METHODOLOGY IN MAMMALIAN GENETICS 3EeSQGBBBBBQQBQQE8£ Marine Biological Laboratory Library Woods Hole, Mass. Presented by In Memory of Dr. Herbert W. Rand 1872 - I960 METHODOLOGY IN MAMMALIAN GENETICS Sponsored by THE ROSCOE B. JACKSON MEMORIAL LABORATORY THE GENETICS STUDY SECTION DIVISION OF RESEARCH GRANTS AND DIVISION OF GENERAL MEDICAL SCIENCES NATIONAL INSTITUTES OF HEALTH * * Grant RG 7097 5 7 s' ^ METHODOLOGY in MAMMALIAN GENETICS Edited by WALTER J. BURDETTE, A.B., A.M., Ph.D., M.D. Professor and Head of the Department of Surgery and Director of the Laboratory of Clinical Biology, University of Utah College of Medicine; Surgeon-in-Chief Salt Lake County Hospital; Chief Surgical Consultant, Veterans Administration Hospital, Salt Lake City, Utah I HOLDEN-DAY, INC., San Francisco !963 © Copyright 1963 by HOLDEN-DAY, INC. Printed in the United States of America Library of Congress Catalog Card Number 62-2og^6 PREFACE Information recently acquired about the biochemistry of heredity has increased the likelihood of determining precisely how the more formal transmission of genetic information is accomplished in higher organisms and has imposed the obligation to renew the task of controlling changes in composition, propagation, and action of the genetic material. The complexity of mammalian genesis and development is regarded no longer as a barrier to investigation at the molecular level, but more as an opportunity to study mechanisms that do not exist in lower organisms in relation to common hereditary units and to choose between alternate explanations for a given process. Possibly the greatest advantage the laboratory mammal offers is not only the possibility of developing strains of animals having remarkably similar partial or total genome but also the opportunity to breed representatives from diverse strains in a manner appropriate for the elucidation of genetic mechanisms. In addition, these uniform lines are available for comparison of the genetic behavior of cells with known properties in vivo and in vitro. Recent evidence for fusion of mammalian cells in vitro suggests that the analytical advantages of sexual reproduction may be extended to studies of the somatic cell as well. Also, the many hereditary diseases known to occur in inbred mammals and the varied response of different strains to bacterial, viral, and parasitic inoculation offer means to determine parameters that may also be operable in similar diseases in man. A host of methods have been evolved for providing stocks of mammals with uniform genotype suitable for given experimental objectives. The theoretical and practical aspects of insuring this type of control and some indication of how methodology from other scientific disciplines may be used in mammalian genetics are mandatory for an approach to the solution of problems that engage the attention of many con- temporary investigators. The intent of the contributors to this volume is to provide a selected array of methods applicable to mammalian genetics that may prove useful vi PREFACE in implementing the ideas of those striving to solve the intricate problems encountered in investigations concerned with differentiation, homotransplantation, directed mutation, repair of deleterious mutants, genetic determinants in disease, and the like. Walter J. Burdette Chairman, Genetics Study Section Salt Lake City, Utah August, 1962 PARTICIPANTS Thomas Anderson, Ph.D.* The Institute for Cancer Research, Fox Chase, Philadelphia 11, Pennsyl- vania H. B. Andervont, Sc.D. Laboratory of Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland Louis Baron, Ph.D.* Division of Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D.C. Morris K. Barrett, M.D. National Cancer Institute, National Institutes of Health, Bethesda, Maryland Michael A. Bender, Ph.D. Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Howard A. Bern, Ph.D. Department of Zoology, Cancer Research Genetics Laboratory, University of California, Berkeley, California S. E. Bernstein, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Walter J. Burdette, Ph.D., M.D.* Department of Surgery, University of Utah, Salt Lake City, Utah * Member, Genetics Study Section vii MM PARTICIPANTS C. K. Chai, Ph.D. Battelle Memorial Institute, Columbus, Ohio Herman B. Chase, Ph.D. Department of Biology, Brown University, Providence, Rhode Island Carl Cohen, Ph.D. Battelle Memorial Institute, Columbus, Ohio Douglas L. Coleman, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine James Crow, Ph.D.* Department of Medical Genetics, University of Wisconsin, Madison, Wisconsin Karl H. Degenhardt, M.D. Department of Human Genetics and Comparative Pathology, University of Frankfurt, Frankfurt, Germany Margaret K. Deringer, Ph.D. National Cancer Institute, Department of Health, Education and Welfare, Bethesda, Maryland Margaret M. Dickie, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Donald P. Doolittle, Ph.D. Department of Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania Sheldon Dray, M.D. Laboratory of Immunology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland D. S. Falconer, Ph.D. Institute of Animal Genetics, Edinburgh, Scotland Morris Foster, Ph.D. Department of Zoology, Mammalian Genetics Center, University of Michigan, Ann Arbor, Michigan F. C. Fraser, Ph.D., M.D.* Department of Genetics, McGill University, Montreal, Canada PARTICIPANTS ix John L. Fuller, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Norman Giles, Ph.D.* Department of Botany, Yale University, New Haven, Connecticut Benson E. Ginsburg, Ph.D. Department of Psychology, University of Chicago, Chicago, Illinois Francis B. Gordon, Ph.D.* Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland John W. Gowen, Ph.D. Department of Genetics, Iowa State University, Ames, Iowa Douglas Grahn, Ph.D. Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois Margaret C. Green, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Earl L. Green, Ph.D.* Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Leonard Herzenberg, Ph.D. Department of Genetics, Stanford University, Palo Alto, California Walter E. Heston, Ph.D.* National Cancer Institute, National Institutes of Health, Bethesda, Maryland Warren G. Hoag, D.V.M. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine T. C. Hsu, Ph.D. Section of Cytology, M. D. Anderson Hospital and Tumor Institute, Texas Medical Center, Houston, Texas George E. Jay, Jr., Ph.D. Department of Laboratory Animals, Microbiological Associates, Inc., Washington, D.C. X PARTICIPANTS Nathan Kaliss, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine George Klein, M.D. Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden Joshua Lederberg, Ph.D.* Department of Genetics, Stanford University, Palo Alto, California Edwin P. Les, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Clarence C. Little, M.D. Littlehaven, Ellsworth, Maine Clara J. Lynch, Ph.D. Rockefeller Institute for Medical Research, New York, New York W. B. Mcintosh, Ph.D. Department of Zoology, Ohio State University, Columbus, Ohio Andrew V. Nalbandov, Ph.D. Department of Animal Science, University of Illinois, Urbana, Illinois Ray D. Owen, Ph.D.* Division of Biological Sciences, California Institute of Technology, Pasadena, California H. Ira Pilgrim, Ph.D. Department of Surgery, University of Utah, Salt Lake City, Utah Raymond A. Popp, Ph.D. Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee T. Edward Reed, Ph.D. Departments of Zoology and Pediatrics, University of Toronto, Toronto, Canada Charles M. Rick, Jr., Ph.D.* Department of Vegetable Crops, University of California, College of Agricul- ture, Davis, California PARTICIPANTS xi T. H. Roderick, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Elizabeth S. Russell, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Robert H. Schaible, Ph.D. The Hall Laboratory of Mammalian Genetics, University of Kansas, Lawrence, Kansas William J. Schull, Ph.D.* Associate Professor of Human Genetics, University of Michigan, Ann Arbor, Michigan J. P. Scott, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Willys K. Silvers, Ph.D. The Wistar Institute, Philadelphia, Pennsylvania Herman M. Slatis, Ph.D. Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois George D. Snell, M.S., Sc.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Joan Staats, M.S. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Arthur G. Steinberg, Ph.D. Biological Laboratory, Western Reserve University, Cleveland, Ohio Gunther Stent, Ph.D.* Virus Laboratory, University of California, Berkeley 4, California Wilson S. Stone, Ph.D.* Department of Zoology, University of Texas, Austin, Texas John A. Weir, Ph.D. The Hall Laboratory of Mammalian Genetics, University of Kansas, Lawrence, Kansas xii PARTICIPANTS W. K. Whitten, D.Sc. National Biological Standards Laboratory, Department of Health, Canberra, Australia Katherine S. Wilson, Ph.D.* Genetics Study Section, Division of Research Grants, National Institutes of Health, Bethesda, Maryland Henry J. Winn, Ph.D. Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine Sewall Wright, Sc.D. Department of Genetics, University of Wisconsin, Madison, Wisconsin George Yerganian, Ph.D. Children's Research Foundation, Inc., Boston, Massachusetts CONTENTS PREFACE (Walter J. Burdette, Ph.D., M.D.) v Genetic Stocks and Breeding Methods SYSTEMS OF MATING USED IN MAMMALIAN GENETICS (E. L. Green, Ph.D., and D. P. Doolittle, Ph.D.) 3 METHODS FOR TESTING LINKAGE (Margaret C. Green, Ph.D.) ... 56 GENETIC STRAINS AND STOCKS (George E. Jay, Jr., Ph.D) .... 83 Radiation Genetics MAMMALIAN RADIATION GENETICS (Douglas Grahn, Ph.D.) . . . 127 Physiologic Genetics GENIC INTERACTION (Sewall Wright, Sc.D.) 159 QUANTITATIVE INHERITANCE (D. S. Falconer, Ph.D.) 193 PROBLEMS AND POTENTIALITIES IN THE STUDY OF GENIC ACTION IN THE MOUSE (E. S. Russell, Ph.D.) 217 METHODOLOGY OF EXPERIMENTAL MAMMALIAN TERATOLOGY (F. Clarke Fraser, Ph.D., M.D.) 233 GENETICS OF NEOPLASIA (Walter E. Heston, Ph.D.) 247 GENETICS OF REPRODUCTIVE PHYSIOLOGY (A. V. JValbandov, Ph.D.) 269 BEHAVIORAL DIFFERENCES (J. P. Scott, Ph.D., and John L. Fuller, Ph.D.) 283 xin ^3432 xiv CONTENTS Biochemical Genetics MAMMALIAN HEMOGLOBINS {Raymond A. Popp, Ph.D.) 299 TACTICS IN PIGMENT-CELL RESEARCH (Willys K. Silvers, Ph.D.) . . 323 Immuno genetics MI'THODS IN MAMMALIAN IMMUNOGENETICS {Ray D. Owen, Ph.D.) 347 Host-Parasite Relationships GENETICS OF INFECTIOUS DISEASES (John W. Gowen, Ph.D.) ... 383 Genetics of Somatic Cells GENETICS OF SOMATIC CELLS (George Klein, M.D.) 407 CYTOGENETIC ANALYSIS (George Terganian, Ph.D.) 469 Appendices I. CONTROL OF THE LITERATURE ON GENETICS OF THE MOUSE (Joan Staats, M.S.) 511 II. INTERNATIONAL RULES OF NOMENCLATURE FOR MICE (Joan Staats, M.S.) 517 III. METHODS OF KEEPING RECORDS (Margaret M. Dickie, Ph.D.) . 522 IV. HUSBANDRY, EQUIPMENT, AND PROCUREMENT OF MICE (Warren G. Hoag, D.V.M., and Edwin P. Les, Ph.D.) 538 V. TECHNIQUES FOR THE STUDY OF ANEMIAS IN MICE (Elizabeth S. Russell, Ph.D.) 558 VI. TECHNIQUE FOR THE TRANSFER OF FERTILIZED OVA (Margaret K. Deringer, Ph.D.) 563 VII. CURRENT APPLICATIONS OF A METHOD OF TRANSPLANTA- TION OF TISSUES INTO GLAND-FREE MAMMARY FAT PADS OF MICE (Staff, Cancer Research Genetics Laboratory, University of Calif ornia, Berkeley) 565 BIBLIOGRAPHY 571 AUTHOR INDEX 622 SUBJECT INDEX 627 GENETIC STOCKS AND BREEDING METHODS E. L. Green, Ph.D., and D. P. Doolittle, Ph.D. SYSTEMS of MATING USED in MAMMALIAN GENETICSt Mammalian geneticists use a variety of mating systems, each designed to accomplish a specific purpose. To use the systems effectively, it is necessary to know what each system is, when it can be used, and what its theoretical genetic consequences are. This paper describes seven systems of mating which have passed into general use by mouse geneticists. Each system will be described by means of its mating types and their probabilities through successive generations. In some cases reference will be made to the kinds of genotypes and their probabilities, in particular to the probability of heterozygotes. The theory of systems of matings has been extensively developed by Wright,1442 Bartlett and Haldane,56 and Fisher,375 on whom we have drawn heavily for this exposition. The system later called the " cross-backcross-intercross system" has not been analyzed heretofore; its theoretic consequences are presented here for the first time. We are indebted to Dr. George D. Snell who described the system to us and who has been the first to use it. The following sections outline the analysis of the mating systems after first defining some necessary symbols and describing the general steps of the analytical method. The last section suggests a few practical rules for the breeders of laboratory animals who desire to improve the genetic quality of mice, rats, rabbits, and other mammals for research. | The authors gratefully acknowledge the support of the Sagamore Foundation and the Richard Webber Jackson Memorial Fund. 4 GENETIC STOCKS AND BREEDING METHODS NOTATIONS AND DEFINITIONS Three autosomal loci of diploid, sexually reproducing organisms such as mice will be designated by the symbols: a-locus, ZMocus, and r-locus. The a-locus is any locus whose heterozygosity is in question as a given breeding system advances from generation to generation. The ZMocus is the locus of a dominant mutation ; the r-locus, that of a recessive mutation. The D and r mutations are called the genes of interest. The alleles at these three loci will be denoted as Aja, Djd, and R/r, and the genotypes by AA, Aa, aa; DD, Dd, dd; and RR, Rr, rr. The relative frequency (i.e., probability) of Aa will be denoted by h. The mating types are of four kinds : Incrosses: A A x A A and aa x aa, matings of like homozygotes, Crosses : AA x aa, matings of unlike homozygotes, Backcrosses: AA x Aa and aa x Aa, matings of homozygote and heterozygote, Intercrosses: Aa x Aa, matings of heterozygotes. When the terms incrosses, crosses, backcrosses, and intercrosses appear in lower-case letters, they refer to the locus with questionable heterozygosity. When they appear in small capitals, incrosses, crosses, backcrosses, intercrosses, they refer to the locus of interest. The last three of these terms are in general use. The relative frequencies or probabilities of the mating types will be denoted by p, q, r, . . ., v with the definition varying slightly from system to system. In general, p will be used to denote the frequency of incrosses (AA x A A and aa x aa), the maxi- mizing of which is the objective of all of the systems of breeding, except random mating. A subscript n (or m) denotes generation n (or cycle m). G (or C) will stand for genera- tion (or cycle). G, P, A, etc. are matrices. P will designate probability. The probability of crossing over between the a-locus with questionable heterozy- gosity and the D- or r-locus carrying the mutation of interest will be denoted by c. To avoid a troublesome complication in notation, for any two loci, c will be treated as equal in the two sexes. The probability of heterozygosity at the a-locus in generation n (or cycle m) will be denoted by hn (or hm). In all cases, as pn increases, hn decreases. As will be seen, hn is a function of the probabilities of backcrosses and intercrosses in each system of mating. SYSTEMS OF BREEDING Relatively few of the systems developed by breeders of domestic and laboratory mammals are used frequently enough to warrant exposition here. Parent-offspring inbreeding, line breeding, or systems which use first, second, or third cousins will not be described. The systems included are, with the exception of random mating, all regular systems which permit the development of sequence equations to relate the probabilities of incrosses, etc., of one generation to those of the next. Irregular SYSTEMS OF MATING 5 systems, as well as regular systems, may be analyzed by Wright's method of path analy- sis. The seven systems are of three types: those based upon relationship, those based upon locus control, and those based upon both relationship and locus control. The systems based on relationship are: 1 . RANDOM-MATING SYSTEM, 2. BROTHER-SISTER INBREEDING SYSTEM. Those based on controlling a locus of interest are : 3. BACKCROSS SYSTEM, 4. CROSS-INTERCROSS SYSTEM, 5. CROSS-BACKCROSS-INTERCROSS SYSTEM. Those which use a combination of locus control and relationship are : 6. BROTHER-SISTER INBREEDING WITH HETEROZYGOSIS FORCED BY BACKCROSSING, 7. BROTHER-SISTER INBREEDING WITH HETEROZYGOSIS FORCED BY INTERCROSSING. In the first two systems, no conscious attention is paid to the pheno types, except for the inevitable selection in favor of vigorous, healthy animals. In the third system, in each generation, (G0, Gx,. . .) the mating is a backcross with respect to the locus of interest. While the locus of interest is thus being controlled by a backcross, the locus whose heterozygosity is in question may be undergoing an incross, a cross, or a backcross. In the fourth system the initial generation (G0) and all subsequent even-numbered generations (G2, G4, . . . ) are crosses and all odd- numbered generations (Gl5 G3,...) are intercrosses with respect to the locus of interest. In the fifth system, the initial, third, sixth, etc., generations (G0, G3, G6, . . .) are crosses; the first, fourth, seventh, etc., generations (Gl5 G4, G7, . . .) are back- crosses; and the second, fifth, eighth, etc., generations (G2, G5, G8, . . .) are inter- crosses with respect to the locus of interest. In the sixth system, each generation, (G0, G1} G2, . . . ) is a backcross with respect to the locus of interest with the provision that the mates are related as brother and sister. In the seventh system, each generation (G0, Gl5 G2, . . .) is an intercross with respect to the locus of interest, with the same provision about the mates. Table 1 shows the generation sequence relative to the type of mating. Table 1 Generation sequence relative to type of mating System cross backcross intercross 3. B 0,1,2,... 4. C-I 0, 2, 4, . . . 1,3,5,... 5. C-B-I 0, 3, 6, . . . 1,4,7,... 2, 5, 8, . . . 6. BS-B 0,1,2,... 7. BS-I 0,1,2,... The serial number of the generation Gn given in the table shows the sequence of use of a cross, backcross, or intercross in controlling the locus of interest in five of the systems of mating. 6 GENETIC STOCKS AND BREEDING METHODS For each system, the breeder should know the probabilities of the mating types and in particular the probability p of incrosses with respect to the a-locus. He will also want to know the probabilities of the genotypes, particularly the probability h of hetero- zygotes Aa. To evaluate a system he should know the number of generations required to increase p or to decrease h to any desired level. We shall try to answer these questions about the seven systems. RANDOM MATING The system of random mating is used when preservation of the genetic (i.e., genotypic) variability of a population without change is desired. Random mating will not, in theory, create genetic variability or diminish it. Variability may be created by outcrossing or by mutations. It may be diminished by any scheme of inbreeding. Random mating means that the frequencies of matings of various types are specifiable by the product and addition rules of probability applied to the geno- types. Let the probabilities of the three genotypes at the a-locus in G0 be P{AA) = k0, ?{Aa) = 2/0, P(afl) = m0, where kt + 2/, + mt = 1, i = 0, 1, 2,. . ., n, and P(x) means the probability of x. By definition, the gene or allele frequencies are: r(A) = x0 = k0 + /0, P(fl) = */o = !0 + ™0, where xt + yx = 1 , and i = 0, 1 , 2, . . . , n. The mating-type frequencies in G0 and the genotype frequencies of their progeny in Gl are shown in table 2. The probability Table 2 Mating-type frequencies in g0 and genotype frequencies of their progeny Genotypes in G1 Mating types in G0 AA Aa aa P(AA x AA) = po = k02 k02 — P(aa x aa) = p0" = mQ2 — m02 P(AA x aa) = g0 = 2k0m0 P(AA x Aa) = r0 — 4kQl0 Y{aa x Aa) — s0 — 4/0w0 ?(Aa x Aa) = v0 = 4/02 The genotype frequencies of Gx are related to the mating-type frequencies of G0 in the random- mating system by the laws of Mendelian genetics. — 2k0m0 — ~k0l0 Zk^iQ — — 2l0m0 2l0m0 / 2 '0 2/02 I 2 SYSTEMS OF MATING 7 p0' of the mating AA x AA is k02 by the product rule of probability. The probability r0 of the matings AA x Aa or Aa x ^ is £0-2/0 plus 2/0-£0, or 4£0/0, by the product and addition rules of probability, and so forth. In the table, P(AA x Aa) is the probability of A A x Aa and its reciprocal Aa x AA. The relative frequencies of the three genotypes in Gls obtained by adding the last three columns of table 2, are: ?(AA) = kx = (k0 + l0)2 P(Aa) = 21, = 2(k0 + /0)(/0 + m0) = 2x0y0, P(aa) = mx = (/0 + w0)2 = y02. If random mating ensues, that is, if the probability of any mating type is the product of the genotypic probabilities of the mates, the mating types of Gx and the genotypes of their progeny in G2 can be represented as functions of the initial allelic frequencies, x0 and y0, as in table 3. The genotypic frequencies in G2 obtained by adding the last three colums of table 3 are : P(AA) =k2 = V(V + 2*0*0 + y02) = a-o2, P(Aa) = 2/2 = 2xQy0(x02 + 2xQy0 + y02) = 2x0y0, P{aa) = m2 = y02(x02 + 2x0y0 + y02) = y02. Table 3 MATING-TYPE FREQUENCIES OF Gi AND THE GENOTYPE FREQUENCIES OF THEIR PROGENY Genotypes in G2 Mating types in Gi AA Aa aa ■ P(AA x AA) = p[ = kx2 = V .v04 P(aa x aa) — p'[ = m,2 = y04 y04 P(AA x aa) — q, — 2k1m1 — 2x02y02 2x02y02 P(AA x Aa) = rx = 4kJ, = 4x03y0 2x03y0 2x03y0 P(aa x Aa) — s, = Alxmi — 4x0y03 — 2x0y03 2x0y03 P(Aa x Aa) = v, = \lx2 = 4x0W *oW 2x02y02 x02y02 The mating-type frequencies of Gx and the genotype frequencies of G2 are functions of the allele frequencies of G0 in the random-mating system. These are identical with the genotypic frequencies of Gx. Hence the mating-type frequencies of G2 will also be identical with the mating-type frequencies of Gx. It follows that p'n + 1 = p[; . . . ; vn + 1 = vx. After one generation of random mating, the probabilities of the six kinds of matings remain constant for any number of generations of random mating. Random mating thus preserves the genetic variability of the popu- lation. The restriction — "after one generation" — may be removed if the initial geno- typic frequencies, k0, 2/0, m0 are related to the allelic frequencies, *0 and y0, as /- — X 2 2/0 = 2x0y0, 8 GENETIC STOCKS AND BREEDING METHODS If they are not so related initially, they become so after one generation of random mating as shown above. GENERAL METHOD OF ANALYZING REGULAR MATING SYSTEMS In the regular mating systems there is a definable probability that matings of any given type will yield matings of the same or any other type (for example, that backcrosses yield incrosses) in the subsequent generation. The probability that a mating of type j yields a mating of type i in the subsequent generation will be called gtj. With s mating types, these probabilities can be arranged in an s x s matrix so that the leading diagonal of the matrix contains the elements git, row i contains all of the elements gu,j = 1, . . ., s, and column j contains all of the elements gtj, i = \, . . ., s. Such a matrix will be referred to as the generation matrix G. If, in generation n, the mating types have frequencies/^, ?„,..., vn, the frequencies in generation n + 1 will be Pn + 1 = Mil + ?n?2l + • • • + vnqsl <7n + l = Ai<7l2 + ?n?22 + • • • + yn?S2 Vn + \ = Mis + ?n?2s + • • • + Vnqss. (1) The probabilities pn, qn, . . . , vn can be arranged in an s x 1 vector Pn, Pn =: /Pn' , and by applying this principle repeatedly, we can show that Pn = G"P0. (3) SYSTEMS OF MATING 9 This means that if the G matrix could be raised to the power n, the mating-type fre- quencies could be obtained immediately in generation n. Raising G to the power n is, however, a difficult task. To avoid this difficulty the method of Kempthorne can be used.700 A diagonal matrix is one in which some of the elements of the leading diagonal have values other than 0, while all elements off this diagonal are zero. If the matrix A is an s x s diagonal matrix, A = / Xx 0 X2 A raised to the power n is An = /Xj" X,* 0 Xs" Thus, if G were a diagonal matrix, we could easily obtain Gn. G will not, however, be diagonal for any system of inbreeding; but we can use a transformation which will give us a diagonal matrix to work with. Let us define an s x s matrix, A, such that Vfc = APfc (4) for any generation k, Vfc being an s x 1 vector. Then from equation (2), Vfc = AGPfc_l5 and since it follows that Vfc = AGA-1^-!. (5) But an s x s matrix A exists such that AGA~ 1 is an s x s diagonal matrix. Therefore, Vfc = AVfc_l5 and, since this has the same form as (2), Vn = A"V0. (6) If, then, we can obtain A and A, we can transform P0 to give us V0, raise A to the power n, calculate Vn, and transform to Pn. 10 GENETIC STOCKS AND BREEDING METHODS The problem is to find A such that AGA"1 = A or AG = AA. AG is an s x s matrix, row i of which is a row vector which can be written afi, where a{ is row i of matrix A. AA is also anui matrix, row i of which can be written a^kj, where Xt is the non-zero element in row i of A, and I is an s x s identity matrix, with diagonal elements 1 and all elements off the diagonal equal to zero. Then to satisfy (5), fltG = fliXjI, a(G — fliXjI = 0 a,(G - X,I) = 0. (7) This defines a set of linear equations in the elements of au the solutions of which give the elements of row i of A. If the values of Xt from the matrix A are substituted into (7), the A matrix required to transform P0 to V0 and Vn to Pn is obtained. To obtain this solution, we must first have the A matrix. Equation (7) can be solved if and only if |G - X4I| = 0. (8) If X is subtracted from each of the diagonal elements of G and the determinant of the resulting matrix set equal to zero, s solutions will be obtained for X, which constitute the Xb i = 1 , . . . , s, the diagonal elements of the matrix A. In each of the systems we shall discuss, one of the roots of equation (8) will be 1 . Since the roots in A can be arranged in any order, we will always designate this root as Xs. The largest numerically of the remaining roots Xl5 . . ., Xs_! we will designate Xx. Xx is often called the characteristic root of matrix G and has certain properties which make it of special value to us. Since X1 is the largest of the roots Xl5 . . ., Xs_l5 all of which are less than 1, as the matrix A is raised to higher powers, all of these elements become smaller (except Xs, which remains 1 for any power). Since Xx is the largest of these, it will retain signifi- cance even when the rest of the roots have become negligible. Thus, Xx becomes an approximate measure of the rate of change of frequency of the various mating types. We may also wish to know how many generations are required to exceed a given percentage of incross matings. This will occur (ignoring sampling variations) when Pn > «, (9) if/>n is the proportion of incrosses and a is the desired proportion of such matings. If we assume that the initial generation includes only crosses which maximize the number of generations required to reach a given percentage of incross matings, v0 = ap0=: /r 1, SYSTEMS OF MATING 1 1 Since Vn = A»V0, Pn = A-*V, = A^A^Vo. Since V0 = /1\ , pn = Mi" + bs2*2n + ■■■ + U" where bsi is the element in row s, column i of A 1. For all inbreeding systems, bss = !> V = 1, X2n = X3n = • • 0 < XiB < 1, and bal < 0. Therefore Xs _ j n = 0 for n of any appreciable size, pn ~ 1 + Xj"*,!. From equation (9), it follows that the number of generations n required to exceed the frequency a of incrosses can be approximated by setting X!n£sl + 1 = a. Then n log Xj = log 6sl - log (1 - a), and log6sl - log(l - a) logXj (10) Using the nearest integral value of n, we can calculate the true pn from equation (6). The value of n for which equation (9) is satisfied should be within one or two generations of the approximation derived from (10) in all cases. In summary, then, the frequencies of the various mating types can be obtained in any generation, if the frequencies in the preceding generation are known, by using equation (2). Equation (3) allows the frequencies of mating types to be derived in some advanced generation n from the frequencies in the initial generation. This, however, involves raising the G matrix to the power n, which may be a difficult task. This difficulty may be avoided by deriving a diagonal matrix from the G matrix; it is easy to raise a diagonal matrix to any power. Doing so requires us to obtain the roots of the original G matrix and to derive a new matrix, A, which is used to trans- 12 GENETIC STOCKS AND BREEDING METHODS form the frequencies into multipliers for the diagonal matrix, A, of the G matrix roots. The roots of the G matrix are also useful in that one of them, called the characteristic root, measures the rate of decrease of the frequency of nonincross matings. This root also can be used to calculate the approximate number of generations required to reach a given proportion of incross matings. In the subsequent discussion, these principles will be applied to the six systems of mating mentioned above. BROTHER-SISTER INBREEDING SYSTEM The system of brother-sister inbreeding is used when it is desired to reduce the genetic (i.e., genotypic) variability at all loci. The probability h of heterozygosity Fig. 1. The brother-sister inbreeding system. GENERATION n 0 CO CO CO CO B at any locus a is to become as small as possible. Figure 1 shows three ways of repre- senting the system in a diagram. In the following analysis of the system reference is made only to autosomal loci. In the case of sex-linked loci, brother-sister inbreeding also reduces the probability of heterozygosity in the homogametic sex, and, if there is crossing over between the sex chromosomes, in the heterogametic sex. The analysis differs in details from that for autosomal loci. Assume that Aja are alleles at the a-locus and that the a-locus is autosomal. We SYSTEMS OF MATING 13 desire to know the probability of matings between like homozygotes, i.e., of incrosses in any generation n. Assume further that the probabilities of various types of matings in the generation w(Gn) are: _ (AA x AA\ Incrosses: r | = p., \aa x aa J Crosses: P (AA x aa) = qn, (AA x Aa\ Backcrosses : P (AA x Aa\ \aa x Aa) yaa x Aa} Intercrosses: P (Aa x Aa) = vn, where each mating type also includes the reciprocal mating, if any. The incrosses each produce one type of progeny, AA or aa, like the parents, and following the system of mating brother by sister, these produce one type of mating, incrosses, in the next generation. The crosses produce one type of progeny Aa, unlike the parents, and one type of mating, intercrosses, in the next generation. The backcrosses each produce two types of progeny AA and Aa, or aa and Aa, with probabilities 1 /2 and 1/2, and so produce three types of matings, incrosses, backcrosses, and intercrosses, in the ratios 1/4:1/2:1/4. Finally, the intercrosses produce three kinds of progeny AA, Aa, and aa in the ratio 1/4:1/2:1/4 which yield incrosses, crosses, backcrosses, and intercrosses in the ratios 1/8:1/8:1/2:1/4. Table 4 Generation matrix for the brother-sister inbreeding system Pn qn Pn + 1 qn+i rn + l On + 1 1 0 1/4 1/8 0 0 0 1/8 0 0 1/2 1/2 0 1 1/4 1/4 The probabilities of mating types in Gn + 1 may conveniently be shown as func- tions of the probabilities in Gn in a generation matrix, as in table 4. The probabilities may be written as equations: Pn + 1 =Pn + (l/4)r.+ (l/8)»„ ?» + i = (1/8K, (l/2)rn + (1/2K, vn + i = 1n + (l/4)rn + (1/4X. 14 GENETIC STOCKS AND BREEDING METHODS It may be seen at once that the probability of incrosses will steadily rise, since incrosses are produced by incrosses, by backcrosses, and by intercrosses of the preceding genera- tion. Incrosses act as an absorbing barrier. Once a locus reaches an incross, it re- mains as an incross so long as the brother-sister mating system continues. If brother-sister mating is started after a cross between unlike strains, so that the initial mating is AA x aa or the reciprocal, that is, q0 = 1, the probabilities of incrosses in G0, Gl5 G2,. . . are: 0, 0, 0.125, 0.281, 0.414, 0.525, 0.616, 0.689, 0.748, 0.796, 0.835, 0.866, 0.892,.... After 12 generations, 89.2 per cent of the matings will be the desired type, incrosses of AA x AA or aa x aa. The probability h of heterozygotes is one-half the probability of backcrosses plus the probability of intercrosses : hn = (l/2)rn + vn. In Gl5 G2, . . . , this probability h takes the successive values: 1, 1/2, 2/4, 3/8, 5/16, 8/32, 13/64, 21/128,..., which are the terms of the Fibonacci series. The ratios hn + 1jhn of the probability of heterozygosity in one generation to the probability of heterozygosity in the preceding generation form a series : 0.5, 1.0, 0.75, 0.8333, 0.8, 0.8125, 0.8077, 0.8095, 0.8088, 0.8091, 0.8090, 0.8090, 0.8090,..., which shows that, after brother-sister inbreeding has been in progress for several genera- tions, the amount of heterozygosity in Gn + 1 tends to be 80.9 per cent of the hetero- zygosity in Gn. Or, stated otherwise, the heterozygosity is decreasing at the rate of 19.1 per cent per generation, as first discovered by Jennings662 and established analytically by Wright.1442 Successive values of/>n, hn, and hn + 1/kn up to n — 12 are shown in figure 2. It may be assumed that, after brother-sister inbreeding has been in progress for several generations, the rate of depletion of the crosses, backcrosses, and intercrosses is constant. That is, ?n + l = Mm rn + l = ™n> Z'n + l = to>„, where X is a factor of proportionality. It follows that ~Mn + (1/8X = 0, (1/2 - X)rn + (l/2)oB = 0, qn + (l/4)r„ + (1/4 - X>n = 0. SYSTEMS OF MATING 15 Solving these three simultaneous linear equations for X yields X = (1/4), (1/4)(1 + V5), (1/4)(1 - V5), of which the largest numerical solution is Xx = (1/4) (1 + VI) = 0.8090 or 80.9 per cent. Xx is known as the characteristic root of the determinant made up of the coefficients in the three simultaneous equations and provides a rapid analytical means of finding the Fig. 2. The probability of incrosses for brother-sister inbreeding system. X hn + |/hn ^ 0.6 PROBABILITY 0.4 2 4 6 8 GENERATIONS The probability of incrosses pn and of heterozygosity hn for the brother-sister inbreeding system when q0 = 1 ; and the ratios of successive values of h and hn + x/hn. expected decrease in heterozygosity after several generations of inbreeding. The largest numerical root is the important one as n gets larger and larger because the probabilities qn + 1, rn + 1, and vn¥1 are each functions of X^, X2", and X3n and of the initial probabilities q0, r0, and v0. If X2 and X3 are smaller than X1} X2n and X3n will be of diminishing importance, relative to Xx n, as n increases in determining the values of 9n + i5 rn + u anQl vn + i- Thus for large n, only the characteristic root need be used for a sufficient approximation in computing the probabilities. The above results have been arrived at by direct multiplication of the generation matrix by the frequency of each mating type in Gn to obtain the frequencies of the mating types in Gn + 1, essentially by the repetitive application of formula (1) in the preceding section. The same results can be reached by the more complex algebra of the formulas (3), (5), and so forth. The generation matrix, G, is given in table 4. The roots X{ can be obtained by solving equation (8), |G - XJI - 0, or = 0. 