9.70., 9.71.

Spiess, L. D. 9.71.

Spizizen, J. 3.39.

Spofford, J. B. 4.13.

Springer, R. 9.28.

Squadroni, S. J. J. 11.5.

Srinivasan, P. R. 3.4.

Stadler, D. R. 4.47.

Stadler, J. 5.71, 5.72., 9.52

Stahl, A. 16.27.

Stalker, H. D. 8.26.

Stanescu, V. 16.22.

Steeno, O. 16.19.

Steffensen, D. M. 6.25.

Stein, H. 10.32.

Steinberg, A. G. 15.77.

Steinitz-Sears, L. R. 7.20.

Steitz, E. 10.27.

Stengel, H. 10.40.

Stinson, H. T. 13.75.

St. Lawrence, P. 4.48., 7.8.

Stoekenius, W. 3.30.

Stoletov, W. N. 3.43, 13.35.

Stone, D. 16.45.

Stone, W. H. 11.13.

Stormont, C. 11.10.

Strauss, B. S. 5.2.

Strong, L. C. 14.6.

Stroun, J. 13.29, 13.30, 14.22, 14.23.

Stroun, M. 13.29, 13.30, 14.22, 14.23.

Stroun-Guttieres, L. 14.22, 14.23.

Struck, E. 16.10.

Stubbe, W. 8.2.

Subbiah, K. C. 5.97.

Subodh, K. R. 9.7.

Suemoto, H. 13.99.

Suomalainen, E. 8.21.

Surzycki, S. 2.7.

Suskind, S. R. 4.46.

Sutherland, D. N. 14.48.

Sutton, H. E. 15.81.

Suwa, T. 13.100.

Suzuki, D. T. 7.25.
Suzuki, Y. 11.10.
Swaminathan, M. S. 5.34, 13.64.
Swiezynski, K. M. 2.19.
Sybenga, J. 7.17.
Szeinberg, A. 15.23, 15.60.
Szemere, G. 11.27.

Taber, H. 4.49.
Takahashi, I. 3.40.
Takahashi, R. 13.50.
Takashi, S. 13.100.
Takumi, T. 7.15.
Tanaka, N. 5.94.
Tantawy, A. A. O. 9.72.
Tarsitani, D. 16.20.
Tashian, R. E. 15.2.
Tates, A. D. 5.44.
Taub, S. R. 12.2.
Tavcar, A. 13.103.
Teissier, G. 9.73.
Teodoreanu, N. 14.54.
Ter-Avanesyan, D. V. 13.77.
Terumi, M. 9.58.
Tessman, I. 2.4.
Thanh, L. M. 17.8., 17.9.
Therkelsen, A. J. 16.12.
Therman, E. 16.17.
Thoday, J. M. 9.49.
Thomas, C. A. (Jr.) 3.13.
Thomas, R. 4.2.
Thompson, J. S. 11.24.
Thompson, M. W. 10.51.
Thompson, P. E. 7.27.
Thomson, J. A. 9.74.
Threlkeld, S. F. H. 2.11.
Thurman, W. G. 15.74.
Thwaites, W. M. 4.43.
Ting, Y. C. 7.14.
Tips, R. L. 15.3.
Tirelli, M. 15.1.
Todd, C. W. Jr. 15.4.
Todor, M. 13.53.
Toki-o, Y. 14.14.
Tokita, K. 15.79., 15.80.

TOKUNAGA, C. 10.6.

Toledo, J. S. de, 9.45., 9.46.
Toledo, S. A. de, 9.45.
Tolksdorf, M. 16.24, 16.25.
Tomasz, A. 3.30.

TONKELAAR, E. M. DEN, 16.7.

Torkel, H. 15.62.

Touchberry, R. W. 5.64, 14.46.

Traut, W. 10.13.

Truslove, G. M. 10.44., 10.45.

Trut, L. N. 14.5.

Tsuboi, H. 15.40.

Tsuchiya, T. 7.15.

Tunte, W. 15.13.

Tupy, Jaroscav, 13.12.

Turbin, N. V. 9.21.

Turkel, H. 15.62.

Turner, H. N. 14.55.

Turner, J. R. G. 9.10.

Turral, G. M. 17.1.

Turtoczky, I. 3.41.

Tutomu, H. 9.22.

Tuveson, R. W. 3.47.

Ulrich, H. 5.47.
Underbrink, A. G. 6.5.
Unger, L. J. 15.71.
Ursprung, H. 10.24.