1 - X 0 1/4 1/8 0 -X 0 1/8 0 0 (1/2) - X 1/2 0 1 1/4 (1/4) - X If GENETIC STOCKS AND BREEDING METHODS Solving the determinant, |1 - A1K1/4) - X][X2 - (1/2)X - (1/4)1 = 0, yielding the roots Xx = (1 + V5)/4, X2 = (1 - V5)/4, X3 = 1/4, and X4 = 1. These are the roots found by solving the three linear equations for a proportionality constant, with the addition of the root X4 = 1 . From these the A matrix given in table 5 (a) is derived. Substituting the above values of X* into formula (7), the matrix A, given in table 5 (b) and its inverse, A-1, table 5 (c) are obtained. Table 5 The matrices A, A, and A-1 for brother-sister mating (a) A = (b) A = (c) A'1 = 0.8090 0 0 0 0 -0.3090 0 0 0 0 0.2500 0 0 0 0 1.0000 0 1 0.6545 0.8090 0 1 0.0955 -0.3090 0 1 -0.25 0.25 1 1 1 1 1.3708 -0.0292 0.4000 1 0.0764 0.5236 0.4000 0 0.8000 0.8000 -1.6000 0 0.4945 -1.2945 0.8000 0 Now, if all matings in the initial generation are assumed to be cross mat'^gs. Then and SYSTEMS OF MATING 7 7 Vn = AnV0, and for n = 12, A12 = / 1.835 x 10-1 0 0 0 8.311 x 10-5 0 0 0 1.526 x 1 0 0 0 V12 = /7.859 x 102 6.772 x lO'7 5.960 x 10"8 p12 = A" *V12 =: /0.8923X , i.e., p12 = 0.8923 0.0060 \ q12 = 0.0060 0.0629 I r12 = 0.0629 ^0.0389/ v12 = 0.0389. This corresponds with the value of/>12 in figure 2, and the other values also agree with results gained by repetitive application of equation (2) . To calculate the generations required to obtain a given percentage of incross matings, formula (10) is used, which gives us an approximation: n log Xi ^ log bu - log (1 - a0 for ai = 0.95, ^ log 1.3708 - log 0.05 = " = log 0.8090 ~ ' * Therefore it is estimated that 16 generations will be required to give 95 per cent incross matings. Checking, we find that p16 = 0.9539, and plb = 0.9430. Repeating the calculations for a2 = 0.99, ^ log 1.3708 -log 0.01 _ " = log 0.8090 and p2i = 0.9915, whereas p23 = 0.9895. Thus, .16 generations of brother-sister inbreeding are required to obtain 95 per cent incross matings, and 24 generations to obtain 99 per cent. Dozens of strains of mice have been inbred by means of brother-sister matings. A few examples are A/J, AKR/J, BALB/cJ, C57BL/6J, DBA/2J, and C3HeB/FeJ. For a complete listing, refer to Committee220 and Snell and Staats.1254 18 GENETIC STOCKS AND BREEDING METHODS THE BACKCROSS SYSTEM The backcross system is a means of placing a dominant mutation or a semidominant lethal mutation D on a standard inbred background. It is assumed that the standard inbred strain is A Add or aadd and the mutant bearing stock is — DD or — Dd. All matings are backcrosses dd x Dd with respect to the ZMocus and may be incrosses, crosses, or backcrosses, with respect to the a-locus. The backcross system may also be used to put a recessive lethal or a recessive with low viability or low fertility on an inbred background, by backcrossing heterozygous carriers Rr of the mutation r to a standard inbred strain RR. In this case it is necessary to be able to distinguish the heterozygotes Rr from the homozygotes RR, each suspected carrier R- being tested by matings with known heterozygotes Rr. The backcross system is shown in figure 3. Fig. 3. The backcross system. INBRED STRAIN MUTANT INBRED STRAIN MUTANT MUTANT GENERATION 0 3 co GENERATION 0 CO I OO A. Backcrossing with a dominant or semidominant lethal mutation, D. B. Backcrossing with a recessive lethal mutation, r. SYSTEMS OF MATING 19 The mating types and their probabilities in Gn are : Incrosses : lAdjAd x ADjAd\ [ad/ad x aDjad ) ~ Pn Crosses- (Ad/Ad x aD/ad \ _ Crosses. F y^ x AD/Ad) - <7», Backcrosses p lAdjAd x ADjad\ (first kind) : W/arf x ai)/^/ = : r"' Backcrosses p /Ad/Ad x aD/Ad\ (second kind) : \adjad x AD /ad J ™ n' The standard inbred strain is given on the left, the mutant bearer on the right in each mating type. For the case of a recessive mutation, replace each D by r and each d by R. The probability of crossing over between the a- and the ZMocus or the a- and the r-locus is c, where c = 1 /2 when there is no linkage. The incrosses each produce one kind of mutant-bearing offspring, which, when mated in turn to the standard strain, yield incrosses in the next generation. The crosses produce doubly heterozygous progeny, considering only the mutant-bearing offspring, which, when mated to the standard inbred strain, yield backcrosses of the second kind. The backcrosses of the first kind produce two kinds of mutant-bearing progeny, ADjAd and aDjAd, or aDjad and ADjad, in the ratio (1 — c):c. The next generation of matings with the standard strain are thus of two types, incrosses and backcrosses of the second kind, in the proportions (1 — c):c. The backcrosses of the second kind produce the same two kinds of progeny, but the ratio is c: (1 — c), so the two types of matings in the next generation, incrosses and backcrosses of the second kind, are in the ratio c: (1 — c). These results may be displayed as a set of linear equations, Pn + 1 = Pn + (1 - - c)rn + d1 7n + l = 0> rn+i = °> tn + l = 1 , is K = rn + tn = (1 -f)"-1. The ratio of successive values of h is constant or the probability of heterozygosity in one generation is the fraction 1 — c of the proba- bility in the preceding generation. When c = 1/2, the probability of heterozygosity is exactly cut in half with each additional generation of backcrossing. The generation matrix, G, for the backcross system is given in table 6 (a) . Because of the fact, mentioned above, that only incrosses or backcrosses of the second type occur after the initial generation, a simplified matrix, G*, can be used if Gx is considered as the initial generation. G* is given in table 6 (b) . Table 6 (c) , (d) , and (e) give the A, A, and A~ l matrices for this system. SYSTEMS OF MATING 21 Table 6 Matrices for the backcross system (a) G = / 1 0 1 — c C \ / 0 0 0 ° 0 0 0 ° \ 0 1 c 1 -c J (b) G* = ( 1 0 c 1 - c ) (c) A = ( 1 - c 0 0 1 ) (d) A = ( 0 1 1 1 ) (e) A"* = ( -1 1 1 0 ) To obtain the frequencies of the various mating types in generation n, Pl5 the vector of frequencies in generation 1, is first obtained from equation (2). Pj will be of the form Pi = (A o 0 tx). We then form the vector P* = (px /j, transform to V*, obtain V* and convert to P*. These vectors are the same as the vectors operated with earlier, in equations (2) through (6) ; the * notation is introduced merely as a reminder that we are operating with only two of the original four mating types. Note that the matrix A being raised to the power (n — 1 ) , since one generation has been accounted for already by calculating Px. P* gives only the frequencies pn of incrosses and tn of backcrosses of the second type; the frequencies of the other two types of matings possible in G0 are, as has been demonstrated, zero in Gn. The frequencies pn and tn calculated by this method agree perfectly with those calculated by repetitive application of formula (2). The number of generations required to obtain a given percentage of incrosses can also be calculated for the backcross system. When c = 1/2, 5 generations are required to obtain a frequency of incrosses greater than 95 per cent and 8 to obtain a frequency greater than 99 per cent. When c = 1/10, 29 generations are required to obtain 95 per cent incrosses, and 44 to obtain 99 per cent. It should be noted that if the gene of interest is recessive, test generations will be required, thus increasing the time required to obtain a given percentage of incross matings. 22 GENETIC STOCKS AND BREEDING METHODS The mean length L of heterozygous chromosome on each side of the locus of interest after n generations of backcrossing is /•1/2 L = (1 - c)«-lde = -[1 -(1/2)"] n 1 n if n is large and if ?0 = 1 . This means, for example, that 40 generations of backcrossing will be expected to reduce the mean length of heterozygous chromosome, on both sides of the locus of interest, to about 5 centimorgans or 2.5 centimorgans on each side. Several mutations in mice have been put on standard inbred backgrounds by use of the backcross system. A few examples are: C57BL/6J-/13', C57BL/6J-Ca.S>, C5im,'S]-Mi^h,Cbim,^yWv, BALB/cGnSn-FwC, C3HeB/FeHu-Z^, DBA/10Hu-Z)j. Complete lists of these strains are given by Lane.751 Some strains have been propa- gated by brother-sister matings with forced heterozygosis after 5 or more generations of backcrossing to a standard inbred strain. Examples are: C57BL/6J-Pf, C57BL/6J- Ra, and BALB/c-Ts. THE CROSS-INTERCROSS SYSTEM The cross-intercross system is used to put a recessive, viable gene r on a standard inbred background. The inbred strain is presumably homozygous, A A or aa, for all loci with heterozygosity in question after the onset of crossing. The inbred strain is also presumably RR. Mutant-bearing animals of a stock, inbred or not, are on hand carrying rr at the locus of interest. The objective is to replace the RR of the inbred strain with rr of the mutant stock, while otherwise preserving the homozygosity of the inbred strain. The matings proceed in cycles of two generations. The first mating of each cycle is a cross, RR x rr; and the second is an intercross, Rr x Rr, with respect to the locus of interest (figure 5) . There are three mating types among the crosses and three among the inter- crosses. The mating types and their probabilities in cycle m (Cm) are : Forr-locus: For a-locus: CROSSES . _ [ARlAR x Ar\Ar\ Incrosses P / = p \aRjaR x ar/ar J r Crosses P (ARIAR x arlar \ _ „ Crosses P ^^ ^ ^ _ qm, D . _ (ARIAR x Arlar\ Backcrosses P ' „ ' = rm, \aRjaR x Ar/ar J m' SYSTEMS OF MATING 23 intercrosses Incrosses P lAR\Ar x ARjAr\ \aR\ar x aRjar } BackcrossesP^' * ^/f), \aRjar x aR/ArJ _ /Aff/ar x i4fl/ar\ Intercrosses P ' ' • \aRjAr x aRjAr) In each mating type of the crosses for the r-locus, the standard inbred strain is on the left, the mutant stock on the right. CYCLE m 0 GENERATION n 0 I 2 3 4 Fig. 5. The cross-intercross system. MUTANT Rr INBRED STRAIN CROSS INTERCROSS CROSS INTERCROSS CROSS With respect to the a-locus, the incrosses, with probability pm, in the first generation of Cm, each produce one kind of offspring which yield incrosses in the next generation of the same cycle. These, in turn, contribute exclusively to the incrosses of the first generation of the next cycle, since all progeny other than rr are discarded. The crosses, with probability qm, also each produce one kind of offspring, all doubly heterozygous. These when intercrossed produce three kinds of rr progeny AA (or aa), Aa, and aa 24 GENETIC STOCKS AND BREEDING METHODS (or AA) in the ratio c2:2c(\ - c) : (1 - c)2. These in turn, when crossed to the inbred strain, yield incrosses, crosses, and backcrosses with respect to the a-locus in the ratio c2:{\ - c)2:2c{\ - c). Finally, the backcrosses, with probability rm, in the first genera- tion of Cm, each produce two kinds of offspring, ARIAr and AR/ar, or aR/ar and aRjAr, in the ratio 1/2:1/2. These yield three kinds of matings in the second generation o Gm, incrosses, backcrosses, and intercrosses, in the proportions 1/4:1/2:1/4. The incrosses in the second generation of Cm each produce one kind of rr progeny, with relative frequency 1/4, and thus yield only incrosses in the first generation of the next cycle, Cm+1. The backcrosses each produce two kinds of rr progeny, A A and Aa, or aa and Aa, in the ratio (l/2)f : (1/2)(1 - e) and so yield incrosses and backcrosses in the same ratio in the first generation of Cm + x. The intercrosses produce three kinds of rr progeny, AA, Aa, and aa, or aa, Aa, and AA, in the ratio (l/4)c2: (l/2)c(l - c) : (1/4)(1 - c)2, and so yield incrosses, crosses, and backcrosses in the proportions (l/4)c2:(l/4)(l - r)2: (l/2)t(l - c) in the first generation of Cm + 1. In summary, the backcrosses in the first generation of Cm yield incrosses, crosses, and backcrosses in the first generation of Cm + 1 in the proportions (1/4)(1 +c)2:(l/4)(l - c)2: (1/2)0 -c2). This is so because 1/4 + (1/2V (1/2)0 -c + (l/4)f2 = 0/4)0 + <>2, 0/4)(l -c)2 = (1/4)(1 -c)\ + o/2Mi -c) = (1/2)0 -<-2)- The probabilities of the various mating types in Cm + x may then be represented as functions of the probabilities in Cm by three linear equations: A» + i = Pm + c2qm + (1/4)(1 +c)zrm, 7. + i = (1 ~c)2qm + (1/4)(1 -c)2rm, 2c{\ -c)qm + (1/2)(1 -c2)rm. Again the incrosses are an absorbing barrier; once matings of like homozygotes are reached, they hold their type in succeeding generations. At the same time fractions of the matings of other types yield more incrosses which are thenceforward fixed, the rate depending upon c. By means of these equations, the probability of the desired type of mating (incrosses) may be computed for any number of cycles and for selected values of c. The results for 6 cycles, or 12 generations, are outlined in figure 6, for the case q0 = 1 , that is when breeding starts with a cross AR/AR x arjar, or aRjaR x ArjAr. When c = 1/2 and q0 = 1, the probabilities pm of incrosses, the matings of the desired type, are the successive values 0, 1/4, 19/32, 203/256, 1835/2048,... form =0, 1,2, 3,4 The probability hm of heterozygotes is hm = r„ . SYSTEMS OF MATING 25 which takes the successively smaller values, after the initial generation, 0, 1/2, 5/16, 21/128, 85/1024,... for m — 0, 1, 2, 3, 4, . . ., when c — 1/2 and q0 = 1. The ratios of the probabilities of heterozygotes in successive generations tends toward a constant as m increases. For large m, ^m + i/^m = I — c, approximately. The probability of heterozygosity in any advanced cycle is, approximately, a fraction, 1 — c, of the probability of heterozygosity in the preceding cycle. When c = 1/2, the loss tends to be 1/2. Fig. 6. Probability of incrosses for the cross-intercross system. 1.0 0.8 0.6 PROBABILITY m 0.4 0.2 0.0 2 3 4 CYCLE m The probability of incrosses pm for the cross-intercross system, starting with q0 = 1, for five selected values oft = 1/10, 2/10, 3/10, 4/10, 5/10. After the cross-intercross system has been in effect for several cycles, the incrosses will be increasing at a steady rate exactly balanced by the rate of decrease of the crosses and backcrosses. This rate may be found by finding the characteristic root of the determinant made up of the q and r rows and columns of the cycle matrix (table 7). The desired root is Aj = 1 — c. Table 7 Cycle matrix of the cross-intercross system Pm qm rm /'m-rl <7m + l rm + 1 1 c2 (l - C)2 2c(l - c) (1/4)(1 + cy (1/4)(1 -c)2 (1/2)(1 - c)2 (1 - c) 2c{\ - X = 1 2 c) - a (1/4)(1 (1/2)(1 c, (1/2)(1 - -cy - cy - x cy = 0 26 GENETIC STOCKS AND BREEDING METHODS This result shows that one cycle of the cross-intercross system is genetically equivalent to one generation of the backcross system. The cycle matrix for the cross-intercross system can be obtained by directly reason- ing what proportion of each type of mating a given type will yield in the subsequent cycle, two generations later. It is possible, however, to avoid the confusion attendant on the attempt to reason through two generations. A generation matrix can be con- structed for each generation in the cycle. By multiplying these matrices together in order opposite to that in which they occur, the cycle matrix is obtained. Thus, for a cycle of k generations, C = GfcGfc.!- . .Gl5 where C is the cycle matrix, and G; is the generation matrix for generation i in the cycle. In the case of the cross-intercross system, the matrix for cross generations is while that for intercross generations is G2 = 1 0 1/4 0 0 1/2 0 1 1/4 and C = GaGi. From the matrices given in tables 7 and 8, Pm can be calculated for any m, given P0. Also the number of cycles required to obtain any given percentage of incross matings can be calculated. For both calculations, we must keep in mind that one cycle is the equivalent of two generations. Table 8 The A, A, and A-1 matrices for the cross-intercross system A = /l -c 0 °\ ° \ o (1/2)(1 - c)* 0 ?) A = 6 1 1 1 -(1 1/2 \ - c)/4e\ A-1 = /_(! + 3c)/(\ +c) (1 - 0/(1 + c) \ 4c/(l + c) 2c/ (I + c) 2c/(l + C) -4c/(l + c) 0/ SYSTEMS OF MATING 27 For c — 1/2, 6 cycles (or 12 generations) of the cross-intercross system are required to exceed 95 per cent incross matings, and 8 cycles (16 generations) to exceed 99 per cent incross matings. For c = 1/10, 31 cycles (62 generations) are required to exceed 95 per cent incross matings and 46 cycles (92 generations) to exceed 99 per cent. This system has been used to put a number of mutations in mice on standard inbred backgrounds. These include: C57BL/6J-T/a, C57BL/6J-/?, C57BL/6J-rw, and BALB/cHu-z'y. Complete lists of these strains are mentioned by Lane.751 Snell1238 used this system to isolate single genes or small segments of chromosomes which affect histocompatibility. Examples are AKR-//-2a (syn. AKR-K), C3H-#-2b (syn. C3HSW), and C57BL/10-//-ld (syn. BIOBY). THE CROSS-BACKCROSS-INTERCROSS SYSTEM The cross-backcross-intercross system, like the cross-intercross system, is used to put a recessive viable gene r on a standard inbred background. It is especially useful when the mutant phenotype is not visible and easily detectable, but must be determined by a laboratory test, and when the ease of making up another generation of matings (the backcross) outweighs the cost of determining the phenotype. The system was invented and first used by Snell for developing strains of mice with genes for resistance to tumor grafts on standard inbred backgrounds. The matings proceed in cycles of three generations. The first mating of each cycle is a cross, RR x rr, when rr is the mutant and RR is the genotype of the inbred strain. The second mating is a backcross, RR x Rr, formed by mating the heterozy- gous progeny of the cross with animals of the inbred strain. The third mating is an intercross, Rr x Rr, of the heterozygous progeny of the backcross. The heterozygotes are discovered to be so by observing which matings produce rr progeny. All other matings, 3/4 of the total on the average, are wasted. The rr progeny are then crossed with RR animals of the inbred strain to start another cycle (figure 7). Among the crosses, there are three mating types when any other locus, the a-locus, is considered in addition to the locus of interest. There are two types of backcrosses and three types of intercrosses. The mating types and their probabilities in Cm are: „ (ARIAR x ArlAA crosses Incrosses F _. ' , ) = pm, \aRjaR x arjar J Crosses F (AR/AR x ar/ar \ _ Crosses P ^ ^ x ^^J - qm, Backcrosses P (AR/AR x Ar/ar\ \aR/aR x Ar/ar) „ (ARIAR x AR/AA backcrosses Incrosses F I „.' ' > \aRjaR x aRjar J Backcrosses P I ARJAR x AR[ar\ \aRfaR x aRjAr) 28 GENETIC STOCKS AND BREEDING METHODS intercrosses Incrosses Backcrosses Intercrosses (AR/Ar x ARjAr\ \ aRjar x aRjar /' (AR/Ar x ARjar\ \ aRjar x aR/Ar J I ARjar x AR/ar\ [aR/Ar x «/?/.4r)' In the crosses and backcrosses with respect to the r-locus, the inbred strain is on the left, the mutant-bearing animals on the right. With respect to the a-locus, the CROSS-incrosses with probability pm each yield BACKCROss-incrosses and these yield iNTERCROSS-incrosses, which thus lead to cross- Fig. 7. The cross-backcross-intercross system. INBRED STRAIN MUTANT GENERATION CYCLE m 0 CROSS BACKCROSS INTERCROSS CROSS BACKCROSS INTERCROSS SYSTEMS OF MATING 29 incrosses to begin the next cycle. The CROSS-crosses, probability qm, produce back- CROSS-backcrosses, and these produce intercrosses of three kinds, incrosses, back- crosses, and intercrosses, in the ratio c2:2c(\ —c):(\ — c)2. These in turn yield crosses of three kinds to begin the next cycle, incrosses, crosses, and backcrosses, in the ratio c2(2 — r)2:(l — c)4:2r(l — r)2(2 — c). The CROSS-backcrosses, probability rm, produce BACKCROss-incrosses and BACKCROSS-backcrosses in the ratio 1/2:1/2. The BACKCROSS-incrosses yield iNTERCROSS-incrosses and thence CROSS-incrosses to begin the next cycle. The BACKCRoss-backcrosses yield intercrosses of three kinds, incrosses, crosses, and intercrosses, in the ratio {\j2)c2:c{\ — c) : (1 /2) ( 1 — c)2; and these in turn yield crosses to begin a new cycle with incrosses, crosses, and backcrosses, in the ratio (1/2V2(2 - ^2:(1/2H1 - r)4:r(l-r)2(2 - c). Fig. 8 . Probability of incrosses for the cross-backcross-intercross system. I. Or PROBABILITY Pm CYCLE m The probability of incrosses pm for the cross-backcross-intercross system beginning with q0 = 1," for values of c = 1/10, 2/10, 3/10, 4/10, 5/10. The ratio of the probabilities of heterozygosity in successive generations is constant, hm + 1/hm = (1 — c)2, when m > 1. The probabilities of mating types in Cm + 1 may thus be shown as functions of the probabilities of mating types in Cm : Pm + l = Pm + qn+\ = c2(2 - c)2qm + {(1/2) + (l/2)c2(2 - c)2}rm, (1 -cYqm+ (1/2) (1 -c)*rn, 2r(l _ C)2{2 - c)qm + r(l - c)2{2 - c)rn Again the familiar result emerges. The incrosses, probability pm, increase at the expense of the backcrosses, qm, and the intercrosses, rm. The rate of increase of/?m depends upon c, the probability of crossing over between the a-locus and the r-locus. When the initial mating is a cross, that is, q0 = 1 , the desired types of matings increase as shown in figure 8, for selected values off. When c = 1/2 and q0 — 1 , the successive values of pm are 0, 9/16, 57/64, 249/256,..., 30 GENETIC STOCKS AND BREEDING METHODS for m = 0, 1 , 2, 3, The probability of heterozygosity is the same as the probability of backcrosses among the crosses, i.e., hm = rm. This probability takes the successive values 0, 3/8, 3/32, 3/128, 3/512,..., for m = 0, 1, 2, It may be seen at once that when m > 0, hm+1 = (l/4)*m, when <70 = 1 and c — 1/2. The characteristic root of the determinant formed from the q and r rows and columns of the cycle matrix (table 9) is Xt = (1 - c)\ Pm + l ?m + l rm + l Table 9 Cycle matrix for the cross-backcross-intercross system A. 1 0 0 c2{2 - c)2 (1 - cY 2c{\ - c)2(2 - c) (1/2) + (1/2)C2(2 - c)2 (1/2)(1 -cy c{\ - c)2(2 - c) (i -cY - x (i/2)(i - cy 2c{l - c)2{2 - c) c(\ - c)2(2 - c) - X X = (1 - c)2, 0 = 0 This is the factor of proportionality between successive values of q and r, <7m + l = ^-<7m> rm + 1 = Arm- It also gives the relationship between successive values of A, the probability for hetero- zygosity, since hm = rm. When c = 1/2 hm + 1 = (1 -c)*hm= (1/4)AB> m>0 confirming the result of the preceding paragraph. Again, the cycle matrix may be obtained more easily by multiplying the generation matrices in reverse order to that in which they appear. The cross generation matrix is Gi = /l 0 1/2 (I 0 l/2\ \0 1 1/2J* SYSTEMS OF MATING 31 The backcross generation matrix is g2 = n c2 \ 0 2,(1 -e)\. \0 (\-c)2/ Finally, the intercross generation matrix is G3 = /l c c2 \ 0 0 (1 -*)")■ ^0 1 - c 2c{\ - c)j Thus, the cycle matrix is C = 036^ = /l c2{2 - c)2 (1/2) + (l/2)c2(2 - c)2\ 0 (1-,)* (1/2)(1 - 04 • \0 2c(l - c)2{2 - c) c{\ - c)2(2 - c) J From the matrices in table 1 0 the frequencies of various types of matings in any cycle and the number of generations to reach a given percentage of incross matings can be calculated, remembering that a cycle represents three generations. Table 10 A, A, AND A-1 MATRICES FOR THE CROSS-BACKCROSS-INTERCROSS SYSTEM A = 7(1 - c)2 0 \ 0 0 0 0 3 A = /0 (? 1 1 1 -(1 - 1/2 \ -c)2/2c(2 -c)\ A"1 = lc2 - 2c - 1 (1 - cy \ 2c(2 - c) c(2 - c{2 - -2c{2 c) c) -c) 0/ Under the cross-backcross-intercross system, with c = 1/2, 3 cycles (9 generations) are required to exceed 95 per cent incross matings and 4 cycles (12 generations) to exceed 99 per cent. If c = 1/10, 16 cycles (48 generations) and 23 cycles (69 genera- tions) are required to exceed 95 per cent and 99 per cent incross matings. It should also be noted that approximately four times as many matings as are required to maintain the stock must be made up in the intercross generation to assure a sufficient supply of the types of matings required. The cost of these extra matings should be borne in mind when the cross-backcross-intercross system is considered for use. 32 GENETIC STOCKS AND BREEDING METHODS This system is being used by Snell for the isolation of loci affecting histo- compatibility in the mouse. No strains have advanced far enough to have been mentioned in the literature. BROTHER-SISTER INBREEDING WITH HETEROZYGOSIS FORCED BY BACKCROSSING The system of brother-sister inbreeding with heterozygosis forced by backcrossing is useful when it is desired to put a recessive mutation and its normal allele or a semi- dominant lethal mutation and its normal allele on a common inbred background. Unlike the three preceding systems, the mutant-bearing animals are not crossed with animals of an existing inbred strain, but are mated as brother x sister. With respect to the locus of interest, all matings are rr x Rr, in the case of a recessive viable mutation r, or dd x Dd in the case of a semidominant lethal mutation D. The other homo- zygote is either rejected (RR) or lethal [DD). As before, let A/a be any other locus whose heterozygosity is in question, and let c be the probability of crossing over between the a-locus and the r-locus or ZMocus. There are six types of matings with probabilities denoted as follows: (ArjAr x ARjAr\ backcrosses lncrosses r , J = pn, arjar x aRjar Crosses PI , ' n , . I = qn, \arjar x ARjAr) (Ar/Ar x ARjar\ [ar/ar x aRjAr) ~ *»' (Arjar x ARjAr\ _ \Arjar x aRjar J ~ *n' (Ar/Ar x aRjAr\ [ar/ar x AR/ar) ~ ni _, [Ar/ar x AR/ar\ Intercrosses r I . ' _, . ) = vn. \Ar/ar x aR/Ar) Backcrosses D [Ar/Ar x AR/ar (first kind) Backcrosses D [Ar/ar x AR/Ar\ (second kind) Backcrosses (third kind) If each r is replaced by d and each R by D, the matings refer to inbreeding with a semidominant lethal mutation. The incrosses yield only incrosses in the next generation. The crosses produce two kinds of progeny, aR/Ar and Arjar, or ARjar and Arjar, but under the rule of this system of mating brother x sister, which are rr x Rr, they yield only one kind of mating in the next generation, intercrosses. The backcrosses of the first kind produce four kinds of progeny which yield incrosses, backcrosses of the second kind, backcrosses of the third kind, and intercrosses in the ratio c(l — c):{\ — c)2:c(\ — c) in the next generation. The backcrosses of the second kind also produce four kinds of progeny which yield incrosses, backcrosses of the first kind, backcrosses of the second kind, and intercrosses SYSTEMS OF MATING 33 in the ratio 1/4: 1/4: 1/4: 1/4 in the next generation. The backcrosses of the third kind likewise produce four kinds of progeny and these yield incrosses, backcrosses of the second kind, backcrosses of the third kind, and intercrosses in the ratio c(\ — c) : c2: (1 — c)2:c{\ — c). Finally, the intercrosses produce seven kinds of progeny which yield all six kinds of matings in the ratio (l/2)c(l -*):(l/4) - (1/2M1 - c):{\j2)c{\ - c) : (1/4) : (1/4) - (l/2)c(l - *):(l/4). The linear equations which relate the probabilities of Gn + j to the probabilities of G„ are: Pn+l = Pn + ?n + l = rn + 1 = sn + 1 = *7l+ 1 = 0» + l = where A; = c(\ — c). qn + krn + (1/4K + ktn + (1/2) kvn, (1/4)(1 -2A:)0ns (1/*K + (l/2)^n, ;i -r)2rn + (1/4)j, + c\+ (l/4)vn, c\+ (1 -c)\+ (1/4)(1 -2/t)^, ATn + (1/4K + to; + (i/4)on, Fig. 9. Probability of incrosses with heterozygosis forced by backcrossing. I.Or 1.0 0.8 0.6 PROBABILITY P m 0.4 0.2h 0.0 2 4 6 8 10 GENERATION, n 12 2 4 6 8 10 GENERATION. n 12 Left: The probability of incrosses for four values of c — 0, 1/10, 3/10, 5/10 for the system of brother-sister inbreeding with heterozygosis forced by backcrossing, starting with q0 = 1. Right: The probability of incrosses pn for three values of c = 1/10, 3/10, 5/10, for the system of brother-sister inbreeding with heterozygosis forced by backcrossing, starting with t0 = 1. Matings in the initial generation may be any of the six types. Two of these are slightly more likely to occur in laboratory populations. First, the mutant allele D may be a semidominant lethal in a noninbred line. When inbreeding is started, the least favorable mating with respect to homozygosity at the a-locus is a cross Ad/ Ad x aDjad, or ad/ad x AD /Ad, i.e., q0 = 1. The probability p of incrosses increases relatively slowly (figure 9). Second, the mutant allele r may already exist in one inbred strain .34 GENETIC STOCKS AND BREEDING METHODS and its nonmutant allele R in another. A preparatory cross between the strains will produce double heterozygotes, ARjar (or aR\Ar). When these are mated with the mutant-bearing strain ar/ar (or ArjAr) the initial matings are backcrosses of the third kind, i.e., /„ = 1 . The probability p of incrosses increases more rapidly with this starting point (figure 9). The probability of heterozygosity is less informative than the probability of in- crosses in this system of breeding. When it is desired to compute hn, however, it must be observed that two probabilities are required, one for rr (or dd) and one for Rr (or Dd) animals. They are: P(Aa\rr) = h'n = sn + vn, P(Aa\Rr) = h"n = rn + tn + vn. In general, the probability h'n of heterozygosity at the a-locus among the homo- zygous mutants will be less than the probability h"n of heterozygosity at the a-locus among the heterozygous mutants for all loci linked with the mutant locus. By forcing heterozygosity upon the locus of interest, one also forces heterozygosity upon loci linked with it. The probabilities, h'n and hi, for the first twelve generations, starting with either a cross (q0 = 1) or a backcross (/0 = 1), are shown in figure 10. If c is near zero, and if q0 = 1, the probability is approximately 2/3 that in a given line the heterozygotes Rr will remain heterozygous Aa at the closely linked a-locus. This is so because the probability tn of backcrosses of the third kind approaches 2/3 as n increases, while sn and vn each approach zero. The probability pn of incrosses equals 1 — tn; as n increases />n approaches 1/3. If t0 — 1 and c is near zero, there is practically no chance of getting incrosses, since backcrosses of the third kind with probability tn yield only backcrosses of the third kind; to = h = t2 = ■ ■ ■ = tn = 1 approximately. The determinant formed from coefficients of qn, . . ., vn in the equations for qn+1, . . . ., vn + 1 is expressible as an equation of the 5th order: (i5 - (3 - 2c - 2k) ^ - c/u3 + {(5 - 7c - 2*)(1 - 2k) + 4(1 - 2c)k2}/u2 - (1 - 2c)(\ - 2k)(\ - Ak)fi - 2(1 - 2c){\ - 2k)2 = 0 where ju — 2X. When c = 1/2, the characteristic root is, as expected, the same as for brother-sister inbreeding, i.e., Xx = (1/4)(1 + y/b). The characteristic root rises to one as c decreases to zero. The values of Xj for selected values off are: c = 0.5 0.4 0.3 0.2 0.1 0 Xx = 0.8090 0.8135 0.8270 0.8514 0.8984 1. It has not been possible to solve the equation |G - X,I| = 0 (8) SYSTEMS OF MATING 35 for this system of brother-sister inbreeding with heterozygosis forced by backcrossing. Values of Xx for selected values of c have been estimated from repetitive application of the equation Pn + 1 = GPr (2) for n = 0, 1, . . . , 11. Until a general solution of equation (7) can be derived for these systems, the equation Vn = A"V0 (6) cannot be applied. Using the above estimate of Xl5 however, the number of generations required to obtain a given percentage of incross matings can be estimated. Fort = 1/2, 15 genera- tions of brother-sister inbreeding with heterozygous forced by backcrossing are required Fig. 10. Probability of heterozygosity with heterozygosis forced by BACKCROSSING. hi 4 6 8 10 12 GENERATION, n 4 6 8 GENERATION, n 10 12 Top: The probability of heterozygosity, h'n for rr, h"n for Rr, for selected values of c = 0, 1/10,. 3/10, 5/10, for the system of brother-sister inbreeding with heterozygosis forced by backcrossing, starting with q0 = 1. Bottom : The probability of heterozygosity, tin for rr, h'n for Rr, for selected values of c = 1/10, 3/10, 5/10 for the system of brother-sister inbreeding with heterozygosis forced by backcrossing, starting with t0 — 1 . 561 GENETIC STOCKS AND BREEDING METHODS to exceed 95 per cent incross matings, starting with q0 — 1 ; 22 generations are required to exceed 99 per cent incross matings. For c = 1/10,26 and 39 generations, respectively, are required to exceed 95 per cent and 99 per cent incross matings. It should be noted, however, that when c is not zero this system ensures eventual attainment of animals homozygous and heterozygous at the locus of interest, and otherwise coisogenic, aside from recent mutations. Examples of strains of mice produced by this system of mating are: SEC/IGn — se, FS/Gn -fs, and QV/Gn - qv. See Lane751 for a complete list. BROTHER-SISTER INBREEDING WITH HETEROZYGOSIS FORCED BY INTERCROSSING This system, brother-sister inbreeding with heterozygosis forced by intercrossing, is useful in putting a recessive lethal mutation on an inbred background. Similarly, any recessive mutation with low viability or fertility when homozygous and its normal allele may be put on a common inbred background. If the new recessive mutation has recently arisen in an already inbred strain, this system provides a way of continued inbreeding. All matings are between heterozygotes, Rr x Rr, with respect to the locus of interest. It will commonly be necessary to determine which are Rr x Rr matings by observing which matings of/?- x R- produce rr progeny. The system is also useful for inbreeding with a semidominant lethal. In this case, all matings are Dd x Dd, DD being lethal, and dd being rejected. Let A/a be any other gene pair whose heterozygosity is in question and let c be the probability of crossing over between the a-locus and the r-locus (or ZMocus). Five types of matings may occur with probabilities in Gn denoted as follows : „ I AR/Ar x AR/Ar\ intercrosses Incrosses P I ' ' ] = pn, \aRjar x aR/ar ) Crosses P (AR/Ar x aR/ar) = qn, (AR/Ar x AR/ar\ aR/ar x aR/Ar AR/Ar x aR/Ar aR/ar x AR/ar I Intercrosses 1 AR/ar x AR/ar\ (first kind) [aR/Ar x aR/Ar) = V"' Intercrosses P (AR/ar x aR/Ar) = wn. (second kind) The incrosses yield incrosses exclusively in the next generation. The crosses yield intercrosses of the first kind and intercrosses of the second kind in the ratio 1/2: 1/2 in the next generation. The backcrosses each produce three kinds of progeny and then yield four kinds of matings — incrosses, backcrosses, intercrosses of the first kind, and intercrosses of the second kind — in the ratio (1/4) : (1/2) : (1/4)(1 - 2k):(\/2)k, where k = c(\ — c), in the next generation. The intercrosses of the first kind produce four kinds of progeny and all five kinds of matings in the ratio 2k2:2k2:4k(l — 2k): SYSTEMS OF MATING 37 1 — 4£ + 2k2 :2k2 in the next generation. Finally, the intercrosses of the second kind produce four kinds of progeny and all five kinds of matings in the ratio (1/2) (1 - 2£)2:(1/2)(1 - 2k)2Ak(\ - 2k) :2k2 :2k2 in the next generation. These results may be shown as a set of five linear equations : Ai+i =P»+ (l/4)r„ + 2k2vn + (1/2)(1 - 2k)2wn, <7n + i = 2k2vn+ (1/2)(1 -2k)2wn, rn+i = (l/2)r„ + 4*(1 - 2k)vn + 4A(1 - 2k)wn, vn + 1 = (l/2)yB + (1/4)(L- 2k)rn + (!_« + 2Aa)i/B + 2^n5 (l/2)?n + :i/2)ArB + 2*2*n + 2£2wr Fig. 11. Probability of incrosses with heterozygosis forced by intercrossing. 1.0 PROBABILITY 4 6 8 10 GENERATION, n The probability of incrosses pn for selected values of c = 0, 1/10, 1/2 for the system of brother-sister inbreeding with heterozygosis forced by intercrossing, starting with q0 = 1. The probabilities pn of incross matings, the desired type, for selected values of c are shown in figure 11, assuming that q0 = 1 . This is the case if the mutation of interest arose in a noninbred stock and the initial mating between carriers of the mutation r is AA x aa. This is the least favorable starting point with respect to the value o£pn ; other starting points will produce higher values of pn in fewer generations. The nonlinked loci become fixed at the usual rate for brother-sister inbreeding. Linked loci reach fixation more slowly at rates dependent upon c. When c is near zero, about 2/3 of the matings will remain as intercrosses of the first kind and 1/3 become incrosses of the desired type (vn = 2/3 and^n = 1/3 when n is infinite). The probability of heterozygosity decreases depending upon c, as seen in figure 12, except when c = 0. In general, hn = (l/2)rn + vn + wn. The determinant formed from the generation matrix by omitting the pn + 1 row and the pn column yields the general equation : //4 - (3 - 4/ + 2l2)/n3 + (1 - U + 4l2 - 4/3)//2 + (3 - 10/ + 14/2 - 12/3 + 8/4)/* - 2(1 - 5/ + 10/2 - 10/3 + 4/4) = 0 38 GENETIC STOCKS AND BREEDING METHODS Fig. 12. Probability of heterozygosity with HETEROZYGOSIS FORCED BY INTERCROSSING. PROBABILITY h„ 2 4 6 8 GENERATION n The probability of heterozygosity hn for selected values of c = 1/10, 1/2 for the system of brother-sister inbreeding with heterozygosis forced by intercrossing, starting with q0 = 1 . where // = 2X and / = 2c(l — c). Solutions for selected values oft give the following characteristic roots: c = 0.5 0.4 0.3 0.2 0.1 0 Xj = 0.8090 0.8092 0.8115 0.8235 0.8674 1. Again the equation |G - XI | = 0 (8) has not been solved, so that formula Vn = A»V0 (6) cannot be applied. However, the above value of Xx can be used to estimate the num- ber of generations required to exceed 95 per cent and 99 per cent incross matings. These are, for c = 1/2, 15 and 22 generations, and for c = 1/10, 22 and 33 generations. Again it should be noted that all three genotypes at the locus of interest are produced on the same genetic background. Examples of strains of mice produced by this system are: WB/Re-WT, WC/Re-W, WH/Re-H7, and WK/Re-M'. These four strains in which all matings are Ww x Ww provide four different genetic backgrounds on which to compare three genotypes, WW, Ww, and ww of the dominant spotting locus. GENERAL REMARKS The breeder of laboratory animals for research must face the question of how to produce and propagate animals of the specific types he needs for his specific objective. The choice will depend upon the type of animal, the knowledge of the genetics of the trait of interest, the relative efficiency of the mating systems, the ease or difficulty of determining the phenotype of each animal, and the amount of space in the animal room to be devoted to maintenance of stocks. SYSTEMS OF MATING 39 The following assertions are intended to help mammalian geneticists in the task of choosing a suitable breeding system. 1. Close inbreeding, such as brother-sister, has been successful on a large scale only with the house mouse. There are in existence, however, lines of rats, rabbits, guinea pigs, and hamsters which satisfy the usual working definition of being inbred, that is, they have survived 20 or more generations of exclusive brother-sister inbreeding. The house mouse does not, however, withstand the depressing effects which accompany inbreeding as well as the existing profusion of inbred strains suggests. The existing strains are the successful survivors of, probably, a twofold or threefold larger number of attempts to establish inbred lines. If one were to start to produce a new inbred strain of mice, he should maintain several lines, say five, to insure that one or two can be propagated to 20 generations at least. 2. Any of the systems of mating (except random mating) described in this chapter will increase the probability of incrosses and will decrease the probability of heterozygo- sity at all loci except those closely linked with a locus of interest deliberately forced to remain heterozygous. Table 1 1 shows the probabilities of incrosses after 12 generations for each of the regular systems. For loose linkage (c between 3/10 and 1 12), the methods of crossing with locus-control only are all more efficient than the methods of inbreeding and locus-control, assuming that the inbred strains used in crossing are in fact homo- zygous. For closer linkage, the methods using forced heterozygosis have greater efficiency. Efficiency may be defined by the number of generations required to achieve a given probability of incrosses or by the probability of incrosses achieved with a fixed number of generations. Table 1 1 also shows the number of generations required to obtain a 95 per cent or a 99 per cent frequency of incross matings. Table 1 1 Probabilities of incrosses after 12 generations and numbers of generations required to achieve probabilities of 95 and 99 per cent for incrosses for six mating systems System Probabilities of incrosses at G12 Generations required to obtain a per cent of incrosses = 0.5 0.4 0.3 0.2 0.1 ac = 0.5 0.4 0.3 0.2 0.1 2. Brother x sister 3. Backcross 4. Cross-intercross 0.8922 — 0.9995 0.9964 0.9802 0.9141 0.6862 0.9740 0.9267 0.8282 0.6508 0.3727 0.9932 0.9725 0.9130 0.7718 0.4877 5. Cross-backcross- intercross 6. Heterozygosis forced 0.8922 0.8853 0.8618 0.8097 0.6833 by backcrossing 7. Heterozygosis forced 0.8922 0.8920 0.8890 0.8723 0.7953 by intercrossing 95 16 — — — — 99 24 95 6 7 10 15 30 99 8 11 14 22 45 95 12 14 20 30 62 99 16 20 28 44 92 95 9 12 15 24 48 99 12 15 24 36 69 95 15 15 16 19 26 99 22 23 25 29 39 95 15 15 15 16 22 99 22 22 23 24 33 40 GENETIC STOCKS AND BREEDING METHODS I 3. In the absence of selection, all loci have equal probabilities of becoming homozygous in any one system, except those linked with a locus of interest. Thus alleles with deleterious effects may also become homozygous. It is inescapable that alleles with deleterious effects will be selected against in the simple act of trying to keep the lines in propagation. 4. The backcross, cross-intercross, and cross-backcross-intercross systems are not only efficient means of putting mutant alleles on inbred backgrounds; they also take advantage of prior inbreeding and selection of the inbred line. 5. It is not always possible, and in some instances it may not be desirable, to put a mutant allele on a standard inbred background. That is, the mutant and the avail- able backgrounds may be in some way incompatible. In these cases, the systems of brother-sister inbreeding with forced heterozygosis offer distinct advantages. They allow the background to evolve as inbreeding progresses. The mutant may thus, so to speak, select its own most favorable background from those possible out of the genetic makeup of the mutant generation. 6. A new mutation which arises in an inbred line should be perpetuated within the line of origin in order to keep the new mutant and its nonmutant allele on the same background. Of course, this does not preclude any outcrossing which will be necessary to establish the genetic basis of the new mutation and to explore its interactions with various other alleles and nonalleles. 7. The backcross, cross-intercross, and cross-backcross-intercross systems are effective means of searching for genetic differences between inbred strains. The search is more likely to yield positive results if any two strains having different pheno types with respect to the trait under study are also different by a relatively small number of loci with discrete effects. However, the true situation cannot be known until after the search has been carried out. 8. The systems described, except random mating and brother-sister inbreeding, all yield mice bearing different alleles on common inbred backgrounds. These strains thus constitute the precision tools for genetic, developmental, physiologic, biochemical, pathologic, behavioral, and immunologic studies. They may be used in either of two ways. First, the alleles caused to segregate (the locus of interest) may be chosen in advance because of their known effect on hair, pigment, blood, skeleton, behavior, and so forth; thus the animals bearing the different alleles are suited for studies of hair or pigment or blood or skeleton or behavior. Second, the animals which differ by one allele only may be studied with a view to seeing if this one allele is concerned with some seemingly unrelated property of the organism. Thus dense (Dd) and dilute (dd) mice may be studied for differences in learning, radiation resist- ance, or susceptibility to disease. This may be a needle-in-haystack search, but once a difference is found, the genetic explanation is readily at hand. This second type of study may be contrasted with searching for differences between inbred strains with respect to such characteristics as ability to learn, resistance to irradiation, or susceptibility to disease. Such a search is almost certain to uncover SYSTEMS OF MATING 41 differences between some of the inbred strains. Yet, once found, there may be great difficulty in accounting for the difference in genetic terms. If such an analysis is im- portant, a classical analysis of producing first, second, and third hybrid generations and first and second backcross generations may yield the information that two given strains differ by 8 or 10 or 50 pairs of genes. This information does not, however, easily pave the way for developmental, physiologic, and other studies with precise genetic control. SUMMARY Mammalian geneticists use a number of breeding techniques to produce suitable animals for research. Aside from random mating, the common objective of all of the systems of matings described in this chapter is to reduce the probability of heterozygotes and to increase the probability of matings of like homozygotes with respect to any locus, called the a-locus, which is not being specifically controlled in the mating system. The random mating system is intended to hold constant the probabilities of all genotypes at each locus from generation to generation. The brother-sister inbreeding system will increase the probability of incrosses, that is, of matings of like homozygotes, with each advancing generation. For neutral genes, the probability will exceed 95 per cent after 1 6 generations and 99 per cent after 24 generations. Three systems of mating (the backcross system, the cross-intercross system, and the cross-backcross-intercross system) are all means of placing mutant genes and their non- mutant alleles on inbred backgrounds. The backcross system is especially useful with dominant mutations, but it may be used with recessive viable or recessive lethal mutations. The cross-intercross and cross-backcross-intercross systems were designed for putting recessive viable mutations on inbred backgrounds. The cross-backcross- intercross system is more efficient than the cross-intercross system in that it requires fewer generations to reach a specified probability of incrosses with respect to any neutral locus (a-locus) not being specifically controlled. Two systems of mating combine inbreeding with locus control. These are brother- sister inbreeding with heterozygosis forced by backcrossing and brother-sister in- breeding with heterozygosis forced by intercrossing. The first, or backcrossing system, is useful with a semidominant lethal or recessive viable mutation ; the second, or intercrossing system is useful with recessive lethal mutations. When the neutral a-locus is closely linked with the mutant locus (0 < c ^ 0.2), these two systems are more efficient than the three systems which require locus control accompanied by crossing with an inbred strain. For higher values of crossing over, they are less efficient. DISCUSSION Dr. Burdette: One most qualified to discuss this paper is Dr. Sewall Wright, who has consented to review from a different point of view the problems of breeding. 42 GENETIC STOCKS AND BREEDING METHODS Dr. Wright : There are two main questions which arise in connection with such a paper as that presented by Drs. Green and Doolittle : first, the question whether solution of the mathematical problem is useful in genetics and, second, whether the results given are correct. Dr. Green has brought out so clearly the importance of knowing how many generations are required to bring about reasonable assurance of isogenicity in all neutral genes under diverse systems of mating that no additional attention need be given to this. With regard to correctness, I have found nothing with which to disagree. I did not attempt to repeat the method of attack used by the authors. It seemed best to compare their results with those of a wholly different method, path analysis, which I have been applying to such problems since 1 92 1 . This leads to certain extensions. As applied to mating systems, this method consists in deducing the changes brought about automatically in the correlations between gametes. As it is a correlation method, it deals only with relations between two things at a time. Thus, it can deal with the relative amount of heterozygosis expressed as a function of the correlation between uniting gametes but cannot deal with the changes in frequency of types of mating where this involves relations among four varying gametes. Thus, it does not give as complete a picture as can be obtained from the matrix of mating types (the method of Bartlett and Haldane56 used by Green and Doolittle), but what it can do, it does more simply. It may be noted that after a sufficient number of generations of a regular system of mating in which the number of individuals in the inbreeding group is constant, all diallelic mating types tend to fall off in frequency at the same limiting rate as does heterozygosis, a rate which is easily obtained by path analysis. Because of its relative simplicity, generalizations can be made by path analysis which have not been found practicable in terms of mating types. I will devote most of my discussion to this point. The method applies only to the effects of accidents of sampling and thus only to neutral alleles. The joint effects of sampling, selection, recurrent mutation, and occasional outcrossing have been dealt with in other ways.1417- 1422 It is necessary to assume that genie frequencies remain constant in a hypothetical, total population consisting of all possible inbreeding lines in order to have a constant basis for comparison of the correlations within lines. In the case of the two alleles, it is assumed that the frequency array in both ova (o) and spermatozoa (s) always remains [(1 — q)a + qA] in this total population. The relation between uniting gametes is represented below, in terms of the amount of heterozygosis, h. The values assigned to a and A obviously make no difference in the correlation between them. It is con- venient to assign 0 to a and 1 to A. os a A Total A h/2 q- (h/2) q a 1 - q - (h/2) h/2 1 - q Total 1 — q SYSTEMS OF MATING 45 d = s = q rso(n, and r00(I) with weights of 1/4, 2/4, and 1/4 respectively. r, = (l/4)|/ss(7) + 2rs0(/) + r00(I)\ Similarly rN ~ ( * /4) |/ss(JV) + ^rso(N) 4" roo(.N)\' On tracing the connecting paths in figure 15 it is found that 'sow = F = [(1 - cs){\ - c0) + cscoyN + 0,(1 - c0) + (1 - csK]r;. If d0 is replaced by D0, coefficients c0 and (1 — c0) must be exchanged; and, if Ds is replaced by ds, cs and (1 — cs) must be exchanged. Union of ds with D0 gives the same result as above. rso(n = [(* - «t)0 - co) + WoVi + 0,0 - O + (1 - cs)coyN. SYSTEMS OF MATING 47 Fig. 15. Mating of type Dd x Dd. D F' d r^ D F' d \Di 17 l_(tscs co'"co \i u Ds d0 F = r so(N) l-csc- l-c0 c0 Ds Do D F' d r'M D F'^d \D / N D / v / '■cscscs'"cs '"cs cs V V 1/ Ds ds Ds ss(N) i X^i 'IN D F' d r'N D F' d vP/i n / Ds Ds Ds D F d r'M D F d O / \o/ •V / '*coco(t)'"co '~co co V \l 1/ D0 d0 D0 roo(N) D F' d r'N D F' d i7i 17 V ^ * / Do Do D0 rso(I) ss(I) oo(I) Diagrams for analysis of six types of gametic pairs with respect to linked locus. In the case of correlations between two spermatozoa or between two eggs, the probabilities that they are produced by the same individual [(l/Nm) in the case of two spermatozoa, (l/Nf) in the case of two eggs] and the probabilities that they are pro- duced by different individuals must be taken into account. '«!> = (l/tfmHKl " O2 + ',*] + &.(1 - CS)F'} + [(Nm - l)/JVm]{[(l - cs)2 + c2y + 2cs{l - cs)r'N} rss(N) = (\/Nm){[(\ - cs)2 + c*]F' + 2cs(l - cs)} + \(Nm-l)INm]{[(\ - cs)2 + c2]r'N + 2cs(\ - cy,}. Similarly r00(/) = (1/JV,){[(1 - c0)2 + c02] + 2c0(l - c0)F'} + [(Nf-\)/Nf]{[(i - o2 + c2y + 2c0(i - c0yN} W) = (W){[(1 - O2 + c2]F' + 2c0(l - c0)} + [(Nf-\)INf]{[(\ - c0)2 + c2yN + 2c0{\ - coy}. 48 GENETIC STOCKS AND BREEDING METHODS It is convenient to let ls = 2cs(l - cs), l0 = 2c0(\ - c0), and lm = cs{\ - c0) + (1 - cs)c0 and a = (Nm - l)/JVm, b = (Nf - l)jNf. The equations for rN and r, can be written from the appropriate averages. A further condensation of symbolism is desirable. u = (l - L) V = lm w= (1/4)[(1 -/s)(l -«) + (! -/.)(1 -*)] * = (l/4)[/,(l - a) + /0(1 - A)] y = (1/4)[2(1 - /m) + (1 - /.)« + (1 - O*] z = (l/4)[2/m + /sa + /0A] F = ur'N + vr\ rN = wF' +yr'N + zr\ + x r, = xF' + zr'N + yr\ 4- w. Since the sums of the coefficients are equal to 1 in all cases, the constant terms disappear on substituting r — 1 — P in each case. This leads to the P matrix, the characteristic equation of which follows: = 0 -X u V w y-\ z X z y-\ X3 — X2(2jy) + X[z/2 — z2 — uw — vx] + [uwy + vxy — uxz — vwz] = 0. The largest root gives the ratio of heterozygosis in successive generations. In the case of brother-sister mating (Nm = Nf=\,a = b = 0), this reduces to the following which could have been arrived at much more simply if these assumptions had been made in the first place : X3 - X2(l - lm) - (X/4)[l - (/s + /0)(1 - 2/m)] + (1/8) (2 - ls - l0){\ - 2/m) = 0. It is interesting to note that, if there is random assortment in one sex (lm = 1/2), the equation reduces to that for brother-sister mating with random assortment in both sexes. If there is no crossing over in one sex as in Drosophila (cs = 0, ls = 0, lm = c0), then: X3 - X2(l - O - (X/4)[l - l0{\ - 2c0)] + (1/8) (2 - 0(1 - 2c0) = 0. On substituting X = fif2, this is an exact divisor of the quartic equation arrived at by Bartlett and Haldane.56 The conclusions on the rate of decrease of heterozygosis are thus in exact agreement. If there is equal crossing over in both sexes (cs = c0 = c, ls = l0 = lm = 2c(l — c), then: X3 - X2(l - 1) - (X/4)[l - 2/(1 - 2/)] + (1/4) (1 - /)(1 - 21) = 0. SYSTEMS OF MATING 49 Bartlett and Haldane56 and Green and Doolittle arrived at a quartic equation in terms of ju = 2X from the matrix of mating types in this case. On substituting X = fi/2, the above cubic is found to be an exact divisor, so that again the conclusions on rate of decrease of heterozygosis are in exact agreement. To hold together a line considerably larger than under brother-sister mating, attention is directed to the limiting case of one male (a = 0) and exclusive mating with half sisters (6 = 1) and also equal crossing over in both sexes. The exact recurrence equation for relative heterozygosis is P = (3/2) (1 - l)P' - (1/16)[5(1 - I)2 - 13P]P" - (3/16)(l - I) (I - 2l)P'" With random assortment, c = 1/2, I = 1/2: P = (3/4) P' + (1/8)/"' as given previously.1443 The limiting rate with any value of c is again given by the largest root of the characteristic equation X3 - (3X2/2)(1 - /) + (X/16)[5(l - I)2 - 13/2] + (3/16)(l - /)(1 - 21) = 0. The values of X for several values of c are given in table 1 2 in comparison with those Table 12 The limiting ratio of heterozygosis in successive generations {PIP') UNDER various SYSTEMS OF MATING AND AMOUNTS OF RECOMBINATION 6" ? 0 0.02 0.05 0.10 0.20 0.30 0.40 0.50 1 Dd X 1 Dd 1.0000 0.9629 0.9179 0.8674 0.8234 0.8114 0.8092 0.8090 1 Dd\ X 1 dd-f 1.0000 0.9743 0.9407 0.8984 0.8514 0.8270 0.8135 0.8090 1 dY X 1 Dd 1.0000 0.9900 0.9749 0.9499 0.9013 0.8581 0.8254 0.8090 1 Dd X oo Dd 1.0000 0.9657 0.9334 0.9084 0.8940 0.8910 0.8904 0.8904 1 Dd X' co dd 1.0000 0.9754 0.9495 0.9273 0.9086 0.8984 0.8924 0.8904 1 dd X oo Dd 1.0000 0.9851 0.9657 0.9413 0.9146 0.9017 0.8972 0.8904 1 dY X oo Dd 1.0000 0.9949 0.9872 0.9745 0.9497 0.9264 0.9060 0.8904 t Matings of type dd<$ x Z)rf$ give the same results as those above for the reciprocal type. Equal crossing over in both sexes is assumed. under brother-sister mating (matings Dd x Dd) and various other cases. Whereas mating one male with many half-sisters is only slightly more than half as effective as full brother-sister mating for loci with random assortment, it approaches equal effective- ness for loci closely linked with the gene of interest. Green and Doolittle (and also Bartlett and Haldane) consider the case of brother- sister matings of the type Dd x dd at a locus of interest. This can also be dealt with by path analysis. First the more general case of a population of Nm males, Nf females will be considered. All males are assumed to be Dd and all females dd, so that only recombination in spermatogenesis (here c) is involved. Letting S and s in subscripts represent spermatozoa carrying D and d respectively, there are six kinds of correlations to consider (rSo, rso, rss, rSs, rss, r00). There is no difficulty constructing gametic diagrams in each case from which the formula for the correlations can be written from 50 GENETIC STOCKS AND BREEDING METHODS inspection (figure 16). In this diagram, F1( = rSo) and F2( = rso) are the inbreeding coefficients of males and females respectively. The coefficients are as follows: r'so r'so rSS rSs rss rao • (l/2)(l-c) (l/2)c 0 (l/2)(l-c) 0 (1/2)* 0 l/2)(l-c) 0 (1/2). 0 (l/2)(l-0 (1/2)(1 -c) (1/2)C / 1 - / I 0 0 0 0 1/2 0 (1 - c)2a (\l2)la c2a 0 0 0 0 0 (1/4)6 % v2 \i V Fi = rs, Fig. 16. Mating type Dd x dd. DsDd dcO d, c l-c 1/ 1/ (2 /2 \J f/ F2 =rso o c2a (1 - /)(1 - a) (l/2)/fl /(l - a) (1 - c)2a (1 - /)(1 - a) (1/4)6 (1/2)(1 - b) ( JVJ W 'o fo'o ft jy2V 'W2 do uo Too Dsdd0 DsDd0 IV' ) •' X • -c c l-c ci-cc \ , V \i W Ds DS DS — — —'' v y rss I rSs ds □ d0 dsd a0 i , x . c l_c c l-cxc l-c \ / rSs" Diagrams for analysis of six types of gametic pairs with respect to linked locus. The P matrix is the same except for elimination of the constant terms. The characteristic equation is thus a rather complicated sextic equation. X6 - X5/4[4(l - c) + 4(1 - c)2a + b] - (X4/4){(c + 2(1 - e)(l - I) - 4(1 - c)3a - (1 - c)b) + (X3/16)(l - 2c){2(3 - 2/) 4- 4(1 - c)\c 4- 2(1 - c)2]a + (1 - 2c)b + 4(1 - l)a2b) 4- (X2/16)(l - 2c) {2(1 - /) - 2(1 - c)2(3 - Ac)a 4- 2cla2 - (1 - l)b 4- 2clab - 4(1 - c){\ - l)a2b) - (X/16)(l - 2/){2(l - c)2a - [1 - 2(1 - c)2]a2b} + (1/16)(1 - 2l)2a2b= 0. SYSTEMS OF MATING 51 Under random assortment [c = (1/2), / = (1/2)], this reduces to X2 - (X/2)[l + (a + b)/2] - 1/4[1 - (a + b)/2] = 0, which agrees with the exact recurrence formula given previously,1442 P = [1 - (Nm + Nf)/4NmNf]P' + [(Nm + Nf)/8NmNf]P". There is considerable simplification in the case of one male (a = 0) . The limiting case of an indefinitely large number of females (b = 1) will be considered. X4 - (X3/4)(5 - Ac) - (X2/4)[l - 2(1 - c)l] + (X/16)(l - 2c) (7 - 10c + 8c2) + (1/16)(1 - 2c)(l - I) = 0. The case in which all males are dd and all females Dd is given by interpreting a as (Nf — l)INf, b as (JVm — l)/Nm, c as recombination rate in oogenesis, and / = 2c(l — c) in this sense. The case of one dd male and indefinitely many Dd females is more complicated than the converse. X5 - X4(l - c){2 - c) - (X3/4)[c + 2(1 - c){\ - I) - 4(1 - c)3] + (X2/16)(l - 2c) [2 (3 - 21) + 4(1 - c)[c + 2(1 - c)2] + (X/16)(l - 2c) [2(1 - I) - 2(1 - c)2(3 - Ac) + 2cl] - 1/8(1 - 2/)(l - c)2 = 0. Both of these reduce to the equation X2 - 3/4X - 1/8 = 0, X = (1/8) [3 + Vl7] = 0.8904, as shown previously, if c = (1/2). In the case of brother-sister mating (a = 0, b = 0), X4 - X3(l -c) - (X2/4)[> + 2(1 - c){\ - I)] + (X/8)(l - 2c) (3 - 21) + 0/8)0 - 2c) (1 - I) = 0. The equation obtained by substituting X = ju/2 is again an exact divisor of the equation (quintic) derived by Bartlett and Haldane56 and by Green and Doolittle from the matrix of mating types. The exact recurrence equation for P in terms of preceding P's is given as usual by replacing the highest power of X by P, the next power by P', and so forth. The results are again in exact agreement. The limiting values of the ratio of heterozygosis in successive generations, X, are shown for several values of c in table 1 2 in the cases of male Dd x female dd (or the reciprocal), male Dd and indefinitely many females dd and the converse. They are less efficient in reducing heterozygosis than if both parents are always Dd. The last is the least effective. 52 GENETIC STOCKS AND BREEDING METHODS The case in which the gene of interest is sex linked is of some interest in mice. In the case in which the males are dY and the females Dd, there are six types of corre- lations to consider. The gametic diagrams are shown in figure 17. In the cases of two spermatozoa or two eggs, the alternative possibilities, production by the same individual (probabilities l/Nm, 1/Nf) and by different individuals (Nm - l)/Nmi (Nf — \)jNf are both shown. Fig. 17. Mating type of dY x Dd. rOo m \j c l-c cl-c c l-c W H V Do D„ Do d0D y dsO p \ J / I l-c c \. 1/ c l-c V V V 00 d0a y d0DY A 1 \ "s X ■ss dso'/?°F'd*6> / 1 / I / l-c c l-c c l-c c 4/ v V V '00 '0o 00 Diagrams for analysis of six types of gametic pairs with respect to sex-linked locus. The formulae for the correlations can be written from inspection. The co- efficients are listed below : rsO ?so roo rOo roo ?ss I r,o = F = 0 C 0 1 - c 0 0 0 rso 0 1 - C 0 c 0 0 0 roo I 0 (1 - c)2b 0 0 c2b (1 - Z)(l - b) rOo 1 - / 0 c{\ - c)b 0 0 c{\ c)b 1(1 - b) roo I 0 c2b 0 0 (1 — c )H (1 - 0(1 - b) rss 0 0 0 0 a 0 1 - a The characteristic equation is thus a rather complicated sextic. In the case of SYSTEMS OF MATING 53 random assortment (c = 1/2), rs0, and rso become the same and so do r00, r0o, and r0 The characteristic equation reduces to: 1/2) - X 1/2 0 1/2 (1/4)4 - X (1/4)4 0 a -X = 0 X3 - X2[(2 + 4)/4] - (X/8)(2 - b + 2ab) + (\/8)ab = 0. This agrees with the exact recurrence equation given previously for P as follows :1426 (Nf+l) _ (Nf-l){Nm-l) _ 8Nf { } 8NmNf { J' In the case of a single male (a — 0, rss = 1), the last two rows and columns in the P matrix drop out, and the characteristic equation becomes: X4 - X3(l - c)[\ + (1 - c)b] - X2(l - c)[(l - /) - (1 - c)2b] + X(l - 2c)[(\ - I) + (1 - c)3b] - (1 - 2c)2(\ - c)2b = 0. The limiting ratios of heterozygosis in successive generations are given for various values of c in table 12. Fixation of sex-linked genes under forced heterozygosis of females is a very slow process. With brother-sister mating (both a = 0, b — 0), the characteristic equation reduces to the form given by Bartlett and Haldane.56 X3 - X2(l - c) - X{\ - c){\ - I) + (1 - 2c) (1 -0=0. Fixation is again interfered with by forced heterozygosis of a gene of interest much more than in the case of autosomal genes. Details of the various important systems of the backcross type discussed by Green and Doolittle will not be elaborated. The gametic diagram can easily be constructed and coefficients can be assigned to each path under the necessary assumption of a hypothetical, symmetrical, total population in which gene frequencies remain constant. The recurrence formulae for P present no difficulty. Under simple backcrossing P = (1 — c)P'. Under the cross-intercross system, the result is the same except that P' here refers to the uniting gametes of the preceding intercross, two generations back. Under the cross-backcross-intercross system, P = (1 — c)2P' in a cycle of three genera- tions from P' to P. In these cases the proportions of incrosses in the crosses to the iso- genic lines are readily found by path analysis since they involve only two varying gametes. The results all agree with those of Green and Doolittle. Dr. Pilgrim: There is no question concerning the logic of the breeding methods discussed, but the mathematics is based on the assumption of randomness, and these conclusions are only valid in the absence of selection. In the case of selection for the homozygote, homozygosity may be achieved in much less than the predicted time; and, in the case of high selective value for the heterozygotes, it is conceivable that 54 GENETIC STOCKS AND BREEDING METHODS homozygosity may never be obtained. I question whether there are very many genes in mammals which behave in a random fashion with no selection for either homo- zygosity or heterozygosity. Dr. Gowen : A factor, generally ignored, is the part chance plays in any quanti- tative estimates of inbreeding as measured by various coefficients. Sampled numbers of genotypes are almost universally small compared to those of the total population. Chance in "random selection" of parents each generation introduces so much variation in the real rate changes in homozygosis that, individually, the calculated coefficients may be quite misleading even though their average may measure approximately the trends in infinite populations of evolutionary dimensions. Dr. Burdette : Dr. Pilgrim, do you wish to clarify your question ? Dr. Pilgrim : I wonder how many of these so-called chance elements are really random. How often do we actually select a particular coat color because it happens to be pleasing? There is always selection for breeding performance; otherwise it would be impossible to maintain an animal colony. Dr. Ginsburg : The probability of mutation should also be taken into account in terms of the eventual effect in stabilizing homozygosis in considering the questions about selection. Dr. Mcintosh: In using the matrix-generation method, can repetitive multiplica- tion be avoided and a final result obtained without going through all the intervening steps ? Dr. Doolittle : In answer to Dr. Mcintosh's question, yes, one can skip a series of generations in a single step, having the basic matrices. Dr. Burdette: Dr. Green, would you close the discussion, please? Dr. Green : I am intrigued by Dr. Wright's demonstration that the method of path-coefficient analysis yields the same results as the matrix method in the cases where the matrix method can be used. We should emphasize again that there are irregular breeding systems used by breeders of laboratory animals which cannot be analyzed by the matrix method but which can be analyzed by the method of path- coefficients. I am glad that the two methods, so different in nature and approach, give identical results where both methods are applicable. Each method has its limitations. Dr. Wright pointed out those of path analysis. The chief limitation of the matrix method is that it cannot be used with irregular systems of mating. With respect to the other question which was raised, we have to appreciate that selection occurs whenever animals are being propagated. We cannot propagate animals in a vacuum in the absence of selection nor in the absence of mutation nor in the absence of chance. When it comes to dealing with actual animals, these elements are ever present. On paper, a model population can be propagated with any amount of selection or any amount of mutation one chooses. In the method of analysis described, we made deliberate choices, namely, selection zero, mutation zero. Other choices increase the complications. If there were selection for homozygotes, 100 per cent SYSTEMS OF MATING 55 probability of incrosses would be approached more readily. If there were some selection for heterozygotes, incrosses would be obtained less readily, but could be achieved with brother-sister inbreeding. We know, in other words, what would be the direction of the change when these other factors are taken into consideration, but we have not worked out the probabilities in detail. Dr. Crow: It is relevant to point out in this connection that, if an organism has something like 10,000 genes, one cannot apply a strong selection to each of them. If the effects of inbreeding on decreasing heterozygosity of any particular locus are, say, 25 per cent per generation, this means that even intense selection cannot counteract the increased homozygosity of more than a small fraction of these in any one population. Dr. Wright: It should be emphasized that these methods have nothing to do with lethals or semilethals. Dr. Slatis has worked out a different coefficient dealing with lethals in bison. The average rate of fixation of loci as derived by these methods applies strictly only to neutral alleles but applies approximately when one allele has a selective advantage of lower order than the reciprocal of ten times the effective size of the inbreeding group. This includes most of the loci, under close inbreeding, for reasons indicated by Dr. Crow. With stronger selection, the rate for the more favorable allele increases while that for the unfavorable one decreases, with only slight change in the total, unless the selection is rather severe. With strong selection for the heterozygote over both homozygotes, the total rate is, of course, decidedly lowered just as it is under enforced heterozygosis of a strongly linked gene, the problem investigated by Green and Doolittle. Dr. Gowen: This approach is really based on an infinite population of gametes, breeding entirely at random. From an evolutionary point of view perhaps the assump- tions may be right, but actual breeding experiments with guinea pigs, mice, Drosophila, and so forth never meet the conditions which have been postulated. Selection continu- ally occurs, however random it may be, with the consequence that individually, inbreeding coefficients may be quite misleading. Margaret C. Green, Ph.D. METHODS for TESTING LINKAGEt Methods for detecting and measuring linkage have been summarized and the theory has been explained in an excellent monograph by Mather.858 Tables of scores which greatly simplify the calculations for the more common types of data have been published by Carter and Falconer172 for the detection of linkage and by Finney364 and Allard7 for the estimation of recombination frequencies. Carter and Falconer172 have also discussed the design of stocks for the detection of linkage and have suggested a particular set of such stocks for the mouse. It is the intent of this paper to provide only a brief introduction to methods of linkage useful for laboratory mammals, and to direct the reader to the above and other sources for a more detailed discussion of the methods and their rationale. The linkage testing stocks of the mouse maintained at the Jackson Memorial Laboratory will be described and the latest linkage map of the mouse presented. Linkage tests fall into two general classes, those in which the main interest is in detecting linkage, and those in which the main interest is in measuring more exactly a linkage already known to exist and determining the order of the two linked genes with respect to other genes in the same linkage group. Positive tests of the first type, of course, give preliminary information on the closeness of the linkage observed, but usually more extensive tests using different stocks will be desirable. Whether the purpose of the mating is detection or estimation, only certain types of matings will yield information of any kind about linkage. The necessary requirement is that one of the mated animals should be heterozygous for each of the two loci with f Part of the work on which this paper is based was supported by research grants NSF G-6200 and NSF G-7023 from the National Science Foundation and ACS E-162 from the American Cancer Society. 