331

Index

Vakhtiu, J. B. 16.32.
Valencia, J. I. 5.87., 16.3.
Valeva, S. A. 5.100.
Valkenburg, H. A. 15.58.
Van Abeelen, J. H. F. 14.4.
Van Arkel, G. A. 3.19, 5.6.
Van Bolhuis, J. 16.26.
Van Brink, J. M. 8.22.
Van Delden, W. 9.39.
Van Duijn, P. 16.7.
Van Gamble, A. N. 16.5.
V\N DEN Berghe, H. 16.19.

Van den Bosch, J. 15.42.
Van de Pol, J. H. 3.19., 5.6.
Van der Putte, P. 5.20.
Van Loen, A. 14.40.
Vann, D. 11.3.
Van Rotterdam, C. 3.16.
Van Sickle, R. 5.15.
Van t Hof, J. 5.98.
Van Valen, L. 9.29.
Varga, M. 10.39.
Vartapetvan, V. V. 13.94.
Velasquez, R. S. 12.11.
Veldhuisen, G. 3.29.
Vereiskaya, V. N. 14.18.
Verresen, H. 16.19.
Vickery, R. K. (Jr.) 9.11.
Vissac, B. 14.41.

VlTAGLIANO TADINI, G. 10.54.

Vogt, D. W. 14.43.
Vojtiskova, M. 5.65, 11.23.
Von Borstel, R. C. 7.23, 9.27.
Von Halle, E. S. 1.3.
Von Rosen, G. 5.107.
Vries, A. de, 15.60.

Wagenaar, L. E. B. 6.31.
Wahl, R. 5.2.
Wahrman, J. 7.24.
Walen, K. H. 6.16.
Walker, G. W. R. 13.106.
Walker, J. T. 13.40.
Wallace, B. 5.56.
Watanabe, T. 4.18.
Watkins, W. L. 15.74.
Watney, M. J. 10.43.
Watson, J. H. 14.57.
Wattiaux, J. M. 9.75.
Wehrli, M. 14.27.
Welch, R. M. 10.5.
Welshons, W. J. 1.3.
Weninger, M. 17.16.
Went, L. N. 15.47.
Wermer, P. 15.44.
West, G. B. 11.29.
Weijer, D. L. 6.6, 16.30.
Weijer, J. 6.6, 16.30.
Whiting, P. W. 1.11.
Whittington, W. J. 13.82.
Wiebe, G. A. 13.56.

Wiedemann, H. R. 16.24, 16.25.
Wilczok, T. 16.34.
Wildervanck, L. S. 15.52.
Wilgram, G. F. 4.33.
Wilkie, D. 2.22.
Williams, L. F. 13.66.
Williams, N. D. 13.48.
Williams, W. 10.30.
Wills, A. B. 13.108.
Wilson, J. A. 15.77.
Wilson, J. F. 10.23.
Winge, H. 5.63, 8.24, 9.59.
Wit, F. 13.4.
Withers, R. F. J. 15.34.
Wittmer, G. 5.96.
Wolf, B. E. 7.22.
Wolff, G. L. 10.37.
Wolff, S. 2.10., 5.81.
Wolsky, A. 11.5.
Wolsky, M. De Issekutzi. 11.5.
Wong, P. 15.65.
Woodward, V. W. 4.44.
Woong-Jik, L. 7.7.
Wright, J. E. 11.4.
Wright, S. 9.13.
Wright, T. R. F. 4.31.
Wu, H. K. 6.32.
Wurgler, F. E. 5.48.

Yalvac, S. 8.23.

Yamada, Y. 14.21.

Yamamoto, T. O. 14.14.

Yamasiki, Y. 13.100.

Yanushkevich, S. I. 5.102.

Yasuzumi, G. 6.1 1.

Yerganian, G. 6.13.

Yi-Yung Hsia, D. 15.59., 15.63.

Yong, K. P. 9.61.

Yoon, C. H. 3.1.

Yoshiko, S. 11.10.

Yoshito, Y. 13.100.

Yost, M. Y. 10.36.

Young, G. O. 11.17.

Yuasa, A. 6.7.

Yuh Lin, H. 2.15.

Yukio, Y. 14.21.

Zaitlin, M. 3.20.
Zakharova, G. M. 13.26.
Zambruni, L. 10.25.
Zamenhof, S. 3.35.
Zanen, J. 15.9.
Zarubailo, T. Ya. 13.83.
Zellweger, H. 16.28.
Zimmermann, F. K. 5.11.
Zinder, N. D. 3.17.
Zohary, D. 13.63.
Zorrilla, N. B. DE, 8.30.
Zubareva, L. A. 14.30.
Zubay, G. 3.3.

332