56 METHODS FOR TESTING LINKAGE 57 linkage in question. Consider that two loci A, a and B, b are to be tested for linkage. The double heterozygote is then either ABjab or AbjaB, depending on whether the recessive alleles, a and b, came to the heterozygote from the same parent or one from each parent. In the former case the linkage is said to be in the coupling phase, in the latter in the repulsion phase. Taking the coupling phase as an example, one can see that four kinds of gametes may be formed by the double heterozygote, AB and ab, the parental combinations, and Ab and aB, the recombinations. In the absence of linkage the parental combinations and recombinations should occur with equal frequency and the proportion of re- combination should be 1/2. In the presence of linkage the parental combinations should occur in excess of the recombinations and the proportion of recombination should be less than 1/2. The purpose of a linkage mating is to provide a way of estimating the proportion of recombination occurring during gamete formation in the double heterozygote, and of determining whether the proportion so estimated differs significantly from 1/2. There are three kinds of matings which provide this information, the double backcross, the single backcross or mixed cross, and the intercross. If there is dominance at both loci, all three kinds of matings produce offspring of the same four phenotypes, A-B-, aabb, A-bb, and aaB-. If these types are represented by the numbers 1, 2, 3, and 4 respectively, the outcome of the various matings can be represented as in table 13. Table 13 Kinds of offspring produced by recombinant and nonrecombinant gametes in three kinds of matings 1 = AB, 2 = ab, 3 = Ab, 4 = aB. Heterozygous parent Other parent Genotype Gametes Double backcross aabb Single backcross Aabb Ab ab Intercross AaBb AB ab Ab aB AaBb AB ab Ab aB The upper two rows represent the parental combinations if the heterozygous parent is in the coupling phase, and the recombinations if the heterozygous parent is in the repulsion phase. In the double backcross and mixed cross, recombination cannot be detected in the other parent, but in the intercross the other parent is also a double heterozygote and produces detectably different parental and recombinant gametes. 58 GENETIC STOCKS AND BREEDING METHODS The two left columns of the intercross are the parental combinations for coupling and the recombinations for repulsion. It can be seen that in the double backcross the parental or recombinant type of the gamete can always be recognized from the phenotype of the offspring. The proportion of recombination in the gametes can therefore be estimated directly from the proportion of recombinant phenotypes among the offspring. In the single backcross and inter- cross, however, some of the phenotypes among the offspring result from both parental and recombinant gametes. A direct estimate of recombination is therefore impossible and more elaborate methods become necessary. USE OF CHI-SQUARE FOR TESTING SEGREGATIONS AND DETECTING LINKAGE Before any effort is expended to detect linkage or estimate recombination, the segregations of the individual genes should be examined to determine whether they are in accordance with expectation, since disturbed segregations may lead to serious errors in estimating recombination. The most useful method for this purpose is that of %2. Since explanations of yf are widely available in textbooks on statistical methods, this discussion will be confined to the special formulas useful with linkage data. Let a be the observed number of individuals in a class and m be the expected proportion, such that mn is the expected number when n is the total number of in- dividuals. The general formula for %2 is then 2 = V (fl ~ mn) * ^-> mn An algebraically equivalent form which is easier to compute is X2 *-> \mn) If there are two classes of individuals, denoted by 1 and 2, and if the numbers in the two classes, ax and a2, are expected to occur in the proportion k:\, %2 can be more easily calculated from the formula {ax - ka2)2 X = kn An example is the segregation of the gene for ruby eye, ru, in the mouse, reported in a cross by Fisher and Snell.379 An intercross gave the following segregation: H — ruru Total Observed number 117 40 157 Expected proportion 3/4 1/4 „ (117-120)2 9 nMn y = — = = 0 019 1 3 x 157 471 For one degree of freedom, the deviation from expectation is not significant. METHODS FOR TESTING LINKAGE 59 In a linkage test, two pairs of genes will commonly be segregating at the same time. Deviations from expectation may be due to faulty segregation of either or both pairs or to failure of random assortment (linkage) between the two pairs. The total ^2, which for four classes has three degrees of freedom, can be partitioned into three in- dependent parts, each with one degree of freedom, one for segregation at each of the two loci, and one for linkage. Table 14 gives the formulas for partitioning the total £2 for the three common kinds of matings. Table 14 Formulas for calculation of x2 for segregation at individual loci, y^-A and x2B> AND FOR LINKAGE, %2L, FOR THREE KINDS OF MATINGS Mating AB Ab aB ab Double backcross 1/4 1/4 1/4 1/4 Z2J = (< X2B = X*L = Intercross 9/16 3/16 3/16 1/16 X*A = X2B = X2L = Single backcross A 3/8 3/8 1/8 1/8 2A = intercrossed (*1 n — a2 + a3 — a4)2 (*1 n — a2 - a3 + a4)2 (*1 n + a2 — 3a3 — 3a4)2 («1 2>n — 3a2 + a3 — 3a4)2 (fll 3n - 3a2 - 2>a3 + 9a4)2 K 9« + a2 - 3a3 - 3a4)2 («i 3n - a2 + a3 - a4)2 (fli n — a2 — 3a3 + 3a4)2 X2B = The data of Fisher and Snell379 on the linkage of ruby, ru, and jerker, je, in the mouse may be taken as an example again. From an intercross in repulsion the following data were obtained : + 4- +je ru + ruJe Total Observed number 86 31 35 5 157 - Expected proportion 9/16 3/16 3/16 1/16 (86 4- 31 - 105 - 15)2 9 X2m = Wl 47T-°-019 60 GENETIC STOCKS AND BREEDING METHODS 2;„ _ (86 - 93 + 35 - 15)2 169 = Q^ XJe 471 471 0T (86 - 93 - 105 + 45)2 4489 %L = I4T3 = T4T3 = 3-177 The £2's for the segregation of ru andje are very small. That for linkage is larger, 3.177, but still below the 5 per cent level of significance for one degree of freedom. The data thus do not give evidence for linkage. It should be noted that the sum of the three values of y2 calculated above, 3.555, is equal to the total %2 calculated by the standard formula. This will always be so if the arithmetic has been correctly performed. If the deviations of the single factor segregations from expectation are significant because of reduced viability, reduced penetrance, or any other disturbing factor, the above formula for the linkage %2 does not give a true estimate of the significance of the deviation due to linkage. It will be better in this case to calculate a contingency %2 to determine whether the two pairs of genes are recombining at random, regardless of their individual segregation ratios. This can be done by the standard method, calcu- lating the expected frequency in the four classes from the marginal totals. A somewhat simpler method is to use the special formula applicable to two-by-two tables, which eliminates the necessity for calculating the expected frequencies. By this formula, 2 «(aifl4 - Q2Q3)2 X = ~ K + «2)(fl3 + ai)(al + a3)(a2 + a4) where au a2, . . . , have the same meaning as in table 14. This %2 has one degree of freedom. Its use can be illustrated by the following example.549 The segregation of ogligodactyly, ol, and albino, c, in the mouse in an intercross in repulsion was as follows : + + +c ol + olc Total 334 188 107 13 642 The segregation of both ol and c is very abnormal as shown by %2 {%2ol — 13.6, y^c = 13.6), there being a deficiency of ol and an excess of c. When the data are arranged in a two-by-two table, it appears that there is an excess in the +c and ol+ classes, as would be expected in the case of linkage. + c Total + 334 188 522 ol 107 13 120 Total 441 201 642 To assess the significance of this excess we calculate %2 by the above formula. 642(4342- 201 16)2 _ z 522 x 120 x 441 x 201 zo-/u^ For one degree of freedom this is highly significant. METHODS FOR TESTING LINKAGE 61 In cases such as this where segregations are abnormal, the methods of measuring recombination which will be considered below may not be applicable. For methods dealing with disturbed segregations the reader is referred to Mather,858 Fisher and Bailey,376 Bailey,45 and Carter.160 USE OF MAXIMUM-LIKELIHOOD METHODS FOR THE DETECTION AND ESTIMATION OF LINKAGE The methods most widely in use for the estimation of linkage are those based on Fisher's maximum-likelihood method of estimation. A simple example will illustrate the principle of the method. Consider a coupling single backcross ABjab x Ab\ab. If p is the probability of recombination, the expected proportions or probabilities of the four types of offspring in terms of p can be determined from table 15. Combining the like phenotypes, we obtain AB Ab aB ab K2-/0 \{\-p) iP i(i-/>). Table 15 Expected proportions of different genotypes among offspring of a single backcross , Expected Ab ab Gametes proportions 1/2 1/2 AB AABb AaBb i(l - P) i(i - p) i(l - P) Ab AAbb Aabb \P iP kP aB AaBb aaBb iP IP IP ab Aabb aabb i(l -P) i(l ~P) i(l - P) The expected proportions of offspring for the other types of matings are given in table 16. If we represent the expected proportions or probabilities of the four classes by mls m2, m3, and ra4, and the observed number of offspring in the corresponding classes by a1} a2, a3, and a4, then the probability, P, of getting such a set of observed numbers from such a mating is proportional to the product of the probabilities for each in- dividual, or P = Cm1alm2a2m3a3mi,a*. 62 GENETIC STOCKS AND BREEDING METHODS Table 16 Expected proportions of classes of offspring in linkage crosses Phase Phenotypes of offspring Mating AB Ab ) 1/- 1(1 ~P) 10 ip -P) Intercross C i[2 + (1 - P)2] i[l-(l- -/o2: 1[1 - (1 - P)2] 4(1 -P)2 R 1(2 + P2) 1(1 -^2) 1(1 - P2) l/>2 C = Coupling, R = Repulsion The value of/? which maximizes the probability P is the maximum likelihood estimate of/&. It can be found by differentiating the above expression with respect to p, setting the derivative equal to zero, and solving for p. The above expression is very difficult to differentiate but the task can be simplified by taking the logarithm L of the expres- sion, differentiating the logarithm with respect to p, and proceeding as before. Since P is maximum when the logarithm of P is maximum the same value ofp will be obtained. Thus, L = log P = C" + ax log mx + a2 log m2 + a3 log m3 + a4 log m4 and dL d\ogm1 dlogm2 dlogm3 d log m± TP = ai ~^p— + a* —dp- + a* ~^p— + a* —dp— = °- For the coupling single backcross, L = C + ax log 1(2 - p) + az log 1(1 + p) + a3 log \p + a4 log 1(1 - p), and dL + _^L_ + ~-l _ ^^ = o. dp 2 - p \ + p p 1 - p The variance of/>, Fp, can be found from the relationship first shown by Fisher370 that the second derivative of the likelihood equation is equal to the negative reciprocal of the variance of/>, when the expected number of progeny (mri) in each of the four classes is substituted for the observed number (a) and the calculated value of/> is used, or d2L 1 ^ / d2 log m\ METHODS FOR TESTING LINKAGE 63 For the coupling single backcross, therefore 1 m-ji m2n m3n m^n vp (2-pr (i+p)2 p2 (\-py _ nt 1 1 1 1 \ 4\2 -p+ I +p+ p+ I -p) 2p(l -p){\ +p)(2-p) »(1 +2p - 2p2) The solution of maximum-likelihood equations of estimation may be quite tedious in some cases. The use of maximum-likelihood scores first introduced by Fisher372 and further developed by Fisher,369 Finney,364 Carter and Falconer,172 and others, has greatly simplified the arithmetic. Their use in both detection and estimation can be illustrated with the previous example. The equation of estimation for the coupling single backcross can be written : The exact solution of p substituted into the equation will give a value of dL\dp equal to zero. A provisional value of p substituted into the equation will give a value of dL\dp whose deviation from zero, D, is a measure of the deviation of the exact estimate of p from the provisional value. If the provisional value of p is one half, for instance, the calculated value of dLjdp will be greater, the greater the evidence for linkage in the data. For the detection of linkage, then, the values of —1/(2 — p), 1/(1 +/>), etc., when p = 1/2 can be calculated in advance and used as scores by which a1} a2, etc., are multiplied. For the case illustrated the scores are: AB Ab aB ab -2/3 2/3 2 -2 and ^ 2 2 Q n — = -- ax + ^ a2 + 2a3 - la± = B. For the detection of linkage we need to know whether D differs significantly from zero, and for this purpose we need the variance of D. It has been demonstrated363 that VD = \\VV We could calculate Vp by the method already shown. A more convenient method which makes use of a score calculated in advance is available, however. The concept, due to Fisher,370 of the amount of information must now be intro- duced. The greater the amount of information in a body of data, the greater the precision of the estimate of a parameter calculated from the data, or the smaller the variance of the estimate. It is therefore convenient to speak of the reciprocal of the 64 GENETIC STOCKS AND BREEDING METHODS variance as the amount of information /, or \jVp — Ip. If Ip is the amount of informa- tion about p contributed by a whole body of data, then Ipjn = ip may be designated as the amount of information contributed by a single individual. We have already seen that 1 d2L sr ( d2 log m\ Therefore L = nir 2 1 d2 log m\ 2 1 d2 log m\ \m-djr\ and which can be shown to be equivalent algebraically to The quantity ip is a constant for any particular kind of mating and value of/? and can be calculated in advance and used as a weight by which to multiply the total number of progeny to obtain Ip. The test of significance is therefore _ _D_ D D WD VTp Vnip with probability obtainable from a normal table, or alternatively, 2 & D and Ip have the useful property of additivity such that they can be summed for different types of matings to give a test of significance based on all appropriate matings available. Table 17 (adapted from Carter and Falconer172) gives scores and values of ip for the detection of linkage for a number of different types of linkage matings. A numerical example will illustrate their use for this purpose. Table 18 gives the data of Fisher and Snell379 on the linkage of ruby (ru) and jerker (je) in the mouse. The calculations of D and Ip are set out in the table. The X2 test for significance of the deviation of p from one half is . (-140.889)' 1 2655.111 METHODS FOR TESTING LINKAGE Table 17 65 Scores of maximum likelihood and amount of information per individual calculated for p = 1/2 for different types of linkage-testing mating + Phenotypic cl; iss Kind of mating AABB AABb AaBB AaBb AAbb A abb aaBB aaBb aabb h A and B semi- dominant Intercross -4 0 0 0 4 0 4 0 -4 4 A semidominant V v ' Intercross -4/3 0 4 0 4/3 -4 8/3 Single back- cross A~\ -2 0 2 0 2 -2 2 AA masks B, b distinction Intercross 0 0 0 0 4/3 -4 4/3 Single back- cross A] AA inviable Intercross 0 0 0 0 2 -2 1 ■v 0 0 4/3 -4 16/9 Single back- cross Af 0 0 2 -2 4/3 A and B fully dominant Double backcross -2 2 2 -2 4 Intercross -4/9 4/3 4/3 -4 16/9 Single back- cross Af -2/3 2/3 2 -2 4/3 aa masks J5, b v J distinction "*■ Double backcross -2 2 0 2 Intercross -4/9 4/3 0 4/9 Single back- cross A'f -2/3 2/3 0 1/3 Single back- cross Z?f -2/3 2 0 2/3 A indistinguishable from B Double backcross V J Y 2/3 -2 4/3 aa indistinguishable from bb Intercross I. j <. j -4/9 Y 4/7 16/63 f Gene intercrossed X A and B represent the dominant alleles of the two genes in the test, a and b their recessive alleles, irrespective of which are the mutant alleles. The genes are in coupling when A and B enter the test together from the same parent. The signs given are for coupling; they should be reversed for matings in repulsion. Adapted from Carter and Falconer. 172 66 GENETIC STOCKS AND BREEDING METHODS Table 18 Calculation of D and Ip for detection of linkage between ru ANDJe in the mouse (Using data of Fisher and Snell.379) ... Offspring Mating — JJ = Parents type + + +je ru + ruje 2 ns Total Ip I C n 151 54 45 20 270 s -4/9 4/3 4/3 -4 ip 16/9 ns -67.111 72.000 60.000 -80.000 -15.111 480.000 1R » 86 31 35 5 157 s 4/9 -4/3 -4/3 4 ip 16/9 ns 38.222 -41.333 -46.667 20.000 -29.778 279.111 B C n 120 103 89 122 434 s -2 2 2-2 ip 4 ns -240.000 206.000 178.000 -244.000-100.000 1,736.000 B R n 9 9 10 12 40 s 2-2-2 2 ip 4 7u 18.000 -18.000 -20.000 24.000 4.000 160.000 -140.889 2,655.111 I = intercross, B = double backcross, C = coupling, R = repulsion, n = number, s = score The deviation is highly significant and linkage must therefore be said to exist. It is desirable now to estimate p. A fortunate property of D and Iv allows them to be used to obtain a first estimate of/>. For small values of D it can be shown that / = D + + + + ruje ruje +je +je ru + ru + + + ruje ruje ruje +je ruje ru + ruje P ~Po and D p = p0 + y> approximately, where p0 is the value of p assumed in calculating D, and p is the exact maximum likelihood estimate of p calculated from the same data. In the above example the improved estimate of/) is therefore p = ■= + Djlp = 0.500 - 0.053 = 0.447- The standard error of this estimate is SE' - W ~ 5T53 " °-019- Recombination between ru andjV is thus 44.7 + 1.9 per cent. The quantity p estimated in this way is very close to the exact estimate for depart- ures from one half of this magnitude. For large departures from one half it will be necessary to obtain a more exact estimate. For this purpose the first estimate of p could be used to calculate a second set of scores and ip which could then be used to get METHODS FOR TESTING LINKAGE 67 a more exact estimate. It is clear that this labor would be greatly facilitated by the availability of tables of scores for various kinds of matings and values of/). Several such tables have been published, notably by Allard7 and by Finney.364 Allard's Table 19 Scores and information per individual for Aa/Bb x aajbb (Finney364) p iP P h 0.01 101.010 0.15 7.843 0.02 51.020 0.20 6.250 0.03 34.364 0.04 26.042 0.25 5.333 0.05 21.053 0.30 4.762 0.06 17.730 0.35 4.396 0.07 15.361 0.40 4.167 0.08 13.587 0.09 12.210 0.45 4.040 0.10 11.111 0.50 4.000 If the heterozygous parent is AB/ab, score aB and Ab offspring as ip, others as zero; for AbjaB parent, interchange these scores. Table 20 Scores and information per individual for AB/ab x Aaj bb (Finney364) p AB aB Ab ab t'p 0.01 -0.2463 100.2563 1.2464 -0.7538 25.626 0.02 -0.2425 50.2625 1.2429 -0.7579 13.126 0.03 -0.2388 33.6022 1.2397 -0.7621 8.961 0.04 -0.2351 25.2751 1.2367 -0.7665 6.878 0.05 -0.2313 20.2815 1.2339 -0.7712 5.629 0.06 -0.2276 16.9545 1.2312 -0.7760 4.797 0.07 -0.2239 14.5799 1.2288 -0.7810 4.203 0.08 -0.2202 12.8007 1.2266 -0.7863 3.758 0.09 -0.2164 11.4183 1.2246 -0.7918 3.413 0.10 -0.2127 10.3137 1.2228 -0.7974 3.137 0.15 -0.1935 7.0137 1.2166 -0.8295 2.313 0.20 -0.1736 5.3819 1.2153 -0.8681 1.910 0.25 -0.1524 4.4190 1.2190 -0.9143 1.676 0.30 -0.1293 3.7923 1.2282 -0.9696 1.530 0.35 -0.1036 3.3596 1.2432 -1.0360 1.436 0.40 -0.0744 3.0506 1.2649 -1.1161 1.376 0.45 -0.0404 2.8269 1.2944 -1.2135 1.344 0.50 0.0000 2.6667 1.3333 -1.3333 1.333 If the heterozygous parent is Ab\aB interchange columns 2 and 4 and also columns 3 and 5 in this table. 68 GENETIC STOCKS AND BREEDING METHODS Table 21 A. Scores and information per individual for ABjab x ABjab (Finney364) AB aB, Ab ab 0.01 0.3339 100.4958 -1.0219 99.831 0.02 0.3345 50.4915 -1.0442 49.829 0.03 0.3352 33.8205 -1.0670 33.161 0.04 0.3359 25.4828 -1.0903 24.826 0.05 0.3366 20.4784 -1.1141 19.824 0.06 0.3374 17.1405 -1.1383 16.489 0.07 0.3382 14.7550 -1.1631 14.106 0.08 0.3390 12.9646 -1.1885 12.318 0.09 0.3399 11.5710 -1.2143 10.927 0.10 0.3409 10.4551 -1.2408 9.815 0.15 0.3465 7.0970 -1.3821 6.473 0.20 0.3535 5.4040 -1.5404 4.798 0.25 0.3624 4.3763 -1.7189 3.791 0.30 0.3733 3.6806 -1.9216 3.118 0.35 0.3865 3.1742 -2.1538 2.638 0.40 0.4025 2.7860 -2.4223 2.278 0.45 0.4217 2.4765 -2.7369 1.999 0.50 0.4444 2.2222 -3.1111 1.778 B. Scores and information per individual for AbjaB x Ab/aB AB aB, Ab ab 0.01 0.0200 -0.0100 200.0100 1.000 0.02 0.0400 -0.0200 100.0200 1.001 0.03 0.0601 -0.0300 66.6967 1.002 0.04 0.0801 -0.0400 50.0402 1.004 0.05 0.1002 -0.0499 40.0503 1.006 0.06 0.1204 -0.0599 33.3939 1.009 0.07 0.1407 -0.0698 28.6423 1.012 0.08 0.1610 -0.0797 25.0813 1.016 0.09 0.1815 -0.0896 22.3141 1.020 0.10 0.2020 -0.0995 20.1025 1.025 0.15 0.3069 -0.1483 13.4919 1.057 0.20 0.4167 -0.1961 10.2206 1.103 0.25 0.5333 -0.2424 8.2909 1.164 0.30 0.6593 -0.2871 7.0389 1.241 0.35 0.7977 -0.3298 6.1822 1.337 0.40 0.9524 -0.3704 5.5820 1.455 0.45 1.1285 -0.4086 5.1643 1.600 0.50 1.3333 - 0.4444 4.8889 1.778 METHODS FOR TESTING LINKAGE 69 Table 22 Scores and information per individual for ABjab x ABjab with A semidominant (Finney364) p AAB AAb AaB Aab aaB aab h 0.01 0.9824 201.0024 0.0126 99.9923 100.4999 -1.0178 100.241 0.02 0.9646 101.0046 0.0254 49.9842 50.4996 -1.0362 50.232 0.03 0.9468 67.6735 0.0387 33.3093 33.8326 -1.0550 33.562 0.04 0.9285 51.0087 0.0519 24.9670 25.4985 -1.0747 25.216 0.05 0.9102 41.0104 0.0655 19.9578 20.4976 -1.0948 20.209 0.06 0.8917 34.3455 0.0796 16.6150 17.1634 -1.1155 16.870 0.07 0.8732 29.5853 0.0940 14.2243 14.7815 -1.1367 14.484 0.08 0.8543 26.0153 0.1086 12.4284 12.9945 -1.1586 12.692 0.09 0.8353 23.2389 0.1236 11.0289 11.6043 -1.1811 11.297 0.10 0.8160 21.0180 0.1389 9.9069 10.4917 -1.2042 10.180 0.15 0.7173 14.3576 0.2219 6.5144 7.1504 -1.3287 6.828 0.20 0.6151 11.0317 0.3175 4.7817 5.4762 -1.4683 5.159 0.25 0.5098 9.0431 0.4277 3.7098 4.4717 -1.6236 4.172 0.30 0.4032 7.7292 0.5562 2.9673 3.8077 -1.7946 3.542 0.35 0.2967 6.8087 0.7060 2.4131 3.3455 -1.9825 3.127 0.40 0.1927 6.1451 0.8819 1.9784 3.0201 -2.1883 2.863 0.45 0.0928 5.6658 1.0884 1.6254 2.7984 -2.4150 2.714 0.50 0.0000 5.3333 1.3333 1.3333 2.6667 -2.6667 2.667 For matings Ab/aB x Ab/aB, interchange columns 2 and 6 and also columns 3 and 7 in this table. Table 23 Scores and information per individual for ABjab x ABjab, with A and B semidominant (Finney364) AABB AABb, aaBb AAbb 3 p aabb AaBB, Aabb AaBb aaBB h 0.01 -0.0204 100.9897 0.0002 201.9998 199.980 0.02 -0.0416 50.9788 0.0009 101.9992 99.959 0.03 -0.0637 34.3006 0.0020 68.6648 66.605 0.04 -0.0867 25.9550 0.0036 51.9967 49.917 0.05 -0.1105 20.9421 0.0058 41.9948 39.895 0.06 -0.1353 17.5952 0.0086 35.3257 33.207 0.07 -0.1610 15.2000 0.0121 30.5610 28.423 0.08 -0.1876 13.3993 0.0163 26.9863 24.829 0.09 -0.2153 11.9948 0.0213 24.2048 22.028 0.10 -0.2439 10.8672 0.0271 21.9783 19.783 0.15 -0.4027 7.4405 0.0711 15.2836 13.002 0.20 -0.5882 5.6618 0.1471 11.9118 9.559 0.25 - 0.8000 4.5333 0.2667 9.8667 7.467 0.30 -1.0345 3.7274 0.4433 8.4893 6.076 0.35 -1.2844 3.1112 0.6916 7.5068 5.121 0.40 -1.5385 2.6282 1.0256 6.7949 4.487 0.45 -1.7822 2.2582 1.4581 6.2986 4.120 0.50 -2.0000 2.0000 2.0000 6.0000 4.000 70 GENETIC STOCKS AND BREEDING METHODS tables are intended chiefly for use with plant material and do not include single back- crosses, but they do include double backcrosses and intercrosses, with several kinds of epistasis and degrees of dominance. The scores are listed for all useful values of/? at intervals of 0.01. Finney's tables, which are reproduced in tables 19 to 23, give scores for five kinds of matings often encountered in animal genetics. Finney's scores are slightly different from those described above. They are based on maximum-likelihood methods, but are calculated so that, instead of giving a correction to be applied to the provisional value of/), they lead directly to the revised estimate, thus eliminating one step in the arithmetic. They are tabled at intervals of 0.01 for values of/? from 0.01 to 0.10 and at intervals of 0.05 for values from 0.10 to 0.50. The use of Finney's scores is shown with data published by Carter167 on the linkage of luxate (/*) and viable dominant spotting (Wv) (table 24). A recombination value of 0.13 can be calculated directly from the backcross, which suggests the use of 0.15 as a provisional value. Using tables 19 and 20, the appropriate scores (A) and values of ip can be found, proceeding as in the previous example: 343.76 A1CQ ' = 2TIT57 = 0-163' **> = VT = ^ = °-°218- This approximation of/? will usually differ very little from the exact estimate. In the present case, rescoring at/? = 0.163 and recalculating gives a value of/? = 0.1624, which is so small an improvement as not to be worth the effort. ESTIMATION OF HETEROGENEITY One may wish to discover whether the several bodies of data which contribute to the estimate of the recombination fraction are homogeneous with respect to the prob- ability of recombination. Fisher371 has pointed out that the sum of D2/I calculated separately for each body of data, using the value of /? estimated from the total, is distributed as %2. Such a %2 includes a part derived from the deviation of the total D from zero. When this is subtracted, the resulting %2 for n — 1 degrees of freedom, where n is the number of separate bodies of data, measures the heterogeneity among the several bodies of data. The data on the linkage of ru and je again may be taken as an example. We would like to calculate D for each of the four kinds of matings for a value of/? close to 0.447. Finney's tables do not give D directly, but D can be obtained from them by a simple arithmetic transformation as follows: p=p0 + D\I, = A\I, D = A- p0Iv METHODS FOR TESTING LINKAGE o2 C u <£ 05 (J S^ 0 £ ° o O rt 2 W P.S w o h <: o < bo G Oj CO Tf — ' CO rh CO m co CO — ' o — CO CM CO •^ o o c") co Oi — i ■* m CO CD co co CO CM tO m O — ' CD CM cm r^ 00 CM © CO f^ CO CM CM tO CO — i "* CM CO co r^- — — < co co O -« CD co m © CD co co CD C£> -H CD O CO CO CM — < CO o o CM lO O t^> CD CD CM CO CO CO O "* i co O O If) IT) Ifl CO CO CM CD t^ o to I I CM (O CM m co co — < to CM CM ^1 «^ K^^ '■' V > t) n-S (73 fin + + + + + + ■i5 Ms + «s +I-S + + * .* » fc -S + + 72 GENETIC STOCKS AND BREEDING METHODS w w h « « Z — ' O u g "" 3 e s — o 3 o ^ o z O H < P o ►J < U l bo a o be C CM Ol O CT) o M U3 CO CO CO cm -^ i m -* m o •* t~^ r~ O LO O ■* o CO "f Tt< o CM cr> o o CM ■* CO Tf o lO CO ^H °2 iO -* "* in t^. CO m cx> r^- lO M if) tO CM O O CM m m m m to "f <£> CM CO CO t^ ^ o ■* -* ■* — . to to CO CO to o to Tj< tO O CM CO o o O -f CM — . O — — « r^ r-~ to m o o LO -h tO CO CO —i CM -^ cm r- cm m ^h ■* to — o O CO -H l*i tO Oi ^ U ^3 JO P« II ^1 CT) O O tJ< tO O CO N e O pq ^ =2 (* METHODS FOR TESTING LINKAGE 73 The calculation of D using the tabled values of A for p = 0.45 is set out in table 25. The heterogeneity %2 can then be determined as follows : Mating f = D2\I 4f I C 0.2338 1 I R 1.1683 1 B C 0.1014 1 B R 0.9090 1 Sum 2.4130 4 Total 0.0187 1 Heterogeneity 2.3943 3 > 0.05 There is thus no significant heterogeneity among the matings. The estimate of the heterogeneity %2 is not accurate unless the total deviation is small. Tables 19 to 23 may not be sufficiently detailed when an accurate value of %2 is needed, as when the heterogeneity is on the borderline of significance. Allard's tables may supply the appropriate scores, or it may be necessary to calculate them. Table 26 gives formulas for maximum-likelihood scores and values of ip for several common kinds of matings. In cases for which no tables of scores are available, it may sometimes be relatively easy to derive maximum-likelihood scores but may be somewhat more difficult to derive formulas for the amount of information per individual, ip. Fortunately there exists an arithmetic method which gives an approximate estimate of the total amount of information, Ip, given by the results of any mating or group of matings. Since Ip is equal to the second derivative of the logarithm-likelihood expression, it is also equal to the first derivative of the equation, dLjdp = 0. Ip is therefore the rate of change of dLjdp with respect to p. If dLjdp has been evaluated for two values of p (p± and p2) close to the exact estimate, to give two values of D, then Ip = (D2 — D1)j{p2 — pi). The value of Ip calculated in this way will not be identical to the value calculated from the formula — ][ {mn[(d2 log m)ldp2]}. The arithmetic method gives the actual amount of information realized in the particular body of data on hand, whereas the formula gives the expected amount of information in bodies of data of this size and value of p. LINEAR ORDER OF LOCI Having determined that a mutant is linked with another mutant known to be a member of a particular linkage group, one may wish to determine the linear order of the two mutant loci with respect to another locus in the linkage group. For this purpose it may sometimes be sufficient to determine the distance of the new mutant from the other locus. The recombination for A-B, say, should equal either the sum or difference of the recombinations for A-C and B-C, and the linear order easily follows. If, however, any two of the three loci are closely linked, the errors of estimate 74 GENETIC STOCKS AND BREEDING METHODS X> H ex o a C5 Dq + ^ + + e, c -ft w ■ft. S| 1 1 + CM — ft, CM |- ■»■ « ■H-T+ +— H I I I E £s J ■W—H 1 II J q- - * £ a -a §£g ro w 10 ■>±> +— H ** ZO .c^ L.IJI ■ I <\| tO_ f- 5 ■ w a. c a)g"o m iik i ii ii ^— f B* S^i I I a. 55: a > 5 3 I I I I io r~ in to METHODS FOR TESTING LINKAGE 79 distances are recombinations and which are the result of subtractions. The linear order of many of the genes is subject to some degree of uncertainty. Those indicated by symbols in italics have not been critically tested to determine their position relative to all other genes in the linkage group. The order of the other loci is established with a fairly high probability of correctness. Many experiments disagree in the estimate of recombination obtained. In combining estimates from different experiments the usual procedure has been to weight the recombination fraction by the amount of information in the body of data contributing the estimate. In linkage groups VI and XIII, how- ever, where there are striking differences in amount of recombination between the sexes, weighted averages for each sex separately were calculated, and the unweighted average of the sexes was taken. There are differences between the sexes in other regions of the linkage map also, but the differences are either small enough or the estimates from the two sexes well enough balanced, so that the unweighted method of averaging would have made very little difference. In general when sex differences occur recom- bination is higher in females than in males, but in linkage group VI and between sh-2 and wa-2 in linkage group VII the reverse is true. Following is a list of the names of the genes used in the map. The numerous alleles at the histocompatibility loci and at the t locus have not been listed. For the other multiple allelic series, the mutants which have been given names are listed. The references cited all give information on linkage, with the exception of a few, cited for members of a multiple allelic series, which contain the original description of the allele. Where no reference is cited, a description has been given by Griineberg.507 Symbol for Gene Name of Gene References fi frizzy 344 ol oligodactyly 549 H-\ Histocompatibility- 1 1238, 1244 Hb Hemoglobin pattern 1017 sh-l shaker- 1 344, 425, 498, 500 264. 353. 498, 1017 c-series cch chinchilla 340. 344. 1267 Ce extreme dilution ch himalayan 487 c albino 143, 180. 183, 263. 297, 425, 500, 518. 549. 1238, 1244 1466 hf hepatic fusion 143 HA Histocompatibility-^: 1244 /^-series Pd dark pink-eye 162 P pink-eyed dilution 143, 180. 183. 263, 264, 297. 340, 344, 353, 359 p-sterile 425. 493, 498, 500, 518. 1112, 1244, 1267, 1466 594 80 GENETIC STOCKS AND BREEDING METHODS Symbol for Gene tp qv da pu lu d-series d Name of Gene taupe quivering dark pudgy II luxoid dilute References 359. 493. 1112 1466 340 1267 182. 426. 454. 1231. 1234. 1240 se sv du fin s <*g hr-series hr hrrh wl pi W-series W Wv w* wa Ph le Ix rl r si fig av Ra dm H-3 kr bp a-series A" d a dilute-lethal short ear 1189 182. 426. 454. 490. 491, 1231. 1234. 1240 SneWs waltzer ducky 490 491, 1234 III pugnose piebald 707 167, 296, 301, 369, 694, 707, 796. 799, 1240 agitans 589 hairless 167, 276, 428, 753. 1240 rhino 589 wabbler-lethal 753 pirouette 275. 276 Dominant spotting 296, 301, 369, 428, 694. 796, 799 Viable dominant spotting 167, 275, 338. 509. 707 Jay's dominant Ames dominant Patch spotting spotting 1106 592 509 light ears luxate 755 167, 338 reeler 338 IV rodless retina 691 silver 343, 691, 1163 Pygmy Ames waltzer 343 1163 V Ragged diminutive 176, 757. 991 838 Histocompatibility-^ kreisler 1238, 1251 549. 755 brachypodism 1089 118, 377. 978, 991 Yellow 169. 1060, 1089 White-bellied agouti 157. 169. 755 black and tan 157, 169, 173. 176. 755, 1355 nonagouti extreme nonapm iti 157. 173. 176. 336 1238, 1251 595 . 368, 549. 755. 757. 838. 1060 METHODS FOR TESTING LINKAGE Symbol for Gene Name of Gen( un undulated we wellhaarig mg mahogany pa pallid ro rough fi fidget Sd Danforth's short tail VI N Naked Ca Caracul hi hair-loss Ht Hightail bt belted VII Re Rex Al Alopecia ti tipsy Tr Trembler sh-2 shaker-2 vt vestigial tail wa-2 waved-2 VIII m misty Pt Pintail b-series Bl Light b ■ brown bc cordovan an anemia vc vacillans wi whirler IX t-series T Brachyury t tailless Fu-series Fu Fused Fuki Kinky tf tufted H-2 Histocompatibility -2 X V waltzer ji jittery References 157, 336, 368, 377, 978, 1089 336, 377, 549. 755, 978, 991 757 118, 169. 176, 336, 377, 755, 978, 1060 336 169. 173. 1355, 1356 118, 176, 336. 1355. 1356 227, 845, 926 227. 593. 845. 926. 1267 593 1267 593. 845, 926, 1267 171, 177, 266. 337. 345, 755. 870, 871. 936, 1190a 266. 755 1190a 345 177, 266. 337. 345, 378, 561. 755, 870, 872. 936, 1253, 1412 870. 871, 872, 1190a 171. 177, 266, 378, 561, 872, 1190a, 1253, 1412 753. 1212, 1404 596, 753 833 549, 596. 753. 1213. 1212. 1404 877 549 1212. 1213 753 12, 302. 303, 305, 816. 819, 1043. 1246 302, 305. 819 11, 12, 302, 303, 304, 458. 1043. 1246 12. 298. 302. 303. 304. 305. 1246 298, 816 819 11, 12, 458, 1246 1241 1241 ob XI obese 82 GENETIC STOCKS AND BREEDING METHODS Symbol for Gene Name of Gene Reference wn'-series Miwh White 142. 174, 272. 508. 1002 mi microphthalmia 508 px postaxial hemimelia 163 wa-\ waved- 1 142, 272 Lc Lurcher XII 174. 1002 ruby eye 268. 339, 379. 1356a ru je jerker XIII 268. 339, 379. 1356a dr dreher 817 Lp Loop tail 1252 py Polydactyly 374, 990 Dh Dominant hemimelia 161. 817 In leaden 161, 274. 374, 990. 1252 sp Splotch 990, 1252 fz fuzzy XIV 274, 990. 1255 cr crinkled 709, 1004 ch congenital hydrocephalus 1004 f flexed tail XV 709. 1004 Tw Twirler 818 ax ataxia XVI 818 Va Varitint-waddler 233 de droopy-ear XVII 233 sa satin 1268 bg beige XVIII 1268 Hk Hook 489 Os Oligosyndactylism 488. 489 tg tottering XX 488 Bn Bent 336, 418, 754, 1001 Bio Blotchy 1112 Ta Tabby 334. 342. 336, 1001. 1112. 1382 Mo-series Mo Mottled 336. 342 Mobr Brindled 336, 342 To Tortoise 269, 754 Dp Dappled 1003 JP jimpy 1001 sf scurfy 1382 George E. Jay, Jr., Ph.D. GENETIC STRAINS and STOCKS INTRODUCTION In this era of rapidly expanding biomedical research, the need for current informa- tion on all aspects of laboratory animal resources is particularly important and will become even more so in the future. From time to time in the past, various listings of laboratory animals have been compiled and made available to the scientific public. The Institute of Laboratory Animal Resources, National Research Council, has prepared a catalogue of commercial sources of laboratory animals in the United States;639 the Mouse News Letter,903 an informal publication prepared by the Laboratory Animals Centre, Medical Research Council, England, provides a continuing listing of inbred mouse strains from all parts of the world,f and the Committee on Standardized Nomenclature for Inbred Strains of MiceJ has recently published a revised listing of inbred strains of mice.219 These listings are of vital importance to the research worker, for they provide information on the location and status of laboratory-animal strains and stocks. However, the listings indicated above have not adequately covered the extensive genetic resources existing for other mammals such as the rat, guinea pig, hamster, f Dr. G. D. Snell and Miss Joan Staats of the R. B. Jackson Memorial Laboratory, Bar Harbor, Maine, now prepare a supplement to the Mouse News Letter, listing the inbred strains of mice. It is anticipated this supplement will appear every two years. X Committee members: G. D. Snell and Joan Staats, R. B.Jackson Memorial Labora- tory, Bar Harbor, Maine; M. F. Lyon, Harwell, Berks, England; L. C. Dunn, Columbia University, New York, New York ; H. Gruneberg, University College, London ; P. Hertwig, Biolojisches Institut, Halle; and W. E. Heston, National Cancer Institute, Bethesda, Maryland. 83 84 GENETIC STOCKS AND BREEDING METHODS rabbit, and so forth. Consequently, a compilation of this information would be most timely and of considerable interest to all concerned. The publication of Billingham and Silvers," the Catalogue of Uniform Strains of Laboratory Animals Maintained in Great Britain,742 and the more recent questionnaire of the Committee on Maintenance of Genetic Stocks, Genetic Society of America, provide some information on the where- abouts of such material, but it is believed that many unlisted stocks of genetic interest are being maintained by various investigators. Thus, the Genetics Study Section, Division of Research Grants, National Institutes of Health, thought it most pertinent to develop a composite listing of genetic material for mammalian species most commonly used in laboratory experimentation, and to include such listing in this volume. This material is the initial effort to bring together all the information alluded to above, plus the results of an extensive inquiry to scores of investigators and organizations around the world. It is hoped that this compilation is complete, but it is realized that some investigators were no doubt inadvertently overlooked. I apologize for this oversight and hope their material will be included in the planned revision. In developing this listing, many sources of information were utilized as well as the talents of many individuals in the United States and abroad. To all of these sincere thanks and appreciation are extended for permission to use their material and for their efforts and interest. Thanks are also due to Mrs. Clarice Overath and Mr. Robert Dettman for their untiring help in assembling the data and preparing the manuscript, and various others at the National Cancer Institute who have been helpful. LISTING ARRANGEMENT The arrangement of this composite listing is by sections according to species as follows: Section I, Mice; Section II, Rats; Section III, Guinea Pigs; Section IV, Hamsters; Section V, Rabbits; Section VI, Peromyscus sp. Each section contains a brief introduction, followed by tables listing (1) established inbred strains, (2) strains in development, and (3) stocks of particular genetic interest. The distinction between established inbred strains and strains in development was made on the basis of the definition of an inbred strain as recommended in Stan- dardized Nomenclature for Inbred Strains of Mice,219 that is, twenty or more consecutive generations of brother x sister or parent x offspring (provided the mating in each case is to the younger parent) . Two exceptions to this rule were made for rat strains MR and MNR, since the generations of inbreeding presently reported are appro- ximately the theoretic twenty generations. It is likely that by the time these listings are published twenty generations of inbreeding will have been completed. Thus, the strains in development are those exhibiting various generations of inbreeding from Fj to something less than F20. In the case of certain strains of rabbits (table 38), when matings of brother x sister or parent x offspring have not been consistently followed, GENETIC STRAINS AND STOCKS 85 the amount of inbreeding is indicated by an inbreeding coefficient (F), devised by Wright.1447 The use of the terms strain and substrain, again, are used according to the definitions recommended by the Committee on Standardized Nomenclature for Inbred Strains of Mice. However, some latitude has been exercised in this regard, for the term strain has been used to designate those populations in the process of being inbred by brother x sister, parent x offspring, or some other system of inbreeding. The term, stock, has been used to designate those populations that are maintained for their identified genes or identifiable genetic characteristics of genetic interest, with or without inbreeding. In the case of mice, only the established strains recently compiled by the Committee on Standardized Nomenclature for Inbred Strains of Mice are included. Stocks containing identified genes or identifiable genetic characteristics are numerous and with a global distribution. The Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine, is probably the largest repository of murine genes in the world, and along with this the laboratory has developed, and is continuing to develop, isogenic strains and linkage stocks for testing purposes. Elsewhere in this volume, the linkage stocks at this laboratory are discussed by Dr. Margaret C. Green. Material of this kind for species other than mice is extremely limited. Similarly, no attempt was made to ascertain the number or kinds of inbred strains in development for mice, whereas this information has been compiled for the other species. It is strange that even though some of the earliest mammalian genetics was done in such species as the rat, guinea pig, and rabbit, there is a paucity of such material today. Fortunately there are indications of renewed interest in these species. As indicated above, information on the location and status of the various strains and stocks came from previously printed lists, publications, and a questionnaire. From these, arid my own knowledge of the whereabouts of strains and stocks, a mailing list of more than a hundred names was compiled. At the present time similar surveys are being made in Japan, Canada, Italy, and Germany. All the information obtained has been arranged in a contributor-strain cross-file. This file will be kept active by sending out periodic requests for bringing the status of strains and stocks up-to-date. No information was solicited or obtained (except in a few cases) regarding the environ- mental conditions under which the animals are maintained, the disease conditions existing (unless of genetic significance), husbandry practices followed, nutritional status, and so forth. It might be of considerable value for future listings to take into consideration such exogenous factors, since certainly some genetic manifestations are subject to environmental modifications. I. MICE As indicated above, only the established inbred strains of mice compiled by the Committee on Standardized Nomenclature for Inbred Strains of Mice219 and the list of contributors are included in this section. The material is presented as published, 86" GENETIC STOCKS AND BREEDING METHODS with only those editing changes necessary to fit the format of this publication. It is realized there are probably many strains now in the process of being developed that will eventually be listed in future revisions by this Committee, or that will be indicated in future editions of the Mouse News Letter.90* The importance of this species in mam- malian genetic research is well recognized, and for this reason communication on the location and status of murine material is much better organized than for the other rodent species. The far-sighted organization of a committee for the purpose of bring- ing order out of approaching chaos has been a tremendous asset, and consequently there is little to add to the splendid work already done. Other kinds of useful informa- tion about murine strains, such as composite-gene stocks, linkage stocks, and isogenic stocks are available through such organizations as the Roscoe B. Jackson Memorial Laboratory in this country and the various research groups in England that have done an outstanding job in collecting such material and maintaining it in usable form. Table 27 Established strains of micej Name or Symbol Synonym(s) Remarks A/Crgl/2 A/Crgl/3 A/He A/Jax A/LN A Lilly inbr (St): 131. genet: aabbcc. H-2a. origin: Strong, from cross made in 1921 of albino from Cold Spring Harbor and Bagg albino. The majority of sublines trace to a stock which Bittner obtained from Strong in 1927. charac: mammary tumor incidence high in breeders, low in virgins. Produces 5-10% young with cleft palate. High incidence of renal disease in old mice, maintained by: Bcr, Br, Cam, Fa, Fn, Ge, Go, Gr, H, Ha, Icrc, Lab, Mr, Not, Rl, Sn, Sp, Ss, St. inbr (Crgl): 26. genet: aabbcc. origin: from a nontumorous $ of A/He-Jax. charac: 30-50% mammary tumors in breeders at 12-13 months. Originally very low incidence, maintained by: Crgl. inbr (Crgl): 18 since mutation, origin: mutation of MTI in 1951 at F84 + 4. charac: over 90% mammary tumort in breeders at 8 months. Approx. 11% in virgins at 16 months, maintained by: Crgl. inbr (Jax) : 111. genet: aabbcc. origin: see strain A. Strong to Bittner 1927, to Heston 1938, to Jax 1948 at F77. charac: mammary tumors 74% in breeders. Primary pulmonary tumors 30% in $?, 51% in o*6*- Mast-cell content of spleen very high in old mice, especially 6\J- maintained by: Anl, Crgl, Cam, Cbi, Chr, De, Gif, Ha, He, Jax, Mas, Mr, Ms, N, Per, Pi, Rd, We. inbr (Jax): 107. genet: aabbcc. origin: see strain A. Strong to Cloudman 1928, survivors of Bar Harbor fire to Jax 1947 at F73. charac: 28% mammary tumors in breeding $$. Primary pulmonary tumors 41% in $$, 49% in o*6*- Lower percentage granulocytes than other A sublines, maintained by: Anl, Bu, Chr, Fr, Jax, Jb, Ks, Mas. inbr (N): 110. genet: aabbcc. origin: see strain A. Strong 1921, to Bittner 1927, to W. Murray, to Eli Lilly & Co. 1941, to Jax 1948, to NIH 1951 at F84. charac: resistant to some tumors from other A sublines, maintained by: N. t A list of abbreviations used is given at the end of this table. GENETIC STRAINS AND STOCKS 87 Table 27 — Continued Name or Symbol Synonym(s) Remarks AfB Af/MySp A/Gr, (RIII^/Pu A/Fa- + c ABG A/Be-CRe REB A-H-2" A.BY A-H-2fFu A.CA A-H-2* A.SW AB Bluhm- Stamm, Agnes-Bluhm AK/n • Ak-n AKR AK, AKm, Afb, R.I.L., Rockefeller Inst. Leuke- AKR-//-2a AKR.K AKR-//-2m AKR.M AU A2G BALB/c BalbC, C inbr (Gif ) : 32. genet: aabbcc. origin: from Netherlands Cancer Inst., a litter of A taken by cesarean section and fostered on C57BL. charac: no mammary tumor, since no MTI. Low percentage of pulmonary tumors, maintained by: Gif. inbr (Sp) : ? + 16. genet: aabbcc. origin: Murray, from A taken by cesarean section and fostered on strain BD. charac : low mammary tumor, high pulmonary tumor, renal disease, maintained by: Sp. inbr (Mr): F17 since fostering, genet: aabbcc. origin: one litter of A/Gr mice fostered on RIII;B/Pu, May 1952, without suckling own mother, charac: no mammary tumors, being free of milk factor, otherwise same as strain A. maintained by: Mr. inbr (Fa) : N16. genet: aabb + c. charac: c segregates, maintained by: Fa. inbr (Be): 20 + . genet: aabbReRe, and either cc or +c origin: from A/Fa circa 1948 by addition of C and Re through five generations of backcrossing to A/Fa; then b x s with forced segregation at the c locus, the parents being always cc $ x +c $. maintained by: Be. inbr (Sn) : G12F15. maintained by: Kl, Sn. inbr (Sn) : G12F16. maintained by: Kl, Sn. inbr (Sn) : G12F18G4F1. maintained by: Kl, Sn. inbr (Kn): 53. genet: cc. origin: Dr. Agnes Bluhm, Kaiser Wil- helm Inst., circa 1930; b x s until 1943. Breeding history disrupted by war. This stock from Prof. Kaufmann, Univ. Marburg, in 1950. Inbreeding continued, charac: no mammary tumors or sarcomas except after radiation treatment; low rate of pulmonary tumors and lymphomas, maintained by: Kn. inbr (Ka): 20+ by Gs + F9. genet: aacc. origin: Furth, to Gross Nov. 1945; Gross to Kaplan, 1956, and Ontario Cancer Inst., 1957. charac: high leukemia; source of virus that induces leukemia in C3H/BiGs mice, maintained by: Gs, Ka, Oci. inbr (N) : 64. genet: aacc. H-2k. origin: carried by Furth as high-leukemia strain from 1928 to 1936. Then random bred at Rockefeller Inst, for several generations, followed by F9 by Mrs. Rhoads and 30+ by C. Lynch, charac: high leukemia, main- tained by: Am, Gif, Jax, Jb, Lab, Lhm, Lw, Ms, Mv, N, Pa, Rd, Rho, S, Sp, We. inbr (Sn): G12F11G4F7. inbr (Sn): G12F17. inbr (Ss) : 27. genet: aaUU. origin: Fisher; Medawar (London) to Silvers in 1957 at F23. charac: $? do not reject 6*c? isografts. MAINTAINED BY: Ss. inbr (Gif): 47. genet: albino, origin: cross of A with noninbred albino, charac: useful for endocrine work especially gonadotrophin assay (Lab). Can carry Salmonella without showing signs of it (G). Good reproduction (Gif). maintained by: G, Gif, Lab, Mr. inbr (Rr): 98. genet: bbcc. H-2d. origin: albino stock acquired by Bagg in 1906, to Little, to MacDowell in 1922; b x s inbreeding started by MacDowell in 1923. Prior inbreeding uncertain. Trans- ferred from MacDowell to Snell in 1932 at F26 and subsequently 88 GENETIC STOCKS AND BREEDING METHODS Table 27 — Continued Xame or Symbol Synonym(s) BALB/Gw Ba, B alb, Bagg albino BALB'MySp B alb, Bagg albino BALBf Si BALB/C/ C + BALB/c- CFU CFu BAMA BD Bd BDP BL Bagg L, BALB/R BRS BrS, Br-S, Br-s BRSUNT BrSunt BRVR BSVS BUA Remarks widely distributed especially via Andervont. charac: low incidence of mammary tumors, but high incidence when milk agent is intro- duced. Some ovarian and adrenal tumors. Susceptible to chronic pneumonia. Primary pulmonary tumors, 26% in $$, 29% in 6\?- maintained by: Anl, Crgl, De, Di, Gif, Go, Jax, Jb, Ka, Lab, Mc, Ms, N, Pi, Rl, Rr, S, Sp, We. inbr (Gw) : ? + 62. genet: bbcc. origin: see BALB/c. MacDowell to Gowen in 1932 at F27. charac: low resistance to Salmonella typhi- murium. Radiation susceptible, maintained by: Gw. inbr (Sp): 47. genet: bbcc. origin: see BALB/c. Murray to Simp- son in 1948 at F23. charac: mammary tumor, pulmonary tumor. maintained by: Sp. inbr (Sp): ? + 20. genet: bbcc. origin: from BALB born by cesarean section and fostered on strain BD. Murray to Simpson in 1948. charac: low mammary tumor, some pulmonary tumor. maintained by: Sp. inbr (Sp) : 47. genet: bbcc. origin: from BALB/cAn fostered on C3H. Andervont to Simpson in 1952 at F3S. charac: mammary tumor, pulmonary tumor, maintained by: Sp. inbr (Rr): N12. genet: bb + cFu + . origin: from BALB/cSnRr. charac: 95% penetrance of Fu. maintained by: Rr. inbr (Sp) : ? + 18. genet: cc. origin: derivative of A ? x MA q*. charac: low mammary tumor, maintained by: Sp. inbr (Sp): ? + 16. genet: aa. origin: Murray, DBA x C57BL, N8 to C57BL, then b x s. Warner to Simpson 1950. (In view of the origin of this strain, it should be classed as a subline of C57BL.) charac: low mammary tumor, maintained by: Sp. inbr (Jax): 38 + . genet: aabbsejdsepprdrd. origin: Gates, inbred since 1926. charac: frequent mammary tumors, ovaries hemorrhagic and necrotic, nervous behavior, maintained by: Jax. inbr (De): 68. genet: aabbcc. origin: Lynch, from Bagg stock via Strong, maintained at Rockefeller Inst, as distinct strain since 1921. Some of original animals obtained from Strong carried agouti. This stock from Lynch in 1951, at F46. charac: low mammary tumor; some pulmonary tumors in old mice, maintained by: De. inbr (St): 55. genet: aabb. origin: Strong, from a branch of NH treated for eight or more generations with methylcholanthrene. charac: gastric lesions, adiposity, maintained by: St. inbr (N): 64. genet: aabb. origin: Strong, a branch of BRS con- tinued without further methylcholanthrene treatment (UNT = un- treated), charac: gastric lesions, adiposity. maintained by: N, St. inbr (Kp) : 57. genet: cc. origin: from H. A. Schneider, Rocke- feller Inst., at F57 in 1959. charac: susceptible to bacteria and to Arbor "B" viruses, maintained by: Kp. inbr (Kp): 57. genet: cc. origin: same as BRVR, at F56 in 1959. charac: susceptible to Salmonella and viruses, particularly hepatitis. maintained by: Kp. inbr (Wi) : 36. genet: albino, origin: albinos of unknown pedigree at Brown Univ. maintained by random breeding for unknown length GENETIC STRAINS AND STOCKS Table 27 — Continued 89 Name or Symbol Synonym(s) Remarks BUB BUC BUE cinnamon CBA XXXIX CBAf ABC, CBAfC3H CBA-a . CBA-a( CSA-da CBA-//-2? CBA-/) CEA-se CC57BR BR CC57W W of time. Inbreeding started in 1945. charac: selected for good growth and reproductive performance. No known spontaneous tumors, maintained by: Wi. inbr (Wi) : 44. genet: aacc. H-2q. origin: same as BUA. charac: same as BUA. maintained by: Bn, Wi. inbr (Wi) : 36. genet: aacc. origin: same as BUA. maintained by: Wi. inbr (Wi) : 19. genet: albino, origin: cross betweeen BUD at F18 (now discarded) and BUA at F14. charac: selected for circling now 20% penetrance. Onset 14-20 days. Audition and vestibula- tion present and apparently normal. Circlers vicious fighters; not good mothers. Very high metabolic activity, maintained by: Cu, Wi. inbr (St): 86. genet: bb. H-2". origin: Strong (see C3H/St). charac: moderate mammary tumor incidence. Tendency to bi- furcation of seminal vesicles, maintained by: Ao, St. inbr (Jax) : 106. genet: 4-. H-2k. origin: (see C3H/St). charac: mammary tumor incidence variously reported l.l-22.2%.742 Some hepatomas. Long-lived. Dietary supplements needed to maintain reproductive efficiency. Absence of lower third molars in about 18%. Few skeletal variants. Moderately resistant to skin cancer induction by chemical carcinogens, maintained by: Br, Cbi, Fa, Gr, H, Jax, Lab, Lhm, Ms, No, Rij, Ss, St. inbr (Pa): 28. genet: 4-. origin: Paterson, from CBA mice received in 1949 from Carr and fostered on C3H. charac: spon- taneous mammary cancer, maintained by: Pa. inbr (Be): 20 4-. genet: aa. origin: Carter outcrossed CBA to aa and backcrossed to CBA to N6. B x s matings thereafter, main- tained by: Be. inbr (H): N5 + F24. genet: +al x a1 a1, origin: CBA/Fa. MAINTAINED BY: Fa, H. inbr (Fa): ? 4- 31. genet: +da, dada. origin: Dark (da) arose in CBA/Fa circa 1955. charac: like CBA. maintained by: Fa. inbr: ? + 16. genet: +. origin: from Bonser to Miihlbock, to Rijswijk in 1953. charac: not H-2k, but an as yet unknown H-2 type, maintained by: Rij. inbr (Cam): ? 4- 16. genet: +p x pp. origin: mutation in 1948 or 1949 in CBA/Ca. maintained by: Cam. inbr (Gn): 30. genet: +se x sese. origin: mutation in 1948 or 1949 in CBA/Ca stock, charac: high incidence of hydronephrosis in sese animals, maintained by: Cam, Gn. inbr (Mv): 41. genet: brown, origin: see CC57W. The same cross produced both CC57BR and CC57W. charac: do not develop spontaneous mammary tumors; 55% tumors after administration of milk agent to newborns. 1 % pulmonary adenomas in old mice, 15% after urethan treatment. Probably differs from CC57W in leukemia incidence and H-2 allele, maintained by: Mv. inbr (Mv) : 40. genet: albino, origin: C57BL and BALB/c mice received in Moscow, Sept., 1943, from Nat. Inst. Health (USA). DO GENETIC STOCKS AND BREEDING METHODS Table 21 —Continued Name or Symbol Synonym (s) Remarks The one living BALB/c $ was mated to a C57BL d*; selection and in- breeding led to development of the CC57BR and CC57W mice. charac: do not develop spontaneous mammary tumors; 55% tumors after administration of milk factor to newborn, 15% pulmonary adenomas in old mice, some leukemia and skin cancer appear; 100% pulmonary tumors after urethan treatment. Viability and fertility good. MAINTAINED BY: Mv. CE Cd, ce, ce inbr (Jax): ? + 25. genet: AwAwcece. origin: wild mutant trapped in 1920 in Illinois by J. E. Knight. Detlefsen studied genetics of the color type. Inbred by Eaton at least 15 generations, some sent to Woolley prior to 1940. Woolley to Speirs, back to Woolley and Jax in 1948. charac: low incidence of mammary tumors, 33% ovarian tumors in old age, some sarcomas, wide range of tumor types, high adrenal cortical carcinoma incidence after neonatal gonadectomy, poor breeders, have relatively few litters but eight to ten per litter. maintained by: Di, Hu, Jax, Lab, Pi. CFCW inbr (Rl): 29. genet: ccCaCa. origin: from Carworth Farms in 1948. MAINTAINED BY : R 1 . CFVV inbr (Rl): 25. genet: cc. origin: Webster to Carworth Farms; distributed by them, charac: 20% mammary tumors in breeders 9-10 mo.; susceptible to many viruses, maintained by: Hd, Ms, Rl. CHI inbr (St): 93. genet: +. H-2". origin: Strong (see C3H/St). charac: similar to C3H/St. maintained by: St. CT inbr (Ch): 24. genet: aabbddpp. origin: Chase, developed for color testing, maintained by: Ch. C3H/An inbr (Wi): 82. genet: +. origin: see C3H/St. A litter of 4 ? and 2 o* sent to Andervont in Oct., 1930. Selected in early generations for high and early mammary tumor incidence, charac: over 90% incidence of mammary tumors in breeders and virgins. Higher mam- mary tumor and hepatoma incidence and lower pulmonary tumor incidence than St subline. Mammary tumors 94% at 8-10 months, almost 100% at 14 months. Hepatomas: $$ over 1 year 10%, C3H/He-.S7 Stock 800, Steel C3H/N-W" Stock 93, W C3HA inbr (Ks): 28. genet: +. origin: Strong, 1920. This stock from C3H mice of unknown pedigree which survived the Bar Harbor fire, charac: resistant to some C3H transplantable tumors. This strain appears to fall into Bittner-Strong group of sublines on basis of vertebral counts, but serological tests place the strain in the Andervont- Heston group, maintained by: Jax, Ks. inbr (St): 114. genet: +. origin: Strong, 1920, from a cross of? Bagg albino x o* DBA. Strains C, CBA and CHI originated from this cross, charac: mammary cancer 70% at 14 months, and almost 100% in those living to 16 months. St and Bi sublines have pre- dominantly 5 lumbar vertebrae, maintained by: Cbi, Fn, Ha, Sp, St, Wi. Others maintaining C3H, subline not given: Ber, Ge, Ka, Pe, S. inbr (He): 39 since fostering, genet: +. origin: Heston, from litter of C3H born by cesarean section and fostered on strain C57BL, 1945. charac: high susceptibility to MTI. Mammary tumor incidence 2% in virgins, 38% in breeders, 20% in males treated with diethylstilbestrol, none in untreated <$$. Heptomas 23% in diethyl- stilbestrol-treated o*H PHL pHL, low blood-/>H PL PL(B), Princeton leukemia, LII PLA RF RFM, Rf, Rfa Paris, R3 Radium In- stitute (Paris) line III RHI/Wy R3 RIIIX inbr (St): 56. genet: aabbpp. origin: Strong, from methyl- cholanthrene-treated NH mice, charac: mammary tumors in breeders, maintained by: St. inbr (We): 28. genet: aalnln. origin: from MacArthur outbred stock, obtained from Butler, 1949. Selected for high blood pH, with b x s mating, charac: blood pH 7.47; sex ratio 55% <$<$. Sex ratio now 52% gg. Mean litter size at weaning 6.5. Over-all fertility low. maintained by: We. inbr (We): 24. genet: aabblnln\alalbblnln (leaden brown, and leaden brown-and-tan families), origin: same as PHH, selected for low blood pH. charac: blood pK, 7.42 ; sex ratio 45% <$<$, now 42% £<$. Mean litter size 5.7. Over-all fertility high, maintained by: We. inbr (Ly) : 43. genet: cc. origin: from noninbred "Princeton" strain started in 1922 from 200 mice purchased from dealer. Inbred by Lynch, giving rise to a high leukemia line B ( = PL) and a second line A ( = PLA) with lower incidence, charac: few mammary tumors, 80-90% leukemia, maintained by: Jax, Ly, Sp. inbr (Ly): 43. genet: cc. origin: see PL. maintained by: Ly. inbr (Jax): ? + 18. genet: cc. H-2k. origin: Furth, 1928, from unknown stock at Rockefeller Institute. He transferred stock to Oak Ridge; to Jax at Oak Ridge's F14. charac: low incidence of leukemia, higher after radiation, maintained by: Gif, Jax, Jb. inbr (Gw) : 48. genet: aabbcc origin: Gowen, from Webster's resistant stock in 1936. charac: quite resistant to Salmonella typhi- murium; unreliable to poor breeders, maintained by: Gw. inbr: 31 + . origin: Korteweg. maintained by: not reported. inbr (Rl) : 31. genet: a'a'ruruss. origin: from Holman Jan. 1948 (F4 from Dunn's BT ruby x Holman's black piebald, shaker-1). MAINTAINED BY: Rl. inbr (Crgl) : ? + 25. genet: cc. origin: Dobrovolskaia-Zavadskaia, Paris, 1928. charac: animals have milk agent; mammary tumor in 84% of breeders, 40% of virgins. Nonchromosomal resistance factor against leukoses. Susceptible to pulmonary infection. Good reproducers but bad mothers (cannibalism frequent), maintained by: Crgl, Mr, Per, Rd, Rho. inbr (Jax) : 3 1 . genet : cc. origin : Dobrovolskaia-Zavadskaia. Sent to Eisen, Eisen to Jax 1948. charac: fairly high incidence of mam- mary tumors. Resistant to RHI/Wy transplantable tumors. Sus- ceptible to skin ulcerations, maintained by: Gif, Jax. inbr (Jax): 48. genet: cc. H-2'. origin: Dobrovolskaia- Zavadskaia, to Curtin and Dunning, to Woolley before 1947. One pair survived Bar Harbor fire; to Snell in 1950, to Jax in 1957 at F42. charac: fairly high mammary tumor incidence, resistant to skin ulcerations found in RHI/Jax. RHI/Wy transplantable tumors will not grow in RHI/Jax. maintained by: Jax. inbr (Pu): 58. genet: cc. origin: RIII fostered on C57BL. charac: does not have milk agent, but very sensitive to it. Mammary tumors in breeders 3%. Fertile but poor mothers, maintained by: Pu, Rd. inbr (Fa): ? + 29. genet: cc + ro. charac: ro segregates, main- tained by: Fa. GENETIC STRAINS AND STOCKS .93 Table 27 — Continued Name or Symbol Synonym(s) Remarks SEA Ab, Sese-Ab SEAC SEAC/c SEC C, Sese-C inbr (Gw) : 65. genet: cc. origin: Schott, obtained from dealer (Schwing) in 1927. Subsequently selected for resistance to Salmonella typhimurium and inbred by Gowen. charac: resistant to S. typhi- murium and to radiation, maintained by: Gw. inbr (Gn) : 61. genet: bbd + /dse. origin: Green, from a cross of BALB/c x P. maintained by: Gn, Rl. inbr (Gn): 26. genet: aabbcchcchd + j +se. origin: Green, from a cross of SEA x SEC/2, maintained by: Gn. inbr (Gn): N12 of se (Carter) to SEAC. genet: aabbcchcchd +/ +se. origin: Green, by backcross of Carter's se from CBA/5e into SEAC. maintained by: Gn. inbr (Rl): ? + 27. genet: aabbcchcch + +jdse. origin: E. and M. Green, from cross of NB x BALB/c. Gave rise by crossover at F18 to SEC/1 — +se and SEC/2 — +d. maintained by: Rl. inbr (Gn) : 56. genet: aabbcchcch + se. origin: Green, charac: multiple pulmonary cysts, especially in sese. maintained by: Gn. inbr (Re): 60. genet: aabbcchcchSeSe. origin: E. S. Russell, from SEC/1, maintained by: Re. inbr (Gn): 55. genet: aabbcchcch + sejdse. origin: Green, main- tained by: Gn. inbr (Cu): 20. genet: albino, origin: outcross of BUD/Wi to CFW at Merck Institute; inbred with continuous selection for circus movement and vigor for 13 generations at Merck, 7 more at Seton Hall College of Medicine, charac: 30% penetrance of circus move- ment, good fertility and maternal care, maintained by: Cu. inbr (Cu): 20. genet: albino, origin: separated from SHC/a at its F13. charac: same as SHC/a. maintained by: Cu. inbr (Ms): 40. genet: cc. origin: K. Tutikawa, Nat. Inst, of Genetics, Misima; derived from one of several lines of SMA (q.v.) at F10, selected for high leukemia; to H. Baba, Kyushu University, 1954, at F16. charac: low mammary carcinoma; leukemia 54.5% in o*6\ 10% in $$; lymphatic leukemia, myeloid leukemia, reticulo- sarcoma, lymphatic leukemia with a conspicuous myeloid reaction were found in this strain, maintained by: Ms, Kyushu Univ., Kyoto Univ. inbr (Jax) : 30. genet: Awa. origin: MacArthur to Runner in 1948. charac: small body size, renal amyloidosis, leukopenia. maintained by: Jax, Rr. inbr (Ms): 39 (26 at Ms), genet: cc. origin: S. Makino to Hok- kaido Univ., 1944; to Misima, 1951, at F15. charac: good breeder, low incidence of mammary and pulmonary tumor in old age, 15-20% kinked tail variants in each generation, maintained by: Ms. inbr (Jax): 56, genet: aabbcc. H-2k. origin: Engelbreth-Holm, to Heston at F23, to Jax at F25. charac: 1-2% leukemia including some plasma-cell leukemias. Other tumors, principally pulmonary adenomas and mammary carcinomas, about 3%. maintained by: Jax. inbr (N): 52. genet: aabb. origin: Strong, to NIH in 1951 at F28. maintained by: N. SEC/1 Sese-Cl. SEC/ 1-5* SEC/2 Sese-C2 SHC/a SHC/b SL S/L SM SMA ST STR S, SM Street 100 GENETIC STOCKS AND BREEDING METHODS Table 27— Continued Name or Symbol Synonym(s) Remarks STR/1 SWR Swiss, Swiss-R, Swiss-8 VVB WC WH WK WLL White Label, Kreyberg, White Label Leeds YBR YS ZBR IV/B XVII Zavadskaia Brachy 17 inbr (N): 50. genet: aabbss. origin: piebald branch of STR originating at NIH in 1951 at F29. maintained by: N. inbr (Jax) : 62. genet: cc. origin: a strain of Swiss mice from A. de Coulon of Lausanne, Switzerland, inbred by Lynch, charac: 44% pulmonary tumors, 19% mammary tumors in breeding $$. main- tained.by: De, G if, Jax, J b, Ly, Ms. inbr (Sp) : 20. genet: cc. origin: noninbred Tumblebrook stock in 1948, inbred thereafter. Mice were submitted to a single cutaneous application of methylcholanthrene in benzene for seven consecutive generations, F6 through F12. charac: high incidence of mammary tumors, maintained by: Sp. inbr (Re): genet: aaWw. origin: heterozygous mice carrying Ww were sent by S. Waelsch and S.J. Holman to Russell in 1948. Following several intercrosses and outcrosses to C57BL/6, b x s inbreeding with forced heterozygosis of Ww began in 1949. Separate lines were main- tained which gave rise to four distinct strains, WB, WC, WH, and WK. charac: homozygotes (WW) are anemic, die at approx. 11 days. maintained by: Re. inbr (Re): 29. genet: aaWw. origin: same as WB. charac: anemics die at approx. 14 days, maintained by: Re. inbr (Re): 29. genet: aaWw. H-2d. origin: same as WB. charac: anemics die at approx. 5 days, maintained by: Re. inbr (Re): 27. genet: aaWw. origin: same as WB. charac: anemics die at approx. 10 days, maintained by: Re. inbr (Ge) : 53. genet: cc. origin: Kreyberg, to Bonser at Leeds in 1934. charac: 20-33% mammary tumors in breeders, main- tained by: Br, Ge. inbr (Wi): 27. genet: A", origin: Wilson, from noninbred stock called "a group of mixed ancestry carrying the yellow gene" received from Danforth, Stanford, in 1948. maintained by: Wi. inbr (He): 70. genet: Ayabb. H-2d'. origin: Little, to Andervont, 1936; to Heston, 1946; to Wilson, 1948; to Hauschka, 1951. main- tained by: Ha, He, Ly, Wi. inbr (Ch): 40. genet: Ayass. origin: original Y from Jax. Spot- ting introduced, then crossed with C57BL in 1948, then inbred. maintained by: Ch. inbr (Gw) : 56. genet: cc. origin: Gowen, from Swiss mice of the Yellow Fever Lab. of the Rockefeller Inst, charac: medium sus- ceptibility to typhoid. Medium susceptibility to radiation. Capable of having many litters, but raises few. maintained by: Gw. inbr (Co): 21. genet: ssT+. origin: from Dobrovolskaia- Zavadskaia in 1930; outcrossed in succession to several Columbia stocks, maintained by: Co. genet: albino, origin: from N. Dobrovolskaia-Zavadskaia to Centro per lo studio e la cura dei tumori, Busto Arsizio, in April, 1948. charac: mammary tumor incidence 28%, early breeding and pregnancy increase incidence. Susceptible to multiple tumors. MAINTAINED BY: Baz. inbr (Rd): 28. genet: cc. origin: Institut du Radium, 1930. Rigorous inbreeding since 1951. charac: mammary tumors 1-5%. GENETIC STRAINS AND STOCKS 101 Table 27 — Continued Name or Symbol Synonym(s) Remarks 101 129 lOlBAg \29-Fu 129/Sv-CP 129/Sv-aCP Sensitive to RIII milk agent (55% tumor incidence after injection of newborns). Spontaneous leukemia less than 2%. Pulmonary tumors 15% in $$ 12—24 mo. Sensitive to pulmonary tumor produc- tion by urethan, methylcholanthrene, or X rays. Relative sensitive to Gross tumor agent. Good breeders and good mothers, main- tained by: Baz, Rd. inbr (H) : 49 + 17. genet: Au'Aw. origin: Dunn, charac: low mammary tumor, slow to breed, fertility moderate. High suscepti- bility to skin and pulmonary tumor induction by chemical carcinogens, low leukemia, low mammary tumor, high incidence of papillonephritis. maintained by: H, Lhm, Rl. inbr (Rr): 33. genet: AwAwcchpjcp. H-2b. origin: Dunn. charac: useful for ovary transplant and ova transfer studies; 1% incidence of testicular teratomas. Low mammary tumor incidence, not susceptible to MTI. High incidence of venous congestion in adrenals and uterus. Highly sensitive to estrogen at all ages. Ulti- mate source of muscular dystrophy animals, maintained by: Ha, Jax, Jb, Lab, Pi, Rl, Rr, Sv. inbr (Rr) : N13. genet: AwAwcchp\cpFu +. origin: Fu introduced in 129/Rr. charac: 48-50% penetrance of Fu. maintained by: Rr. inbr (Sv): Nil + F6. genet: AwAw. origin: outcross of 129/RrSv to DBA with backcross to 129 to Nl 1, b x s since then, charac: useful for ovarian transplants, maintained by: Sv. inbr (Sv) : Nil + F7. genet: aa. origin: spontaneous mutation from Aw to a detected in F2 of 129/Sv-G>. charac: useful for ovarian transplants, maintained by: Sv. List of symbols for designating substrains of mice A Antoni van Leeuwenhoekhuis, Amster- Be dam C, Sarphatistraat 108, Netherlands (Dr. O. Muhlbock). Am Dr. J. L. Ambrus, Roswell Park Bi Memorial Institute, Buffalo 3, N.Y. Ao Dr. D. B. Amos, Roswell Park Memorial Institute, Buffalo 3, N.Y. Bl An Dr. H. B. Andervont, National Cancer Institute, Bethesda 14, Md. Anl Argonne National Laboratory, Division of Biological and Medical Research, Box 299, Lemont, 111. (Dr. Robert J. Bn Flynn). Ba Institute de Medicina Experimental, Laboratorio de Genetica, Avda. San Bnd Martin 5481, Buenos Aires, Argentina. Baz Centro per lo studio e la cura dei tumori, Busto Arsizio, Italy (Dr. G. Ceriotti). Bo Bcr University of Birmingham, Cancer Research Laboratories, The Medical School, Birmingham 15, England (Dr. Br June Marchant). Dr. R. A. Beatty, Institute of Animal Genetics, West Mains Road, Edin- burgh 9, Scotland. Dr. J. J. Bittner, University of Minne- sota Medical School, Minneapolis 14, Minn, (deceased). Drs. M. & F. Bielschowsky, Hugh Adams Cancer Research Department, University of Otago Medical School, Great King Street, Dunedin C.I., New Zealand. Dr. S. E. Bernstein, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Barnard College, Department of Zoology, Columbia University, New York 2 7, N.Y. (Dr. Reba M. Goodman). Biochemical Department of Cancer Institute, Zluty kopec 7. Brno, Czecho- slovakia. Dr. G. M. Bonser, Department of Experimental Pathology and Cancer 102 GENETIC STOCKS AND BREEDING METHODS Research, School of Medicine, Leeds 2, England. Bs Dr. P. R. F. Borges, Tufts Cancer Control Unit, Tufts College Medical School, 30 Bennet Street, Boston 11, Mass. Bt Dr. N. Batcman, Field Laboratory, Animal Breeding Research Organiza- tion, Drydcn Mains, Roslin, Mid- lothian, Scotland. Bu Dr. VV. J. Burdette, Laboratory of Clinical Biology, Department of Surgery, University of Utah, College of Medicine, Salt Lake City, Utah. C or University of Cambridge, Department Cam of Genetics, 44 Storey's Way, Cam- bridge, England (Dr. M. E. Wallace). Ca Dr. T. C. Carter, c/o Western Chicken Ltd., London Road, Devizes, Wilts, England. Cbi Chester Beatty Research Institute, Insti- tute of Cancer Research, Fulham Road, London, S.W.3, England (Dr. P. C. Roller). Cg Mrs. A. Cohen, Experimental Oncology Laboratory, Radiation Therapy De- partment, Johannesburg General Hospi- tal, Johannesburg, South Africa. Ch Dr. H. B. Chase, Department of Biology, Brown University, Providence, R.I. Chr Children's Hospital Research Founda- tion, Elland Ave. and Bethesda, Cin- cinnati 29, Ohio (Dr. Josef Warkany). Ciu Unidad de Investigaciones Cancerologi- cas, Avenida Cuauhtemoc 240, Ciudad de Mexico, Mexico. CI Mrs. Ruth Clayton, Institute of Animal Genetics, West Mains Road, Edinburgh 9, Scotland. Co Columbia University, Department of Zoology, New York 27, N.Y. (Dr. L. C. Dunn). Cr Professor J. Craigie, Imperial Cancer Research Fund, Burtonhole Lane, Mill Hill, London, N.W.7, England. Crgl Cancer Research Genetics Laboratory, University of California, Berkeley 4, California (Dr. Earl B. Barnawell). Cu Dr. R. L. Curtis, Seton Hall College of Medicine, Department of Anatomy, Jersey City 4, N.J. De Dr. Margaret Deringer, National Can- cer Institute, Bethesda 14, Md. Di Dr. M. M. Dickie, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Dm Dr. L. Dmochowski, M. D. Anderson Hospital and Tumor Institute, Virology and Electron Microscopy, Houston 25, Texas. Eh Dr. J. Engelbreth-Holm, Universitetets patologiskanatomiske Institut, Frederick V's Vej 11, Kobenhavn, Denmark. Fa Dr. D. S. Falconer, Institute of Animal Genetics, West Mains Road, Edinburgh 9, Scotland. Fe Dr. Elizabeth Fekete, Roscoe B.Jackson Memorial Laboratory, Bar Harbor, Maine (retired). Fo Dr. P. Forsthoefel, University of De- troit, Biology Department, McNichols Road at Livernois, Detroit 21, Mich. Fr Dr. F. Clarke Fraser, Department of Genetics, McGill University, Montreal, Canada. Fu Dr. J. Furth, Roswell Park Memorial Institute, Buffalo 3, N.Y. G Glaxo Laboratories, Ltd., Greenford, Middlesex, England. Ge Dr. H. N. Green, Department of Experi- mental Pathology and Cancer Research, School of Medicine, Leeds 2, England. Gi Dr. A. B. Griffen, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Gif Centre de Selection des Animaux de Laboratoire, 5 rue Gustave Vatonne, Gif-sur-Yvette (S. & O.), France (Dr. M. A. Sabourdy). Gl Dr. A. Glucksmann, Strangeways Re- search Laboratory, Wort's Causeway, Cambridge, England. Glw Dr. Salome G. Waelsch, Albert Ein- stein College of Medicine, Department of Anatomy, Eastchester Road and Morris Park, New York 61, N.Y. Gn Drs. M. C. and E. L. Green, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Go Dr. Peter A. Gorer, Guy's Hospital, Department of Pathology, London S.E. 1, England. Gr Dr. H. Griineberg, University College London, Department of Zoology, Gower Street, W.C.I, England. Gs Dr. Ludwik Gross, Cancer Research Laboratory, V. A. Hospital, 130 W. Kingsbridge Road, Bronx 63, N.Y. Gw Dr. John W. Gowen, Iowa State College, Department of Genetics, Ames, Iowa. H Radiobiological Research Unit, Har- well, Didcot, Berks., England (Dr. M. F. Lyon). Ha Dr. T. S. Hauschka, Roswell Park Memorial Institute, Buffalo 3, N.Y. Hd Mr. L. B. Hardy, Roswell Park Memo- rial Institute, Biological Station, Spring- ville, N.Y. GENETIC STRAINS AND STOCKS 103 He Dr. W. E. Heston, National Cancer Institute, Bethesda 14, Md. Hg Professor Dr. P. Hertwig, Biologisches Ln Institut, Universitatsplatz 7, Halle (Saale), Germany. Hn Dr. P. J. Harman, Seton Hall College Lw of Medicine, Department of Anatomy, Jersey City 4, N.J. Ly Ho Prof. W. F. Hollander, Department of Genetics, Iowa State College, Ames, Iowa. M How Dr. Alma Howard, Research Unit in Radiobiology, Mount Vernon Hospital, Northwood, Middlesex, England. Ma Hr Dr. G. Hoecker, Catedra de Biologia, Av. Zanartu 1042, Santiago de Chile. Mas Hu Dr. Katharine P. Hummel, Roscoe B. Jackson Memorial Laboratory, Bar Mc Harbor, Maine. Icr Institute for Cancer Research, 7701 Burholme Avenue, Fox Chase, Phila- delphia, Pa. Md Icrc Indian Cancer Research Centre, Parel, Bombay 12, India (Dr. B. K. Batra). J or Expansion stocks of the Roscoe B. Jack- Jax son Memorial Laboratory, Bar Harbor, Me Maine (under the superivision of M. M. Dickie and E. S. Russell). Jb Dr. Jan H. Bruell, Psychology De- Mi partment, Western Reserve University, Cleveland 6, Ohio. Ka Dr. H. S. Kaplan, Department of Mo Radiology, Stanford University School of Medicine, Stanford, Calif. Ki Dr. A. Kirschbaum (deceased). Mr Kl , Drs. G. and E. Klein, Department of Cell Research, Karolinska Institutet, Stockholm 60, Sweden. Kn Prof. Dr. Fr. Kroning, Medizinische Ms Forschungsanstalt, Pharmakologische Abteilung, Bunsenstrasse 10, Gottingen, Germany. Mv Kp Dr. Hilary Koprowski, The Wistar Institute, 36th at Spruce, Philadelphia, Pa. My Kr Prof. L. Kreyberg, Universitetets Insti- tutt for Patologi, Rikshospitalet, Oslo, Norway. N Ks Dr. N. Kaliss, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. L Eli Lilly and Co., Indianapolis 6, No Indiana. La Dr. A. Lacassagne, Institut du Radium, rue d'Ulm, Paris (retired). Not Lab Laboratory Animals Centre, M.R.C. Laboratories, Woodmansterne Road, Carshalton, Surrey, England. Oci Lhm The London Hospital Medical College, Department of Cancer Research, Ash- field Street, Whitechapel E. 1, England (Dr. F.J. C. Roe). Dr. J. B. Lyon, Department of Bio- chemistry, Emory University, Atlanta, Georgia. Dr. L. W. Law, National Cancer Institute, Bethesda 14, Md. Dr. Clara Lynch, Rockefeller Institute, 66th Street and New York Avenue, New York 21, N.Y. Memorial Hospital and Sloan-Kettering Institute for Cancer Research, 410 E. 68th Street, New York 21, N.Y. Dr. E. A. Mirand, Roswell Park Memorial Institute, Buffalo 3, N. Y. Department of Zoology, University of Massachusetts, Amherst, Massachusetts. Dr. W. B. Mcintosh, Department of Zoology and Entomology, Ohio State University, 1735 Neil Avenue, Columbus 10, Ohio. Dr. E. C. MacDowell, Department of Genetics, Carnegie Institution of Wash- ington, Cold Spring Harbor, New York (retired). Dr. P. B. Medawar, Department of Zoology, University College London, Gower Street, W.C. 1, England. Dr. D. Michie, Department of Surgical Science, University New Buildings, Teviot Place, Edinburgh 8, Scotland. Dr. R. H. Mole, Radiobiological Re- search Unit, Harwell, Didcot, Berks, England. Dr. W. E. Miller, Cancer Research La- boratory, Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne 1, England. National Institute of Genetics, Yata IIII, Misima, Sizuoka-ken, Japan (Dr. Tosihide H. Yoshida). Dr. N. N. Medvedev, Gamaleya Insti- tute of Epidemiology and Microbio- logy, Schukinskaya 33, Moscow, USSR. Dr. W. S. Murray, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Genetics Research Unit, Laboratory Aids Branch, National Institutes of Health, Bethesda 14, Maryland (Dr. D. W. Bailey). Dr. D. J. Nolte, University of the Wit- watersrand, Genetics Laboratory, Mil- ner Park, Johannesburg, South Africa. Cancer Research Laboratory, The University, University Park, Notting- ham, England (Dr. R. W. Baldwin). Ontario Cancer Institute, University of Toronto, 500 Sherbourne Street, Toronto 5, Canada (Dr. A. A. Axelrad). 104 GENETIC STOCKS AND BREEDING METHODS Pa Dr. Edith Paterson, Christie Hospital and Holt Radium Institute, Manchester 20, England. Pe Dr. P. R. Peacock, Royal Beatson Memorial Hospital, 132-138 Hill Street, Glasgow C.3, Scotland. Per Division of Cancer Research, Univ. of Perugia, Italy (Prof. Lucio Severi). Pi Dr. H. I. Pilgrim, Laboratory of Clinical Biology, Department of Surgery, Uni- versity of Utah College of Medicine, Salt Lake City, Utah. Pu Dr. B. D. Pullinger, Royal Beatson Memorial Hospital, Cancer Research Department, 132-138 Hill Street, Glas- gow, C.3, Scotland. Rb Dr. R. G. Busnel, Institut National de la Recherche Agronomique, Labora- toire dc Physiologie Acoustique, Jouy- en-Josas (S. & O.), France. Rd Dr. G. Rudali, Fondation Curie, 26 rue d'Ulm, Paris (Ve), France. Re Dr. Elizabeth S. Russell, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Rho Laboratoire de Recherches de la Societe Rhone- Poulenc, 13 quai Jules Guesde, Vitry sur Seine (Seine), France (Prof. R. Paul). Rij Medical Biological Laboratory, Na- tional Health Research Council TNO, Lange Kleiweg 139, Rijswijk (Z. H.), Netherlands (Dr. D. W. van Bekkum). Rl Drs. W. J. and L. B. Russell, Bio- logy Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, Tenn. Ro Dr. U. Roth, Hamburg 36, Jungiusstr. 6, Zoologisches Institut, Germany. Rr Dr. M. N. Runner, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. S Mount Sinai Medical Research Founda- tion, 2755 West 15th Street, Chicago 8, 111. (Dr. Kurt Stern). Sb Dr. Arthur G. Steinberg, Biological Laboratory, Western Reserve Univer- sity, Cleveland 6, Ohio. Sc Dr. J. P. Scott, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Se Sid Sk Sn Sp Ss St Sv Tn Tu Wa We Wf Wi Ww Wy Prof. Lucio Severi, Istituto di Anatomia e Istologia Patologica dell'Universita de- gli Studi di Perugia, Italy. Stanford University School of Medicine, Department of Anatomy, Stanford, California (Dr. Elizabeth M. Center). Dr. H. E. Skipper, Southern Research Institute, Birmingham 5, Ala. Dr. G. D. Snell, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Dr. William L. Simpson, Detroit Insti- tute of Cancer Research, 4811 John R Street, Detroit 1, Mich. Dr. W. K. Silvers, The Wistar Institute, 36th at Spruce, Philadelphia, Pa. Dr. L. C. Strong, Roswell Park Memo- rial Institute, Biological Station, Spring- ville, N.Y. Dr. L. C. Stevens, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Department of Zoology, University of Toronto, Toronto 5, Canada (Dr. L. Butler). Dr. A. Tannenbaum, Medical Research Institute, Michael Reese Hospital, 29th and Ellis Avenue, Chicago 16, 111. Dr. F. C. Turner, Laboratory for research on the Treatment of Cancer, Box 807, Boulder Creek, Calif. Miss Emelia Vicari, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine (retired). Dr. S. G. Warner (deceased). Dr. J. A. Weir, University of Kansas, Department of Zoology, Lawrence, Kansas. Dr. George L. Wolff, Institute for Cancer Research, Fox Chase, Phila- delphia 1 1, Pa. Dr. J. W. Wilson, Department of Biology, Brown University, Providence 12, Rhode Island. Mrs. E. F. Woodworth, Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. Dr. G. W. Woolley, Sloan-Kettering Institute for Cancer Research, 444 East 68th Street, New York 21, N.Y. II. RATS Except for the previous publication of Billingham and Silvers," this is the first attempt to obtain a comprehensive listing of established inbred strains of rats, inbred strains in development, or stocks with genie markers. Additional information will doubtless be forthcoming with the passage of time. Since the number of established GENETIC STRAINS AND STOCKS 705 inbred strains and strains in development is not yet too great, standardization of nomen- clature and symbols at the present time should avoid the confusion that once existed for mice. Consequently, ( 1 ) the rules recommended by the Committee on Standardized Genetic Nomenclature for Inbred Strains of Mice should be adopted for inbred rat strains, and (2) the symbols used in the following table should be immediately adopted. The international rules of gene and strain nomenclature are discussed in the chapter by Miss Staats. Perhaps the Committee will be asked to extend its jurisdiction to rats. The symbols for strains used in the following listing are based on the rules for mice. Concurrence for the use of these symbols was obtained from most of the contributors maintaining the strains. The descriptions of the strains are similar to those previously used for mice with the exception of identified genetic characteristics which have been included as part of the characteristics, since the genes are unknown except in the genetic stocks maintained by Dr. E. Dempster. This list of contributors at the end of this section is in the same format as the list for mice. The abbrevatiations were checked in order to avoid duplication, and, if a contributor appears on both lists (or all lists), the same abbreviation of the name is used. Table 28 Established strains of rats Name or Symbol Synonym(s) Remarks ACI ACH ALB A990 AXC9935, AXC9935 Irish, Irish AXC9935 Piebald Albany August 990 I, D (brown hooded). inbr: F65. origin: Curtis and Dunning at the Columbia University Institute for Cancer Research, 1926, to Heston, 1945, at F30, to N 1950 at F41; subsequent sublines from either Du or N colonies. charac: life span 21.7 + 0.17 mo., susceptible to estrogen-induced tumors, the N subline exhibits a high incidence of kidney abnormalities (from cystic kidney to kidneys absent, unilaterally or bilaterally), spontaneous Leydig cell tumors (25%) and anterior pituitary tumors (15%) in old animals, a high incidence of uterine abnormalities (horns absent, etc.), some resistance to Bartonella irffection, susceptible to bronchiectasis (40%) and otitis media (60%), will grow trans- plantable tumors M-C961, 969, 970, 972, positive for R-l factor hemagglutinogen B, the Br subline shows low defecation response and high activity response in open-field test of emotional behavior, black agouti, irish. maintained by: Brh, Du, Ko, N, Seg. inbr: F51 + . origin: Curtis and Dunning, 1926, at the Columbia University Institute for Cancer Research, charac: high incidence of spontaneous lymphosarcoma of illeocecal mesentery, will grow transplantable tumors R2788, R2572, IRS6820, B-P839, black agouti, hooded, maintained by: Du. inbr: F2i +. origin: Wolfe and Wright, Albany Medical College, to N 1950; no inbreeding records available prior to the transfer to N; b x s matings since, charac: exhibits some mammary fibro- adenomata, negative hemagglutinogen B, large body size, docile, good reproduction, nonagouti, brown, maintained by: N. inbr: F61. origin: Inbreeding started at Columbia University Institute for Cancer Research by Curtis in 1921. charac: life span 14.03 ± 0.03 mo., resistant to Cysticercus, susceptible to estrogen- 7 Off GENETIC STOCKS AND BREEDING METHODS Table 28 — Continued Name or Symbol Synonym(s) Remarks induced mammary and adrenal tumors; will grow transplantable tumor IRC855, good reproduction; Br subline shows low defecation response and high activity response in open-field test of emotional behavior; black agouti, hooded, maintained by: Brh, Dem, Du. A7322 August 7322 inbr: F64. origin: Curtis, 1925, at the Columbia University Institute for Cancer Research, charac: life span 14.03 + 0.04 mo., spon- taneous mammary tumors frequent, resistant to Cysticercus, will grow transplantable tumors R2857, R2737; positive R-l factor hemag- glutinogen B, agouti, hooded, pink-eye. maintained by: Du. A28807 August 28807 inbr: F37. origin: Curtis and Dunning, 1936 from a half b x s mating of F15 animals of strain A7322. charac: no information submitted, maintained by: Du. A35322 August 35322 inbr: F29. origin: Curtis and Dunning, 1942, from a mutation originating from an aunt x nephew cross in F27 animals of strain A990. charac: vaginal prolapse frequent, will grow transplantable tumor R3280 (bronchiogenic carcinoma), Brh subline shows high defecation response and low activity response in open-field test of emotional behavior, black, nonagouti, hooded, maintained by: Brh, Du. AVO Avon 34968 inbr: F27. origin: Curtis and Dunning, 1941. charac: red-eyed, selfed, poor reproduction, maintained by: Du. B inbr: F69. origin: Dr. P. Swanson from a Wistar stock; to E. Dempster at F43. charac: large body size (weaning weight 68 grams at 28 days), poor maternal instincts, fertile; albino, maintained by: Dem. BDI inbr: F35. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding was started, charac: pink-eyed, yellow, selfed. main- tained by: Dr. BD II inbr: F20. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding was started, charac: albino (carries nonagouti, black, hooded), maintained by: Dr. BD III inbr: F35. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding was started, charac: pink-eyed, yellow, hooded, main- tained by: Dr, Rot. BD IV inbr: F30. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding was started, charac: nonagouti, black, hooded, main- tained by: Dr. BUF Buffalo inbr: F37. origin: Heston, 1946, from Buffalo stock of H. Morris, to N 1950 at F10. charac: low incidence of dental caries; negative R-l factor hemagglutinogen B, will grow Hepatoma 5123 (some enzymes similar to normal liver) and Yoshida ascites sarcoma (16%); spon- taneous tumors of the anterior pituitary (30%) and adrenal cortex (25%) in old animals, susceptible to otitis media (70%), bron- chiectasis (80%), and myocarditis (30%) in old animals, medium repro- duction, albino, maintained by: N, Mor. CAR Hunt's Caries inbr: F38. origin: Hunt, 1937. charac: low incidence of dental Resistant, caries, negative R-l factor hemagglutinogen B, fair reproduction, CA/R albino, maintained by: Hun, N. GENETIC STRAINS AND STOCKS Table 28 — Continued 107 Name or Symbol Synonym(s) Remarks CAS C2331 F344 Hunt's Caries Susceptible, CA/S Copenhagen 2331 Fischer 344, Fischer LEW M520. Lewis Marshall 520, M-520 M14 M17 MR Mi4, L4 Mi7, H7 Maudsley Reactive inbr: F38. origin: Hunt, 1937. charac: high incidence of dental caries, negative R-l factor hemagglutinogen B, poor reproduction, susceptible to respiratory infections, albino, maintained by: Hun, N. inbr: F53. origin: Curtis, at the Columbia University Institute for Cancer Research, 1921. charac: life span 19.08 ±0.18 mo., spontaneous tumors of thymus, resistant to estrogen-induced mammary tumors, susceptible to estrogen-induced bladder tumors, resistant to Cysticercus, will grow (100%) transplantable tumor IRS4337, positive R-l factor hemagglutinogen B, coat color black, agouti, hooded, maintained by: Du. inbr: F75. origin: Curtis at the Columbia University for Cancer Research 1920, to Heston 1949, to N 1950 at F51, subsequent sublines from either the Du or N colonies, charac: life span 12.3 + 0.10 mo., susceptible to Cysticercus, uniform body size (small), susceptible to 2- acetylaminofluorine-induced mammary tumors, will grow transplant- able tumors IRC741, IRS1548, L-C18, IRS9802, R3211, R3230, R3251, R3259, N subline exhibits 15% spontaneous tumor incidence of the anterior pituitary gland, uterus and mammary gland, 30-50% incidence of Leydig cell tumors, granulomatosis lesions of the lymph nodes and spleen, old animals manifest otitis media (40-60%), bronchiectasis (61-70%), nephritis (21-30%), myocarditis (21-30%); negative R-l factor hemagglutinogen B, albino (carries nonagouti, black, hooded), maintained by: Dem, Du, Fu, Gm, Hi, Ko, N, Nl, Rot, Seg, Sy, Ta. inbr: F36. origin: Lewis from Wistar stock, to Aptekman and Bogden, 1954, at F20, to Silvers, 1958, at F31. charac: negative R-l factor hemagglutinogen B; homozygous for red cell antigen C, iso- histogenic, docile, high fertility, will grow transplantable carcinoma # 10, lymphoma # 8; albino, maintained by: Ss, Mai. inbr: F76. origin: Curtis, 1920, at the Columbia University In- stitute for Cancer Research, to Heston, 1949, at F49, to N, 1950 at F51. charac: life span 13.5 + 0.09 mo.; susceptible to Cysticercus; spon- taneous tumors rare in Du subline; N subline exhibits 21-25% incidence of adrenal medulla tumors; susceptible to 2-acetylamino- fluorine induced tumors; medium susceptible to Bartonella; manifests high incidence of otitis media (71-80%), bronchiectasis (60%), nephritis (60%), periarteritis (20%), cecitis (20%), infections of the Harderian gland (100%); will grow Jensen sarcoma, Yoshida ascites sarcoma (80%), hepatoma 7974 (75%), hepatoma 130 (70%); negative R-l factor hemagglutinogen B; albino (carries black, non- agouti, hooded), maintained by: Du, N. inbr: F40 +. origin: A. B. Chapman, 1940, from Sprague-Dawley stock, selection for low ovarian response to pregnant mare serum. charac: low ovarian response to pregnant mare serum; albino. maintained by: Cp. inbr: F40 +. origin: A. B. Chapman, 1940, from Sprague-Dawley stock, selection for high ovarian response to pregnant mare serum. charac: high ovarian response to pregnant mare serum; albino. MAINTAINED BY: Cp. inbr: F18 +. origin: P. L. Broadhurst, 1954, from a commercial Wistar stock; selection for high defecation response, charac: high defecation response in the open-field test of emotional behavior as well as a heightened susceptibility to certain motivating stimuli; albino, maintained by: Brh. 108 GENETIC STOCKS AND BREEDING METHODS Table 28— Continued Name or Symbol Synonym(s) Remarks \1.\R OM PA WN Z61 Maudsley Non-reactive Inbred Osborne Mendel, O-M P.A. King Albino PVC B (black hooded) (PVG/c) PVG/c, P.V.G./c (piebald) R R/A SEL Selfed 36670 WAB Wistar Albino Boots WAG Wistar Albino Glaxo, W.A.G., WAG/C, W.A.G./c A (albino (Wag/G)) WF Wistar/Furth, W/Fu, WR Inbred Wistar Zimmerman 61 inbr: F19 + . origin: P. L. Broadhurst, 1954, from a commercial Wistar stock; selection for low defecation response, charac: low defecation response in the open-field emotional behavior as well as a reduced susceptibility to certain motivating stimuli; albino, main- tained by: Brh. inbr: F30. origin: Heston, 1946, from a noninbred Osborne-Mendel stock obtained from J. White, to N, 1950, at F10. charac: exhibits 70% incidence of adrenal cortex tumors, 21-25% ovarian tumors, 26-30% mammary tumors; susceptible to Bartonella, otitis media (70%), bronchiectasis (80%), myocarditis (30%), positive R-l factor hemagglutinogen B; albino, maintained by: N, Mor. inbr: F162. origin: King, 1909, from Wistar Institute stock, to Aptekman, 1946, at F135, to Bogden, 1958, at F155. charac: positive R-l factor hemagglutinogen B; isohistogenic, vigorous (and vicious), healthy; good reproduction, will grow ascites tumor 9A, leukemia LK2, carcinoma # 5, lymphoma # 6; albino, maintained by: Bg, Nl. inbr: F30 + . origin: Glaxo Laboratories, 1947, from stock received from Virol Ltd. charac: isohistogenic, Br subline shows low defeca- tion response and low activity response in open-field emotional behavior, susceptible to otitis media, black, hooded, maintained by: Brh, Ct, Gor. inbr: F40. origin: O. Miihlbock from a Wistar stock in 1947. charac: albino, maintained by: A, Kl. inbr: F28. origin: W. Dunning, 1948. charac: positive R-l factor hemagglutinogen B, black, nonagouti, selfed. maintained by: Du. inbr: F81 +. origin: From the Glaxo WAG stock in 1926 before it became established as a strain, b x s matings since, charac: albino, no other information available, maintained by: Ad. inbr: F70 +. origin: A. L. Bacharach, 1924, from Wistar Institute stock, charac: susceptible to iron deficiency, Br subline shows high defecation response and high activity response in open-field test of emotional behavior, av. litter size 8.7, albino, maintained by: Bk, Brh, Ct, Gor. inbr: F27 +. origin: J. Furth, 1945, from a commercial Wistar stock, charac: high incidence of mammotropic pituitary tumors and leukemias, tumors 100% transplantable within strains, susceptible to methylcholanthrene-induced mammary tumors, albino, maintained by: Fu. inbr: F34. origin: W. Heston, 1942, from Wistar stock of Nettleship, to N, 1950, at F15. charac: 30-50% incidence of spontaneous mammary tumors, 21-25% incidence of anterior pituitary tumors in old animals, squamous cell metaplasia and hyperplasia of the thyroid manifested, resistant to Bartonella, poor reproduction, negative R-l factor hemagglutinogen B, albino, maintained by: N. inbr: F70. origin: Curtis, 1920, at the Columbia University In- stitute for Cancer Research, charac: life span 11.9 ± 0.17 mo., susceptible to Cysticercus, susceptible to estrogen-induced tumors and 2-acetylaminofluorine tumors, will grow Jensen sarcoma and R92, albino (carries nonagouti, hooded), maintained by: Du. GENETIC STRAINS AND STOCKS 7 05 Table 29 Strains of rats in development Name or Symbol Synonym(s) Remarks BD V BD VI BD VII BD VIII BD IX BD X BN TEC1 TEC2 YOS B.N. c D H hooded HB black mutant ofH NEDH Slonaker NEDH Tecl Tec2 Yoshida 38366 inbr: F10. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: pink-eyed, nonagouti, hooded, main- tained by: Dr. inbr: F5. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: nonagouti, black, selfed. maintained by: Dr. inbr: F5. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: pink-eyed, nonagouti, selfed. main- tained by: Dr. inbr: F?. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: black, agouti, hooded, maintained by: Dr. inbr: F3. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: black, agouti, selfed. maintained by: Dr. inbr: F3. origin: H. Druckrey from animals homozygous for certain hair and eye color genes, b x s mating system from the time inbreeding started, charac: albino (carries nonagouti, pink-eyed, hooded). MAINTAINED BY: Dr. inbr: F9. origin: Silvers and Billingham, 1958, from a brown mutation maintained by H. D. King and P. Aptekman in a pen-bred colony, b x s matings with selection for histocompatibility, charac: histocompatible, segregating for red cell antigens C and D, low fertility, not very docile, non-agouti, brown, maintained by: Ss. inbr: F6. origin: R. Owen, 1958. charac: homozygous for the C blood group allele, albino, maintained by: Ow. inbr: F6. origin: R. Owen, 1958. charac: homozygous for the D blood group allele, nonagouti, irish. maintained by: Ow. inbr: F16. origin: H. Newcombe, 1952 (?), b x s matings with selection for large litters, charac: hooded, maintained by: Ne. inbr: F9. origin: H. Newcombe, 1956 (?), b x s matings with no selection for litter size, charac: black, hooded, maintained by: Ne. inbr: F14. origin: W. E. Knox, 1955, from a Wistar stock, b x s matings with selection for moderate body size, good reproduction, longevity, gentleness, charac: moderate body size, good reproduc- tion, longevity, gentle, albino, maintained by: Kx. inbr: F5. origin: W. G. Downs, Jr., 1958, from a Wistar-Sprague Dawley cross, b x s matings with selection for standardized total and differential white blood cell count, charac: albino, maintained by: Do. inbr: F3. origin: W. G. Downs, Jr., 1959, from a Wistar-Sprague Dawley cross, b x s matings with selection for a standardized response to insulin, charac: albino, maintained by: Do. inbr: F14. origin: W. Dunning, 1953. charac: will grow Yoshida sarcoma, albino, maintained by: Du. no GENETIC STOCKS AND BREEDING METHODS Table 30 Stocks of rats of genetic interest Name or Symbol Synonym(s) M BLACK SELF BLUE DILUTE CHOCO- LATE, CURLY, RED EYED, PINK EYED CHOCO- LATE SILVER COW- LICK- HOODED NOTCH CURLY 2 HAIR- LESS JAUNDICE KINKY RETINI- TIS PIGMEN- TOSA SHAKER WOBBLY YELLOWS Microphthal- mia Black Blue Remarks charac: Microphthalmic or anophthalmia eye to near normal eyes, normal-appearing eyes that are blind due to abberrant pathway of the optic nerve, maintained by: Bw. origin: From Castle's stocks, charac: aaB-C-H-. maintained by: Dem. origin: From Castle's stocks, charac: aa B-C-H-dd. maintained by: Dem. origin: From Castle's stocks, charac: aabb C-Cu-rr or aabbc-Cu-pp. maintained by: Dem. origin: From Castle's stocks, charac: aabbC-se. maintained by: Dem. origin: From Castle's stocks, charac: aaB-C-hnhncwcw. main- tained by: Dem. origin: From Castle's stocks, charac: Cu2-. maintained by: Dem. origin: From Castle's stocks, charac: hrhr. maintained by: Dem. origin: From Gunn's stock via Castle, charac: aaB-hhjj or AaB- hhjj. maintained by: Dem, N. origin: From Castle's stocks, charac: aaB-cckk or aaB-C-kk. maintained by: Dem. origin: Recessive mutation, 1936, in a heterogenous stock maintained by Bemax Laboratories (England), to Dr. D. Campbell, Birmingham Eye Hospital, additional animals added in 1949, gene is maintained by outcrossing and recovering in the backcross, defect ascertained in one eye before breeding, charac: retinitis pigmentosa, stock segregates for various hair colors, maintained by : To. origin: From Castle's stocks, charac: srsr. maintained by: Dem. origin: From Castle's stocks, charac: aaB-hnhnwowo. maintained by: Dem. origin: From Castle's stocks, charac: No information available. maintained by: Dem. List of symbols for designating substrains and stocks of rats A Dr. O. Miihlbock, Antoni van Leewen- hoekhuis, Sarphatistraat 108, Amster- dam C, Netherlands. Ad Dr. S. S. Adams, Boots Pure Drug Co., Ltd., Pharmacology and Physiology Division, Oaksfield Road, Nottingham, England. Bg Dr. Arthur E. Bogden, The Biochemical Research Foundation, Newark, Dela- ware. Bk Dr. D. W. van Bekkum, Radiobiological Laboratory, National Health Research Council T.N.O., Lange Kleiweg 139, Rijswijk (Z.H.), Netherlands. GENETIC STRAINS AND STOCKS 111 Brh Dr. P. L. Broadhurst, Institute of Ko Psychiatry, Animal Psychology Lab- oratory, Bethlem Royal Hospital, Monks Orchard, Beckenham, Kent, Kx England. Bw Dr. L. G. Browman, Montana State University, Missoula, Montana. Mai Cp Dr. A. B. Chapman, Department of Genetics, University of Wisconsin, Madison, Wisconsin. Mor Ct Dr. W. F. J. Cuthbertson, Glaxo Laboratories, Middlesex, England. N Dem Dr. E. Dempster, Department of Genetics, University of California, Berkeley, California. Do Dr. William G. Downs, Tennessee Ne Polytechnic Institute, Cookeville, Ten- nessee. Nl Dr Prof. Dr. Med. Herman Druckrey, Laboratorium D. Chirurg. Univ. Klink., Hugstetterstrasse 55, Freiburg im Breis- gau, Germany. Du Dr. Wilhelmina F. Dunning, Experi- Ow mental Cancer Research Laboratory, University of Miami, Coral Gables, Florida. Rot Fu J. Furth, M.D., Roswell Park Memorial Institute, Buffalo, N.Y. Gm J. P. W. Gilman, D.V.M., Ontario Veterinary College, Guelph, Ontario, Seg Canada. Gor Dr. W. S. Gordon, Director, Agricultural Research Council, Field Station, Com- pton, near Newbury, Berkshire, Eng- Ss land. Hi Dr. Russell Hilf, The Squibb Institute Sy for Medical Research, New Brunswick, New Jersey. Ta Hun Dr. H. R. Hunt, Department of Zoology, Michigan State University, East Lan- To sing, Michigan. Kl Drs. G. and E. Klein, Department of Cell Research, Karolinsha Institute, Stockholm, Sweden. Dr. Henry Kohn, Radiological Labora- tory, University of California Medical Center, San Francisco 22, California. Dr. W. Eugene Knox, Harvard Medical School, Harvard University, Cambridge, Massachusetts. Microbiological Associates, Inc., 4648 Bethesda Avenue, Bethesda 14, Mary- land. Dr. Pablo Mori-Chavez, 779 Sanchez- Carrion, Lima, Peru. Genetics Research Unit (Dr. D. Bailey), Laboratory Aids Branch, National Institutes of Health, Bethesda 14, Md. Dr. Howard Newcombe, Atomic Energy of Canada, Chalk River, Canada. R. L. Noble, M.D., Department of Medical Research, The Collip Medical Research Laboratory, The University of Western Ontario, London, Canada. Dr. Ray D. Owen, Division of Biological Sciences, California Institute of Tech- nology, Pasadena 4, California. Dr. med. W. Rotzsch, Physiologisch- Chemisches, Institut der Universitat Leipzig, Liebigstraat 16, Fernruf 311 14, Leipzig, Germany. Dr. Albert Segaloff, Division of Endo- crinology, Alton Ochsner Medical Foundation, 1520 Jefferson Hwy., New Orleans 21, Louisiana. Dr. W. K. Silvers, The Wistar Institute, Philadelphia 4, Pennsylvania. Dr. K. L. Sydnor, University of Chicago, Chicago, Illinois. Dr. Martha J. Taylor, Pathology Division, Fort Detrick, Maryland. Miss Eva L. Tonks, Research Depart- ment, Birmingham (Dudley Road) Group of Hospitals, Birmingham & Midland Eye Hospital, Church Road, Birmingham 3, Alabama. m. GUINEA PIGS The genetic material for guinea pigs is very limited in this country as well as elsewhere. Apparently only three established inbred strains exist; two of the three are the remaining strains from the thirty-five original ones started by the U.S. Department of Agriculture in 1906 and later developed by Wright,1445- 1447 and the third has been developed by Dr. O. Miihlbock more recently. Several strains are in the process of development, but little is known about them, and, except for the waltzing and silvering stocks, no stocks of genetic significance are maintained. Renewed interest seems to be developing for the use of inbred guinea pigs for immunogenetic work, so the future does hold some promise for the increased use of this species in mammalian genetics. 112 GENETIC STOCKS AND BREEDING METHODS As in the case of the rats, it is suggested that (1) the rules recommended by the Committee on Standardized Genetic Nomenclature for Inbred Strains of Mice be adopted for inbred guinea pig strains, and that (2) the strain symbols herein used be immediately adopted. Also, possibly the jurisdiction of the Committee will be Tabic 31 Established strains of guinea piGsf Name or Symbol Synonym(s) Remarks 13 C/A inbr: F19. origin: O. Miihlbock, 1948. charac: No information submitted, maintained by: A. Family 2, inbr: F26 +• origin: U.S. Department of Agriculture, 1906, to S. Strain 2, Wright, 1915, at Fu; b x s matings for 33 generations (1933), then STR. 2 within strain random breeding until 1940; to Heston, 1940, at which time b x s mating system restored; to N, 1950, at F12; all subsequent sublines from the N colony, charac: fairly resistant to tuberculosis, N subline has median incidence of deciminated calcification occurring in greater curvature of stomach, colon, kidney, striated muscle of the abdominal wall, lung, and aorta of old animals (34 mo.). Medium reproduction, active sexual behavior, small body size, isohistogenic, tricolor (black, red, white), maintained by: Bk, Fn, Ju, N, Ne, Sm, Ss, Wal, Yo. Family 13, inbr: F26 +. origin: U.S. Department of Agriculture, 1906, to S. Strain 13, Wright, 1915, at F13; b x s matings for 33 generations (1933), then STR 13 within strain random breeding until 1940; to Heston, 1940, at which time b x s mating system restored; to N, 1950, at F12; all subsequent sublines from the N colony, charac: less resistant to tuberculosis than Strain 2, medium reproduction, less active sexual behavior than Strain 2, larger body size than Strain 2, isohistogenic, tricolor (black, red, white), maintained by: Bk, Fn, Ju, N, Ne, Sm, Ss, Wal, Yo. f A list of abbreviations follows table 33. Table 32 Strains of guinea pigs in development Name or Symbol Synonym(s) Remarks ICRF OM 3 R 7 R9 ICRF/GP inbr: F12. origin: P. C. Williams, 1954, a heterogeneous (?) stock. charac: albino, some polydactylism. maintained by: Icrf. inbr: F ? origin: J. B. Rogers, 1952, from a random-bred com- mercial stock, charac: no toxemia of pregnancy, no spontaneous tumors, does not reproduce well above 5000 feet, maintained by: Rog. inbr: F ? origin: J. B. Rogers, 1941, from a random-bred stock. charac: toxemia of pregnancy (15% incidence in late pregnancy, during parturition, and 1 week post-partum), no spontaneous tumors, life span 3+ years, maintained by: Rog. inbr: F ? origin: J. B. Rogers, 1941, from random-bred ancestry. charac: 14% spontaneous tumor incidence in animals over 1095 days of age, life span 4+ years, maintained by: Rog. GENETIC STRAINS AND STOCKS 113 extended to include guinea pigs. The symbols for strains used are based on the rules for mice. Since few strains are involved, the changes indicated have been made with- out concurrence from anyone, trusting that the changes will be accepted. The des- criptions of the strains are in the same format as for rats and, like the rats, few, if any, of the actual genetic factors involved are known. The list of contributors at the end of the section conforms to that used for mice, and the abbreviations indicated have been checked against both previous lists to avoid duplication. Table 33 Stocks of guinea pigs of genetic interest Name or Symbol Synonym(s) Remarks Silvering origin: This stock was obtained from S. Wright in 1955. charac: homozygous for silvering (sisi) and segregating for diminished (didi and Didi), C, cd, ca, P, p, e, and ep. maintained by: Re. Waltzing origin: The waltzing manifestation first appeared in 1953 as a probable mutation in a noninbred colony maintained by N. charac: the waltzing condition in typical cases shows a structurally normal hearing apparatus at birth, but shortly thereafter the hair cells of Corti's organ begin to disappear, followed by a gradual atrophy and disappearance of the other cellular elements of the organ; the cochlear neurons atrophy more slowly, with some persisting for more than two years; the trait is dominant, with variable expression, maintained by: Mp, N. List of symbols for designating substrains and stocks of guinea pigs A Antoni van Leeuwenhoekhuis, Am- sterdam C, Sarphatistraat 108, Nether- lands (Dr. O. Miihlbock). Ne Bk Dr. D. W. van Bekkum, Radiobiological Laboratory, National Health Research Council T.N.O., Lange Kleiweg 139, Re Rijswijk (ZH), Netherlands. Fn Dr. Frank Fenner, Department of Microbiology, The Australian National Rog University, Canberra, Australia. Icrf Imperial Cancer Research Fund, Cen- tral Laboratories, Burtonhole Lane, The Sm Ridgeway, Mill Hill, N.W. 7, England. Ju Dr. Clair W. Jungeblut, Department of Ss Microbiology, College of Physicians and Surgeons, Columbia University, Wal New York, New York. Mp Dr. ' Leo Massopust, Physiological Science Department, Southeast Louisi- Yo ana Hospital, Mandeville, Louisiana. N Dr. D. W. Bailey, Genetics Re- search Unit, Laboratory Aids Branch, National Institutes of Health, Bethesda, Maryland. Dr. W. T. Newton, Scfjpol of Medicine, Washington University, St. Louis, Missouri. Dr. Elizabeth S. Russell, R. B. Jackson Memorial Laboratory, Bar Harbor, Maine. Dr. J. B. Rogers, Department of Anatomy, School of Medicine, Univer- sity of Louisville, Louisville, Kentucky. Dr. L. H. Smith, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Dr. Willys Silvers, The Wistar Institute, Philadelphia 4, Pennsylvania. Dr. Roy L. Walford, Department of Pathology, Medical Center, University of California, Los Angeles, California. Dr. W. C. Young, Department of Anatomy, University of Kansas, Law- rence, Kansas. 114 GENETIC STOCKS AND BREEDING METHODS IV. HAMSTERS During the past several years, there has been considerable interest in the use of hamster species in mammalian genetic research. Consequently, several inbred strains have been established, and a number of additional strains have been developed. The Syrian (golden) hamster, Mesocricetus auratus, has been used most, but through the research of Dr. G. Yerganian (see his paper elsewhere in this volume) the Chinese hamster, Cricetulus griseus, has proved to be a desirable animal for research. Information on the location and status of genetic stocks of these two species is included in this section. As recommended for rats and guinea pigs, the adoption for hamsters of (1) the rules recommended by the Committee on Standardized Nomenclature for Inbred Strains of Mice, and (2) the symbols herein used, is urged. Since the basic genetic principles involved in inbreeding hamsters are probably the same as in mice, there is no reason why such definitions and rules cannot be applied equally well. The symbols indicated in the listings conform for the most part to rules for mice. The system of numbers used by Dr. Rae Whitney is not in conformity, but since there has been no opportunity to discuss possible changes with Dr. Whitney, the strains are listed as submitted. Perhaps changes can be made in a revised listing at a later date. Most of the descriptions are abbreviated, probably because not much is known about any of the strains. Future revisions undoubtedly will contain more specific information. The abbreviation of names listed at the end of the section were checked with all the other lists to avoid duplications. Table 34 Established strains of Syrian hamsters f (Mesocricetus auratus) Name or Symbol Synonym(s) Remarks H inbr: F39. origin: O. Muhlbock, 1949, from noninbred stock obtained from Hagedoorn. charac: no information submitted. maintained by: A. HL inbr: F2i. origin: O. Muhlbock, 1955, from noninbred stock obtained from Horning, charac: no information submitted, main- tained by: A. MX MxBU inbr: F32. origin: H. Magalhaes from a noninbred commercial colony, charac: small-medium body size, small litters (4-5 av.), coat color agouti, maintained by: Mg. W WBU inbr: F27. origin: H. Magalhaes from a noninbred commercial colony, charac: small litters (5 + av.), white mottled coat in fe- males only, sex modified lethal (?) trait in males, maintained by: Mg. 5-1 inbr: F23. origin: Department of Biology, Boston University, 1950, from Ingham stock, to R. Whitney at F12, 1956. charac: slow to breed, senile at one year of age, some males show testicular atrophy, some females fail to ovulate, agouti, black eyes, maintained by: Wh. t A list of abbreviations follows table 37. ^c^" GENETIC STRAINS AND STOCKS 115 Table 35 Strains of Syrian hamsters in development (Mesocricetus auratus) Name or Symbol Synonym(s) Remarks CBC CBW LSH MHC MHW CB CB CRB Cream BU, British Cream IGH ICRF/GH IWH ICRF/WH MHA/c MHA MIT MIT(IG) TAB Tawny BU WHB White BU 1.26 1.50 1.97 3.19 3.20 4.1 inbr: F5. origin: W. H. Hildeman, 1958, from a closed (11 years) random-bred colony maintained at the Chester Beatty Institute. charac: isohistogenic, agouti, maintained by: Hi. inbr: F10. origin: W. K. Silvers, 1957, from a closed (11 years) random-bred colony maintained at the Chester Beatty Institute. charac: isohistogenic, agouti, maintained by: Ss. inbr: F10 + . origin: H. Magalhaes from stock received from Cook (England), charac: medium-large body size, agouti, black eyes. maintained by: Mg. inbr: F17. origin: F. C. Chesterman from a heterogenous stock, 1955. charac: agouti, maintained by: Icrf. inbr: F6. origin: F. C. Chesterman from a pair born March, 1959, to a golden hamster containing bilateral orthotropic ovarian grafts from a white hamster, charac: white, maintained by: Icrf. inbr: F9. origin: W. K. Silvers received from the London School of Hygiene, 1957. charac: isohistogenic, agouti, maintained by: Hi, Ss. inbr: F8. origin: W. H. Hildeman, 1958, from a closed random- bred colony maintained at the National Institute for Medical Research, Mill Hill, England, charac: isohistogenic, partial albino (cdcd). MAINTAINED BY: Hi. inbr: F10. origin: W. K. Silvers, 1957, from a closed random-bred colony maintained at the National Institute of Medical Research, Mill Hill, England, charac: isohistogenic, partial albino (cdcd). MAINTAINED BY: Ss. inbr: F4. origin: R. Whitney, 1959, from Ingham stock, charac: susceptible to dental caries on special diet, maintained by: Wh. inbr: F10. origin: H. Magalhaes. charac: medium-large body size; pale agouti, black eyes, maintained by: Mg. inbr: F8. origin: H. Magalhaes. charac: irritable, pink eyes, white with gray ears and pigmented area around tail, maintained by: Mg. inbr: Fu. origin: R. Whitney, 1956, from a Schwentker x LaCasse hybrid, charac: white, black ears, black eyes, maintained by: Wh. inbr: F12. origin: R. Whitney, 1956, from a Schwentker x LaCasse hybrid, charac: white, black ears, black eyes, maintained by: Wh. inbr: F7. origin: R. Whitney, 1958, from Haddow cream x LaCasse white, charac: white and cream, pink eyes, black eyes, black ears. maintained by: Wh. inbr: F14. origin: R. Whitney, 1956, from Schwentker stock. charac: agouti, maintained by: Wh. inbr: F12. origin: R. Whitney from Schwentker stock, 1956. charac: agouti, maintained by: Wh. inbr: F14. origin: R. A. Adams, 1954, from LaCasse noninbred stock, to R. Whitney at F3, 1956. charac: white, black eyes, black ears. maintained by: Wh. 116 GENETIC STOCKS AND BREEDING METHODS Table 35 — Continued Name or Symbol 4.22 4.24 4.39 7.88 7.9 8.9 9.37 X.22 X.3 X.68 Synonym(s) Remarks inbr: F13. origin: R. Whitney from Schwentker stock, 1956. charac: agouti, maintained by: Wh. inbr: Fi2. origin: R. Whitney from Schwentker stock, 1956. charac: agouti, maintained by: Wh. inbr: Fi2. origin: R. Whitney from Schwentker stock, 1956. charac: agouti, maintained by: Wh. inbr: F4. origin: R. Whitney from a Gulf Panda x Haddow Cream hybrid, 1959. charac: white with pink eyes and black ears, or cream (self-spotted) with black eyes and black ears, maintained by: Wh. inbr : Fx + . origin : R. Whitney from Ingham stock, 1 959. charac : "buff," black eyes, pink ears, maintained by: Wh. inbr: F5. origin: R. Whitney, 1959, from a Gulf Panda x Haddow Cream, charac: white with pink eyes and black ears, or cream (self or spotted) with black eyes and black ears, maintained by: Wh. inbr: F10. origin: R. Whitney, 1956, from Schwentker stock. charac: agouti, maintained by: Wh. inbr: F12. origin: R. Whitney, 1956, from Schwentker stock. charac: agouti, maintained by: Wh. inbr: Fi3. origin: R. A. Adams, 1954, from LaCasse stock, to Whitney, 1956, at F3. charac: white, black ears, pink eyes, main- tained by: Wh. inbr: F9. origin: R. Whitney, 1957, from a Schwentker stock x LaCasse stock hybrid, charac: white, black ears, black eyes. maintained by: Wh. Name or Symbol Table 36 Stocks of Syrian Hamsters of Genetic Interest (Mesocricetus auratus) Synonym(s) Remarks White spotted charac: urogenital abnormalities (missing kidneys, uteri, seminal Panda piebald vesicles) — 10% incidence, maintained by: Mg. Table 37 Strains of Chinese Hamsters in Development {Cricetulus griseus) Name or Symbol Synonym (s) Remarks BUY inbr: F1S. origin: G. Yerganian from Schwentker stock, charac: fair-good reproduction, 15% incidence of diabetes mellitus, average mature body weight 33-35 g., fairly docile, exhibits brittle bristle, a sex- linked trait that causes hair follicles to fall and break off at 5-6 months of age. maintained by: Ye. GENETIC STRAINS AND STOCKS Table 37 — Continued 117 Name or Symbol Synonym (s) Remarks HGYA HGYB JBY JBYA JBYB JFY ORY VSY inbr: F6. origin: G. Yerganian from a BUY x VSY hybrid; VSY males for this cross were irradiated with 400—500 r localized testicular X irradiation prior to mating, charac: animals are extremely nervous, diabetes mellitus appears spontaneously, maintained by: Ye. inbr: F6. origin: G. Yerganian from a VSY x BUY hybrid; male BUY animals for this cross were irradiated with 400-500 r localized testicular X irradiation prior to mating, charac: animals are extremely nervous, diabetes mellitus appears spontaneously, main- tained by: Ye. inbr: F9. origin: G. Yerganian from a JFY x BUY hybrid. charac: mature body weight large (45-50 g.), past generations (F6_9) manifested 90% incidence of diabetes mellitus, no evidence in current generations, maintained by: Ye. inbr:F3. origin: G. Yerganian from BUY x JFY hybrids, charac: no information available, maintained by: Ye. inbr:F3. origin: G. Yerganian from JFY x BUY hybrids, charac: no information available, maintained by: Ye. inbr: F13. origin: G. Yerganian from BUY strain, charac: female dominance over male evident, males pugnacious toward each other, mature body weight large (45-50 g.), polyuresis or diabetes insipidis still observed, occasional absence of outer ear in both sexes. maintained by: Ye. inbr: Fji. origin: G. Yerganian from BUY strain, charac: mature body weight approximately 45 g., medium reproduction mildly susceptible to SE polyoma virus in newborn (tumors of various types appear 7-36 months after inoculation), some diabetes mellitus exhibited in earlier generations, maintained by: Ye. inbr: F13. origin: G. Yerganian from BUY strain, charac: late sexual maturity, average body weight 33 g., diabetes mellitus in earlier generations (10% at F8_10). maintained by: Ye. List of symbols for designating substrains and stocks of hamsters A Dr. O. Muhlbock, Antoni van Leewen- hoekhuis, Sarphatistraat 108, Amster- dam C, Netherlands. Hi Dr. W. H. Hildeman, University of California Medical Center, Los Angeles, California. Icrf Imperial Cancer Research Fund, Cen- tral Laboratories, Burtonhale Lane, The Ridgeway, Mill Hill, N. W. 7, England. Mg Dr. Hulda Magalhaes, Department of Ss Wh Biology, Bucknell University, Lewis- burg, Pennsylvania. Dr. W. K. Silvers, The Wistar Institute, Philadelphia 4, Pennsylvania. Dr. Rae Whitney, Bio-Research Con- sultants, Inc., 9 Commercial Avenue, Cambridge 41, Massachusetts. Dr. George Yerganian, Laboratories of Cytogenetics, Harvard University, School of Medicine, Boston, Massa- chusetts. V. RABBITS A formal listing of genetic stocks of rabbits has not been made before, and conse- quently the information received in this survey is of considerable interest. At least two fairly well inbred strains exist in this country, and several others are well on the 118 GENETIC STOCKS AND BREEDING METHODS way to becoming established strains. The stocks bearing marker genes maintained by Dr. C. Cohen are of considerable value for certain immunogenetic work, and it is likely that more populations of this kind will be developed in the future. There is no doubt that a vast reservoir of genetic material exists in the many stocks maintained by fanciers, but unfortunately they are not readily identifiable at this time. It would be most desirable if genetic material for this species, comparable to the murine material, could be collected in one or more places and properly maintained for mammalian genetic research. The standardization of strains and stocks of rabbits has not been done in a manner similar to mice, but the adoption of the same rules for designating and defining strains and substrains and the promulgation of symbols is desirable. However, it may be advisable to amend the rules so that inbred strains can be defined in terms of an inbreeding coefficient (F) in addition to the definition based on matings of brother x Table 38f Strains of Rabbits in Development The amount of inbreeding is expressed either as F generations of brother x sister matings or by the inbreeding coefficient (F). Name or Symbol Synonym(s) Remarks A inbr: F19. origin: M. Lurie, 1932, from a heterogeneous stock obtained from E. L. Stubbs, University of Michigan, b x s matings since the start of inbreeding, charac: resistance to tuberculosis variable (highly resistant up to F8), low fertility, many die of impaction of the large intestine (perhaps faulty peristalsis?), maintained by: Lu. AC inbr: F = 36%. origin: P. Sawin from Rockefeller Institute Dutch stock, charac: black, recessive white marking, aa or A-EdEddu-, occasionally segregating for y and ac, mature female body weight 2200 g. maintained by: Sa. ACEP inbr: F = 70%. origin: P. Sawin from Rockefeller Institute Dutch stock, charac: aa or A-EdEddu-epep (black, recessive white marking, audiogenic seizures), occasionally segregating for v and agonadia, mature female body weight 2400 g. maintained by : Sa. AD inbr: F8. origin: M. Lurie from a Strain A x Strain D cross (Strain D is now extinct), charac: intermediate resistance to tuberculosis, medium fertility, dutch color, maintained by: Lu. AX inbr: F — 36%. origin: P. Sawin from outcross of chinchilla Race V to Races III and X. charac: Achdww, Axas forced heterozygosis, occasionally segregating for du, ep, bu, chm, c, mature female body weight 3500 g. maintained by: Sa. B inbr: F10. origin: C. Cohen from Rockefeller Institute Dutch stock, separated from Strain Y and Strain R at F2, to C. K. Chai at F7. charac: segregating for yellow fat y, and angora (1), albino, mature body weight 2000 g. maintained by: Ci. f A list of abbreviations follows table 39. GENETIC STRAINS AND STOCKS 119 Table 38 — Continued Name or Symbol Synonym(s) Remarks inbr: F18. origin: M. Lurie, 1932, from Swift stock (originally received from Bull and Webster), b x s matings. charac: highly susceptible to tuberculosis, many adenocarcinoma of the uterus, low fertility, nervous, albino, maintained by: Lu. inbr: F8. origin: M. Lurie from an F12 Strain C x Ff Carworth animal, b x s matings since, charac: susceptible to tuberculosis and snuffles, fair fertility, nervous, dutch with occasional albino. maintained by: Lu. CAC DA OS R TA TTC T3F Os III A III III C IIPF III III C III R Race III, T inbr: F = 53%. origin: P. Sawin from a New Zealand white stock (California), 1949. charac: albino (ce A-Ed), mature female body weight 4200 g. maintained by: Sa. inbr: F = 53%. origin: P. Sawin, 1948, from Rockefeller Institute stock, charac: aa or EdEdOsos (black, minimal recessive white marking), occasionally segregating for hydrocephaly, mature female body weight 3200 g. maintained by: Sa. inbr: Fn. origin: C. Cohen from Rockefeller Institute Dutch stock, separated from Strain Y and Strain B at F2, to C. K. Chai at F7. charac: segregating for yellow fat, y, and angora (1), albino, mature body weight 2000 g. maintained by: Ci. inbr: F6. origin: M. Lurie from an F5 Strain III x F14 Strain A, b x s matings since then, charac: medium resistance to tuberculosis, resistant to snuffles, fertile, albino, maintained by: Lu. inbr: F6. origin: M. Lurie from a Strain III x Strain C cross, backcrossed to Strain III, b x s matings since then, charac: inter- mediate resistance to tuberculosis, resistant to snuffles, fair fertility, young animals susceptible to infantile diarrhea, albino, main- tained by: Lu. inbr: F6. origin: M. Lurie from a Strain III x Swift stock cross, backcrossed twice to Strain III, b x s matings since then, charac: high resistance to tuberculosis, dutch agouti with occasional albino. maintained by: Lu. inbr: Fn. origin: C. Cohen from Rockefeller Institute Dutch stock, to C. K. Chai at F7. charac: segregating for yellow fat, y, angora (1), albino, mature body weight approximately 2000 g. maintained by: Ci. inbr: F = 70%. origin: P. Sawin from Castle's New Zealand white stock, 1932, probably all subsequent sublines of this strain have come from this colony, charac : ccAAEdEd (albino) , occasionally segregating for angora, bu, ep, and scoliosis; Lu subline is now F9, highly resistant to tuberculosis and snuffies, docile, maintained by: Lu, Sa. inbr: F = 53%. origin: P. Sawin from Strain III. charac: aaAAEdEd (albino), occasionally segregating for bu, scolosis. main- tained by: Sa. inbr: F9. origin: M. Lurie from Strain III animals that survived a virulent bovine tuberculosis infection, charac: similar to Strain III, resistant to tuberculosis and snuffles, docile, albino, maintained by: Lu. inbr:F=53%. origin: P. Sawin from Castle's small race, charac: aaeebbC{CH2), sooty yellow, occasionally segregating for r2, dk,^ Dw, scoliosis, As, mature female body size 2200 g. MAINTAINED BY: Sa. I '20 GENETIC STOCKS AND BREEDING METHODS sister or parent x offspring, since less stringent systems of mating sometimes have been expedient. Thus, in the case of rabbits, the inbreeding can be expressed either as the number of generations of brother x sister or parent x offspring matings or in terms of the inbreeding coefficient (F) . The listings are prepared in this manner. As for the other species already mentioned, it is recommended that the Committee on Stan- dardized Nomenclature extend its coverage to include the rabbits. Some changes have been made in symbols (according to the rules for mice), and concurrence was obtained from the contributor for the acceptance of such changes. The abbreviations in the contributor list, as in the case of previous lists, were checked to avoid duplication. Table 39 Stocks of rabbits of genetic interest Name or Symbol Synonym(s) Remarks BLOOD GROUP GENES STOCKS origin: C. Cohen, by selection of identified blood group genes. charac: One stock is segregating for HgA, the other four stocks are segregating for HgD, HgF, He, he, He, Hh, hh. maintained by: Cn. List of symbols for designating strains and stocks of rabbits. Lu Ci Dr. C. K. Chai, R. B.Jackson Memorial Laboratory, Bar Harbor, Maine. Cn Dr. C. Cohen, Battelle Memorial Institute, Columbus, Ohio. Dr. Max Lurie, The Henry Phipps Institute, Philadelphia, Pennsylvania. Sa Dr. Paul B. Sawin, R. B. Jackson Memorial Laboratory, Bar Harbor, Maine. VI. PEROMYSCUS SP. Peromyscus sp., as a research animal in mammalian genetics and in medicine, has not been widely used, despite the number of identified genetic traits and the possible importance of some of these traits. For this reason, the locations of Peromyscus sp. stocks are few. It is believed, however, that additional stocks do exist in the United States, but applicable information had not been submitted by the time this list was closed. It is hoped that future revisions will include such stocks. The stocks herein listed are given as submitted by the contributors. Since there apparently are no inbred strains as such, but only stocks with identified genes, no changes were made in stock designation. The descriptions given are very brieff but sufficient to illustrate the kind of material available. Because no inbred strains exist, and, therefore, no problems have yet been created with respect to strain definition, strain symbols, and so forth, it is probably not necessary to establish a standardized system at this time. However, it is likely that such strains t A more detailed description of these genes can be obtained by writing to Dr. Elizabeth Barto, Mammalian Genetics Center, University of Michigan, Ann Arbor, Michigan. GENETIC STRAINS AND STOCKS 121 will eventually be developed, so these problems may occur some day. If the Committee on Standardized Nomenclature does promulgate rules for rats, guinea pigs, rabbits, and hamsters, possibly they will also consider the potential problem of Peromyscus sp. Table 40 Mutant stocks of Peromyscus maniculatus Name or Symbol Synonym(s) Remarks Albino Blaze * Boggier Brown Brown-tip CNV Con- vulsive Dilute EP (Sound induced) Convulsive Flexed Tail Gray Hairless Ivory Pink Eyed Dilution Platinum* Rotator* Gray-bond origin: Wild population, California, genet: c. charac: linkage group I, apparently identical to albino phenotype of the laboratory mouse, maintained by: Bar, Mc. No information available on origin, genetics, or characteristics. maintained by: Bar. origin: No information submitted, genet: bg. charac: no informa- tion submitted, maintained by: Bar. origin: Wild population, New Mexico, genet: b. charac: linkage group IV, very similar to the cinnamon phenotype of the laboratory mouse, sepia pigment granules brown, reduced in size, yellow pigment granules not visibly affected, maintained by: Bar, Mc. No information submitted on the origin, genetics, or characteristics. maintained by: Bar. origin: Wild population, New Mexico, genet: d. charac: linkage group IV, similar in appearance to dilute or leaden genes in the laboratory mouse, variable expression, maintained by: Bar, Mc. origin: No information submitted, genet: e. charac: no informa- tion submitted, maintained by: Bar. origin: Wild population, Oregon, genet: /. charac: linkage group I, caudal vertebrae variously malformed, tail usually visibly kinky, knobby at the end, or shortened, highly variable, no evidence of associated belly spotting or anemia, maintained by: Bar, Mc. origin: Wild population, New Mexico, gene frequency high in certain localities, genet: g. charac: superficially resembles chinchilla in the laboratory mouse, maintained by: Bar, Mc. origin: Wild population, California, genet: hr. charac: linkage group III, very similar to hairless in the laboratory mouse, moulting pattern irregular, fertility of females reduced, maintained by: Bar, Mc. origin: Wild population, Oregon, genet: i. charac: similar to albino, but hair retains a moderate amount of sepia pigment in juvenile pelage, slight amount in adults, maintained by: Bar, Mc. origin: Laboratory stock, genet: p. charac: linkage group I, apparently identical to pink eyed dilution phenotype of the laboratory mouse, dilution and modification of the shape of sepia pigment granules, maintained by: Bar, Mc. origin: Laboratory stock, genet: pt. charac: similar to dilute and silver, but almost always with high-grade expression, hair bases vary from gray to almost white, maintained by: Bar, Mc. No information available on origin, genotype, or characteristics. maintained by: Bar. 122 GENETIC STOCKS AND BREEDING .METHODS Table 40— Continued Name or Symbol Synonym(s) Remarks Silver Spinner Spotting Dominant spot THF (Sound- induced) Convul- sive* Whiteside Wide Band Waltzing Waltzer Yellow origin: Wild population, Oregon, genet: si. charac: linkage group I, very similar to dilute, equally variable in expression, main- tained by: Bar, Mc. origin: Wild population of P. polionotus rhoodsi, Florida, now trans- ferred to P. maniculatus background, genet: sp. charac: whirling behavior and early deafness, whirling expression variable but almost always detectable, maintained by: Bar, Mc. origin: Wild population, Illinois, genet: S. charac: high variable amount of white spotting on head, belly and distal portion of tail, homozygotes variable, probably not distinguishable from hetero- zygotes, some reduction in penetrance, maintained by: Bar, Mc. No information submitted on origin, genetics, and characteristics. maintained by: Bar. origin: No information submitted, genet: wh. charac: No in- formation submitted, maintained by: Bar. origin: Wild population, Nebraska, of P. maniculatus nebrassensis, gene frequency probably high in the Sand Hills region, genet: Nb. charac: linkage group V, longer agouti band and somewhat shorter sepia tip on hair, maintained by: Bar, Mc. origin: Wild population of P. maniculatus hairdi, Iowa, genet: v. charac : linkage group V, similar to Spinner, but deafness not occurring or appearing late in life, maintained by: Bar, Mc. origin: Wild population of P. maniculatus gambeli, California, genet: y. charac: linkage group II, longer agouti band, sepia tip of hair much shortened or occasionally absent, white tip of ventral hair lengthened, nonagouti hairs usually absent, monatricks usually with agouti band, maintained by: Bar, Mc. * Tentative designations; not yet described in publications. Table 41 Other stocks of Peromyscus sp. of possible genetic interest Name or Symbol Synonym (s) Remarks BW/RX BW/RA PO origin : Wild population of Peromyscus maniculatus hairdi, from Washte- naw County, Michigan, charac: no known mutant genes, wild-type stock, all mutant stocks have been crossed into this stock at least twice, maintained by avoiding full and half sib matings. maintained by: Mc. inbr: F3. origin: W. Mcintosh from BW/RX, continuing attempts to inbreed b x s have not produced progeny beyond F5, current attempt now at F3. charac: wild type, maintained by: Mc. origin : Wild population of Peromyscus polionotus from Ocala, Florida. charac: no known mutant genes although an unidentified whirling trait appears occasionally, small size, will cross reciprocally with Peromyscus maniculatus producing fertile hybrids, maintained by: Mc. GENETIC STRAINS AND STOCKS 123 List of symbols for designating stocks of Peromyscus i Barto, Mammalian Gene- Mc Dr. W. B. Mclnto; University of Michigan, Zoology and Entor Ann Arbor, Michigan. University, Columbus, Ohio. Bar Dr. Elizabeth Barto, Mammalian Gene- Mc Dr. W. B. Mcintosh, Department of tics Center, University of Michigan, Zoology and Entomoloy, Ohio State SUMMARY The material on genetic strains and stocks compiled and presented represents an attempt to bring together as much information as possible on the location and status of such strains and stocks. It is hoped that this compilation, although incomplete, will be of value in supplying information on the location and status of laboratory rodent material of medical genetic interest. The six common species of laboratory rodents (mice, rats, guinea pigs, hamsters, rabbits, Peromyscus sp.) are listed. Other species will doubtless be added in the future as their usefulness increases. Recommendations have been made regarding the adoption of rules for defining strains and substrains, for the designation of symbols, and for the extension of the juris- diction of the Committee on Standardized Genetic Nomenclature for Mice to include rats, guinea pigs, hamsters, rabbits, and Peromyscus sp. RADIATION GENETICS Douglas Grahn, Ph.D. MAMMALIAN RADIATION GENETICS The best qualification of the mouse for studies on radiation genetics is that it has certain attributes of our best experimental means to the end, Drosophila, and the experimental end itself, man. Although it may be many years before murine genetics will have the esoteric qualities of Drosophila genetics, the mouse offers us the opportunity of obtaining quantitatively reliable data on radiation effects that can be checked against Drosophila for theoretical consistency and extrapolated to man without zoo- logical inconsistency. Other mammalian species have been and are presently being employed, such as the rat, the hamster, swine, sheep, bovines, and even the monkey. But, for reasons of economy and general genetic and biological background, the mouse will certainly continue to reign supreme for some time in the field of mammalian radiation genetics. Prior to World War II, radiation genetics progressed in an orderly fashion as a relatively subsidiary area of interest in the field of genetics. With the advent of the atomic age and the potentiality of widespread contamination of the biosphere with the by-products of nuclear energy applications, radiation genetics rapidly became a major field of research activity and interest. Fortunately, many different sources of radiation have become available for critical experimental purposes, but as part of the price of technical advancement, many sources of radiation have also become an ubiquitious part of our general environment. The quantitative and realistic evalua- tion of the potential cost of nuclear energy to man's genetic worth has thus become highly important. Those familiar with modern radiation biology are well aware of the complexities of this cross-bred science. In many instances, the combined talents of diverse bio- logical and physical scientists are required. The study of radiation effects is not often 127 128 RADIATION GENETICS suitable for isolated investigators. Anyone anticipating research activity in the field of mammalian radiation genetics is well advised to enlist the consulting services of a radiological physicist and a radiobiologist. RADIATION PARAMETERS As intimated above, there is a wide array of radiation sources available for experi- mental use; and the source or sources should be carefully chosen for their general applicability and, in some studies, for their use as a simple means of inducing injury. The methodology of exposure is also of extreme importance. This involves the correct selection of total doses, dose rates, and other temporal factors that will be discussed below. Before enumerating radiation parameters, it should be stated that accurate and uniform measurement of the absorbed dose is essential. The techniques and instrumentation of modern radiation dosimetry are sometimes quite specialized and the experimental biologist should not hesitate to ask for outside assistance. The best bio- logical measures are of little value when the physical parameters are uncertain or inaccurate. Considerable insight into the questions of dosimetry and radiation sources may be obtained by referring to Hine and Brownell,582 Glasstone,438 Fano,346 and Marinelli and Tavlor.850 SOURCES OF RADIATION A' and gamma radiations. — These need little description as they have been the principal sources of radiation for most genetic studies. A wide range of energy levels is available for both radiations, but, for mammalian studies, it is advisable to avoid energies below 1 20 kev in order to avoid the problem of nonuniformity of absorbed dose.476 When energies appreciably above 500 kev are employed, the specific ionization or linear energy transfer per unit length of the ionization track declines ; and a factor of relative biological effectiveness, the RBE, may be needed to provide comparative analysis with data obtained from more conventional deep therapy X-ray units of 200- 300 kev. The most economical gamma-ray sources available are in the higher energy class: cesium-137 and cobalt-60 with photon energies of 0.66 Mev and 1.25 Mev, respectively. Neutrons. — Neutron energies vary from 0.025 ev for thermal neutrons to over 20 Mev for fast neutrons. Linear accelerators can provide reasonably monochromatic energies, whereas the neutron-energy spectrum from the fissioning of uranium-235 is extremely heterogeneous and has an average energy of about 2.5 Mev. Details of the spectrum can be obtained in Lapp and Andrews.758 The RBE for neutrons is extremely variable and for most biological effects reaches a maximum value at energies of 2-4 Mev. The RBE itself may vary with the biological endpoint and all too little is known MAMMALIAN RADIATION GENETICS 129 about this factor for induction of mutation in mammalian systems, although consider- able work has been done with Tradescantia and Drosophila.222- 317 More accurate knowledge of the nonlinear relation between RBE and energy is critical, since the maximum RBE is in the region of the mean energy for fission neutrons. Unfortunately, neutron sources are generally expensive and require careful monitoring; thus, they are not widely available. On the other hand, the increasing number of training reactors on college campuses may improve neutron source avail- ability for genetic studies. A complete discussion of neutron physics and neutron sources is given in Glasstone438 and Lapp and Andrews.758 Cosmic radiation. — As man prepares to enter the new and rather exciting scientific era of space flight, considerable increase in interest in the estimation of the genetic effects of cosmic radiation can be expected. There are two radiation belts, the Van Allen belts, held in the earth's magnetic field. The outer belt is composed of com- paratively low-energy electrons, predominantly in the 20 to 100 kev range, which can be easily shielded out with aluminum. However, the production of soft secondary X rays may still present some difficulties. The inner belt, which comes to within 600- 700 miles of the earth's surface, contains extremely high-energy protons. The energy spectrum is not fully known, but ranges up to at least 700 Mev. Present data suggest that the dose rate can reach 10 r/hour at altitudes of about 1,600 miles and 10,000 miles for the inner and outer belts. In addition to this trapped radiation, there are the primary cosmic particles. These are composed of about 85 per cent protons, 15 per cent helium atoms, and less than 1 per cent heavy nuclei such as carbon, calcium, iron, and oxygen. The cosmic primaries go into the billion-electron-volt energy range and can penetrate deep into tissue with a very dense ionization track. The iron nucleus, for example, has a maxi- mum ionization density of 100,000 ion pairs per micron and can produce a dose to an individual cell of over 1,000 rep. A comprehensive recent review of space radiation has been presented by Schaefer.1161 Certainly, very few humans will be subjected to these radiations, but their ability to induce severe damage all along the ionizing pathway, their unknown RBE values, and our inability to duplicate the very high-energy heavy nuclei with earthbound machines present an intriguing set of problems. Some progress is being made with high-energy linear-accelerator beams of stripped nuclei and deuterons at the Lawrence Radiation Laboratory, University of California, and at Brookhaven National Labora- tory.201' 234 Internal emitters or internally deposited radioisotopes. — The vast majority of radio- nuclides present no unique genetic problem. Those nuclides with an affinity to bone, for example, are of little genetic concern. Gamma-emitting isotopes that seek the soft tissues, such as cesium- 137, can be treated as any typical external radiation source. The more difficult problems arise from those isotopes that can become incorporated into the genetic materials. Tritium, carbon- 14, and phosphorous-32 are good ex- amples and all emit a beta particle without an associated gamma-photon emission. ISO RADIATION GENETICS Thus, their radiations are highly localized, often to the cell in which they are deposited. Very little mammalian genetic work has been done with these, but certain tech- niques appear to be accurate enough to warrant more study. For example, death of spermatogonial cells has been quantitatively measured following the simple intra- peritoneal injection of tritiated thymidine into mice.664 The results compare excellently with those obtained by external gamma-radiation,952 and offer a straight- forward estimate of comparative toxicity. The importance of carbon- 14 to the problem of long-term genetic hazards to man and animals from past nuclear weapons testing has been emphasized by Totter et a/.1326 and Pauling,993 yet only empiric estimates are available on the relative contribution of the three potentially injurious consequences of radioactive decay of C14 to N14: trans- mutation, atomic recoil, and ionization. Another isotope of considerable interest is deuterium. Though not a radioactive isotope of hydrogen, it is DaO or heavy water that is commonly used as a moderator and coolant in nuclear reactors. Its introduction into biological systems presents many intriguing problems that have been reviewed by Katz et al.689 and Bennett et al.78 Nearly complete sterility can be induced in mice when they are provided a 30 per cent D20 concentration in the drinking water. Lesser amounts of D20 induce partial sterility from which recovery will occur. Since the size of litters remains un- affected, there is no evidence of genetic damage, but deuterium may well be mutagenic when incorporated into the genetic materials. While the above summary of radiation sources is by no means all-inclusive, the brief descriptions do indicate something of the type and energy of radiations that can be employed and a few of the problems that exist. TEMPORAL FACTORS IN RADIATION When one begins to consider the number of permutations and combinations of radiation exposure that can be employed, it soon becomes apparent that it is probably impossible to account for all possibilities experimentally and even operationally important. To start with, the radiation dose can be delivered in the form of a single exposure (often referred to as an acute exposure, which should not be the preferred terminology) , multiple exposures or a fractionated exposure, or a continuous exposure (often called a chronic exposure, again not the preferred terminology). Within any one of these, there can be variation in total dose and rate of dose delivered per unit time, or dose rate. Obviously, for continuous exposures, dose rate and total dose are positively related, although termination of the exposure can be varied to hold total dose constant under different dose rates. This, then, varies the duration of exposure or protraction period. Fractionated exposures may be even more complex. One can vary the number of doses or fractions, the total dose, the dose rate, the interval between doses, the size of the individual fractions, and the total protraction period. Again, a little considera- MAMMALIAN RADIATION GENETICS 131 tion of these factors points out a number of interdependent relationships which can at times result in difficulties when attempts are made to isolate the effect of some one particular factor. Variations in the pattern of exposure have been employed for years by geneticists to assist in understanding the kinetics of chromosomal breakage and restitution. The simple use of paired doses usually has been the method of choice. The effects of a given total dose, delivered in a single exposure for determination of base-line, is com- pared to the effects following the same total dose delivered in two equal parts with a varying interval between halves. The value of this procedure for cytogenetic studies, which have largely been done with plant material and Drosophila, needs no documenta- tion here, but apparently the paired-dose method has not been employed for mammalian genetic studies. Weekly and daily fractionations of dose have been used, however, in a number of studies on the induction of sterility.328, 1085, 1087, 1114 Rather than extend the discussion of exposure pattern, reference will be made to certain applications of these variables at pertinent places in the discussion of genetic tests. The following summary of exposure variables is therefore given. I. Single exposure; with variation in: a. Total dose b. Dose rate II. Fractionated exposure; with variation in: a. Total dose b. Size of individual dose or fraction c. Number of individual fractions d. Dose rate/fraction , e. Interval between fractions f. Protraction period or interval between first and last fractions III. Continuous exposure; with variation in: a. Total dose b. Dose rate c. Protraction period Of course, these factors are fully applicable to the use of external radiations. When internal emitters are employed, some modification is required because of the lack of discreteness to the exposure period. The duration of exposure will depend upon a combination of the radioactive half-life, metabolic activity, and rate of excretion. Continuous exposure to internal emitters at a constant dose rate can be accomplished by the proper adjustment between input of dose increment and decay and excretion rates. TECHNIQUES OF EXPOSURE Although the irradiation of an animal initially may seem a simple, straightforward procedure requiring little more than the placement of the creature under an X-ray 132 RADIATION GENETICS tube and turning the machine on and off, contemporary standards require recognition of a few principles of radiological physics and of problems of variation in machine output. It is standard practice to place the animals on a rotating board during X irradiation. This will randomize variations in the dose field due to slight misalign- ment of the tungsten target of the tube and to unequal absorption of some of the X rays by the target itself. The board, usually a one-half-inch slab of masonite, serves as a back-scattering device to help assure the attainment of electron equilibrium and the elimination of variations in tissue depth dose. Exposure of mice and rats from a single plane, either dorsal or ventral, will usually give a uniform tissue dose, provided that X rays of 120 kev or greater are employed with filtration adequate to remove the lowest energy components of the total spectrum. When photon energies of 1 Mev and above are used (Co60 is an example), the animals should be in a chamber-like device that provides both forward and backward scattering of the secondary electrons. The forward scattering is necessary since high- energy gamma and X rays must penetrate about 3 or 4 millimeters of tissue or tissue- equivalent material to reach secondary electron equilibrium. In the absence of such scattering material, the tissue dose will build up in the animal and lead to an irregular depth-dose curve. Four millimeters of lucite are sufficient to insure equilibrium for the 1 .25 Mev, Co60 gamma photon. When animals the size of guinea pigs, rabbits, monkeys, dogs, and larger are irradiated, it generally becomes necessary to use a bilateral exposure technique. The total dose is delivered in two equal parts, one part to each lateral surface. This prevents sharp changes in depth dosage and accompanying inequalities of exposure of internal organs. Unilateral irradiation of female dogs, for example, could lead to some discrepancy in dose delivered to the two ovaries. Detailed discussions of the pattern of depth dose for different energy radiations in mice, rats, and rabbits are given by Grahn et a/.476 and for the domestic animals and man by Bond et a/.115 A common procedure for genetic studies involves the use of partial body exposure. When doses above the midlethal level are required, local irradiation of the gonads must be done. Simple lead hemispheres can be devised for small mammals which permit full exposure of the gonads with only a limited exposure of surrounding tissue. For obvious reasons, the procedure is simplest for males. Sheet lead one-eighth inch thick provides excellent shielding for 250 kev radiations and below. Partial body exposure with high-energy X and gamma radiation and neutrons cannot be accom- plished through the use of shields alone. Beam collimation is required, although this is not always feasible with all radiation sources. Thus, for some situations, the total dose will be controlled by the survival of the animal rather than by technical manipulations. RADIATION GENETIC ANALYSIS IN MAMMALS Apparently, the first effort to study the genetic effects of radiation in mice was reported by Little and Bagg in 1923 and 1924.801 Although several new mutations MAMMALIAN RADIATION GENETICS 133 were detected, a radiation origin could not be substantiated. Snell 1250 and Hertwig551 did report positive evidence for the induction of gene mutations in mice by X irradiation several years after Muller had reported this for Drosophila. All of the early literature has been completely and excellently reviewed by Griineberg507 and Russell,1128 and anyone considering entering the field of mammalian radiation genetics would be well advised to review the pioneering efforts of G. D. Snell, P. Hertwig, H. Brennecke, and their co-workers. INDUCED STERILITY Prior to any consideration of genetic analysis, the problem of radiation-induced sterility must be faced. Interest in this field traces back to the early part of the cen- tury743 and considerable activity continues to the present. It is, of course, a problem of practical concern to the radiation geneticist, since certain experimental conditions may be precluded by the induction of either temporary or permanent sterility. A certain amount of trial-and-error methodology is still required for many species, since only the male mouse has been studied in fairly complete detail.328, 952- 953, 955, 957 The work of Oakberg can be considered a model for those who wish to study the sterilizing effects of ionizing radiation. He has pointed out the need for careful timing of post- irradiation intervals for sampling, exact identification of cellular type and maturation stage, and the need for correction for architectural distortion due to shrinkage of tubules. Spermatogonial cells of the mouse are extremely sensitive to radiation and have an ld50 dose of 20 to 24 roentgens. An increase in cellular death is even detected at single doses as low as 5 rad of gamma rays or 2 rad of fast neutrons.1142 Johnson and Cronkite 664 used Oakberg's techniques in a recent study to evaluate the effect of tritiated thymidine on spermatogonial cells. Since exact dose rate and total dose from the tritium are not known, a comparison with data from external radiation permits use of the curve for cell-killing as a bioassay. A dose of 1 [xr/gram of body weight, for example, after 60 hours of exposure produced an effect equivalent to something less than 5 r of external, Co60, gamma radiation. The reproductive performance of irradiated males will vary from species to species with respect to dose sensitivity, duration of reduced fertility or complete sterility, and time of recovery to near normal fertility. As a general rule, there are three distinct periods: the pre-sterile period that immediately follows exposure, the sterile period, and the post-sterile period. During the pre-sterile period, fecundity gradually declines as post-meiotic germ cells are cleared through. This period is used for the study of dominant-lethal induction rates, translocation rates, and mutation rates in spermato- cytes, spermatids, and spermatozoa. The post-sterile-period matings are used to study mutations induced in spermatogonial cells. Thus, the stage of the cell at the time of exposure can be quite accurately defined for genetic analysis. The sterilizing effects of radiation of the female have not been as thoroughly investigated as in the male, but there is a tremendous species factor in radiosensitivity. V 134 RADIATION GENETICS The mature female mouse is permanently sterilized by a single dose of 50 r or greater.1087- 1147 The newborn female, however, is extremely resistant and will show normal fertility even after a single dose of 300 r.1126- At several weeks of age, however, a period of extreme sensitivity occurs so that even a low dose rate such as continuous exposure to about 8 milliroentgens per minute for a total dose of85rwill cause complete sterility. This is in contrast to the lesser sterilizing effects of a pro- tracted exposure on the mature female in which doses up to nearly 300 r of X rays may be accumulated at the rate of 10 r/week before complete sterility occurs.1085- 1087- 1114 Reproduction studies have very clearly indicated that no oogonia are present in the mature ovary and that the bulk of the cells are primary oocytes.956 These are most radiosensitive in early stages of development of the follicle. The more mature stages go through to ovulation but are not replaced after a sterilizing exposure. Females of other mammalian species are not as radiosensitive as the mouse to the sterilizing effect of radiation. This phenomenon is not clearly understood and ap- parently no working hypothesis has been set forth. Rats and rabbits are only tem- porarily sterilized by doses above 600 r.743 The female beagle hound that survives an acute radiation syndrome induced by a near midlethal dose of 300 r whole-body X irradiation shows excellent reproductive performance in terms of litter size and estrus activity, with even a tendency to improved lactation.22- 1201 Exposure to fast neutrons is more effective than X irradiation for inducing a form of sterility in dogs, but the data are not adequate enough to determine if an unusually high RBE might be involved.22 In this instance, the dogs bred but were unable to whelp or lactate. The above discussion of the sterilizing effects of ionizing radiation was not in- tended to be complete and a fuller discussion will be found in a report by Oakberg.954 Additional studies have been reported for dogs, rats, and mice, following single, fractionated, and continuous exposure to X rays, gamma rays, and neutrons.179- 846- 847. 937. ii94 Tn summary, the degree and time of sterility varies with species, sex, dose rate, age at exposure, and quality of radiation. However, the pattern of reproduc- tive performance following exposure is employed to control the cellular stage of interest for mutation studies and therefore must be understood by the investigator before genetic analysis can be carried out. QUALITATIVE GENETIC EFFECTS OF RADIATION Hereditary partial sterility. — Some of the offspring of irradiated males were observed to produce consistently small litters by Snell 1250 in one of the early radiation genetic studies in mice. Litter size was reduced by about one-half, and one-half of the progeny of these litters expressed the trait in the next generation. The characteristic, which behaves as a dominant trait, is generally classed as an hereditary dominant partial sterility and has been regularly observed in all studies to date with mice.198, 550, 731- 1132 Snell1232 demonstrated by the use of marker genes that the characteristic definitely involves a reciprocal translocation. The original interpretation was derived from the MAMMALIAN RADIATION GENETICS 135 genetic behavior of the trait, but cytogenetic proof has also been obtained.39, 731 Embryonic deaths appear to occur largely at or soon after implantation.973 Partial sterility resulting from induced reciprocal translocation is almost entirely restricted to the mature-germ-cell stages, possibly only significantly in spermatozoa. It has been suggested that aberrations of this type would not survive meiosis and there- fore the yield in gonial cells would be negligible. This has been challenged by Griffen497 but his data await complete cytologic confirmation. The simple use of reduced litter size as the detector can be misleading, although it is the normal procedure of first screening for semisterile animals. The frequency in offspring from irradiated females is much less than in offspring from irradiated males.1127 Since the germ cells of the mature ovary are largely primary oocytes, this finding indirectly implies that the most mature germ cell is more sensitive to the induction of a transmissible partial sterility ; germ-cell death probably culls out this damage in the less mature cells. The dose-response data are erratic.1128 Theoretically, one would expect the yield of semisterile mice to be linearly related to the square of the dose since a two-hit aberration is involved. The early work, summarized in the above reference, does not appear to demonstrate any clear-cut dose-response relationship. The data obtained by Charles et al.198 do, however, fit a (dose)2 function with a greater reduction of the variance than the simple linear-arithmetic function employed by the authors. In the experiment of Charles, male mice were exposed to four different daily dose levels of X rays but at a constant dose rate. Thus, the physical factors were appropriate for the use of the D2 function. Matings were continually carried out so that the total dose levels were average values and not carefully separated points. This, along with the fact that there was no sure way of knowing what irradiated cell stage produced the trait, detracts from any quantitative test."}" Dominant lethals. — The induction of dominant lethals in male germ cells can be detected by measuring the reduction in litter size produced by the irradiated sire, although litter size at birth is not usually considered the most accurate measure. A careful evaluation requires sacrifice of the pregnant female at about 15-17 days post- conception and the counting of (a) number of live embryos, (b) number of dead embryos, (c) number of corpora lutea, and (d) number of pre-implantation deaths. Most of the losses occur prior to 10-11 days of gestation and pre-implantation losses are closely correlated with dose.1141 Post-implantation losses rise to about a 20-25 per cent representation of the number of corpora lutea at doses below 100 r and remains at that level. Cytologic studies have demonstrated that the lethal action is pre- dominantly due to aberrant cleavage and chromosomal fragmentation.128 f The Charles experiment, carried out at the University of Rochester, was nevertheless the first major concerted effort to evaluate the genetic hazards of radiation in a mammal and was set up under the auspices of the Manhattan Engineering District, predecessor of the Atomic Energy Commission. Preparation of a final report was delayed by the untimely death of Dr. Charles, but the report is now being published as a University of Rochester- Atomic Energy Commission Project Report No. UR-565 and should soon become available to interested geneticists. 136 RADIATION GENETICS The frequency of dominant lethals varies with the stage of the cell at the time of irradiation. Mature sperm cells are less sensitive than spermatids but are more sensitive than spermatocytes.57' 58 The cell-stage factor is relatively easy to control, since it now appears that mice do not store mature sperm and the stage at irradiation can be ascertained by control of the time interval between mating and irradiation.57 Bateman's report includes a timetable relating post-exposure week with cellular stage. For matings carried out in the first week after exposure, the dose for inducing 50 per cent dominant lethals is approximately 700 r for 250 kvp X rays, about 100 r for fast neutrons from a nuclear detonation (average energy ~ 2 Mev), about 100 r for 1 Mev cyclotron fast neutrons, and around 300 r for 14 Mev neutrons produced by a Cockcroft-Walton accelerator.1140- 1142 In all cases the data for survival of embryos fit a simple exponential equation. Bateman57- 59 has made the most recent and thorough theoretical study of the induction of dominant lethals and the relationship between postulated number of chromosomal breaks and time of death of the conceptus. On the basis of his analysis, the dose dependence of dominant lethals proved to be linear for neutron-induced lethals but nonlinear for X-ray damage. This is consistent with the expectation that neutrons will produce more than one hit per ionization track. In a series of papers, the Russells have reported on the induction of dominant lethals in female mice.1123, 1124, 1125 The use of litter size alone is not valid in this sex, since there is a period between 1 and 14 days postexposure when irradiated mice show an excess in ovulation rate. As for studies with males, the pregnant female is sacrificed late in gestation and the number of corpora lutea and living embryos are counted. The ratio of living embryos to corpora lutea is considered an accurate measure of the incidence of dominant lethality. Cellular stage at irradiation is very critical for oocytes and can be ascertained by the interval between exposure and fertilization. A dose of 400 r will induce more than 98 per cent dominant lethality in oocytes in first meiotic metaphase, which occurs 8 to 10 hours prior to fertilization. If the interval between irradiation and fertilization is doubled (16 hours), frequency of dominant lethals drops to less than 20 per cent. For X rays, the dose inducing 50 per cent dominant lethals is about 700 r for primary oocytes, but only about 70 r for the cells in meiotic metaphase.1120 Gene mutations. — The greatest interest and concern has been expressed on the following question: what is the radiation-induced mutation rate in a representative laboratory mammal; how does it compare with Drosophila and how can it be applied to man ? For reasons previously noted, the mouse has been the mammal of choice for this critical area of research in radiation genetics. The problem can be approached in several ways. The search can be for a total, gametic mutation rate or it can be restricted to specific selected loci. The search can be for dominant visibles, recessive lethals, or recessive visibles. To date, the most successful procedure has been the specific-locus test for the induction of recessive visibles and any associated viability effects, although efforts to make separate estimates of the rates of dominant visible and recessive lethal mutations have also been made. MAMMALIAN RADIATION GENETICS 137 The specific-locus test procedure is a standard genetic test system that has been employed in Drosophila genetics for many years. A wild-type mouse is irradiated and mated to an animal from a multiple recessive tester stock. The immediate progeny are then screened for the appearance of a mutation at any of the loci marked in the parent from the tester stock. Since these Fa mice are all heterozygous for the markers, a new mutation at any of the loci should appear in this first generation. Mutants with intermediate degrees of expression can also be detected with some degree of precision. Subsequent tests, of course, can check for such homozygous effects as reduced fertility, lethality, reductions in growth rate, and so forth. The specific-locus procedure is limited by the number of mutants that can be carried in one stock without seriously reducing viability and reproductive performance. Another problem is that of overlapping phenotypes ; the array of mutants must be discrete and separable in their expression. The test stock used in both U.S. and British genetics programs using mice at the Oak Ridge National Laboratory (Dr. W. L. Russell and colleagues) and the Atomic Energy Research Establishment, Harwell, England (Dr. A. G. Searle and colleagues, previously Dr. T. C. Carter), respectively, is the seven-locus stock containing the recessive genes: a (nonagouti), b {brown), cch {chinchilla), d {dilute), p {pink-eye), s {piebald), and se {short-ear). This stock was synthesized by Dr. W. L. Russell at Oak Ridge specifically for studies of mutation rate.1134 The bulk of information on mutation rates in mammals has been developed from this tester stock, an obvious limitation to current knowledge. It can only be assumed that these seven loci are fully representative of mutability and viability of all genes. A certain degree of doubt is raised by the fact that there is a greater than thirtyfold difference in the spermatogonial mutation rates for several of the loci.1138 Alternative methods for detecting the mutation rate for recessive visible genes require three generations of breeding rather than only one for the above procedure. These methods screen the whole genome and require the segregation of the new mutant in homozygous form in the third generation. One method, the backcross method, involves the outcrossing of a son of an irradiated parent and then backcrossing one of his daughters to himself. If the first-generation son carried a mutant, there would be a probability of 0.5 for its transmission to the second-generation daughter. If she carried the mutant, then the third-generation, backcross progeny would have a one-in-four chance of segregating the new mutant in homozygous form. Altogether, the chance of segregation in the third generation is only one in eight, which is not very efficient. The method was employed in the early work of Hertwig549 and also by Carter and Phillips.178 The second method for screening the entire genome requires three generations of full-sibling matings. This procedure is only half as efficient as the backcross method and has been used, apparently only briefly, by Carter and Phillips.175 Aside from the inherent disadvantages of these two methods of gametic analysis with respect to experimental economy, they will not succeed in detecting mutations l^ 138 RADIATION GENETICS that are recessive lethals. Since about three-fourths of the induced gonial mutants reported by Russell are recessive lethals, the yield in the backcross and sibmating procedures may be further reduced by another factor of four. With the exception of the fact that there is no restriction of the number of loci under test, little can be said for these latter methods of genetic analysis. In any of these methods, the mutation rate for either spermatogonial cells or post-gonial cells is isolated by analysis of matings only in poststerile or presterile periods, respectively. Only the rate for oocytes can be obtained for females. Recessive lethal mutations. — Of the three methods described above, only the specific- locus test system will permit an estimate of the frequency of recessive lethal mutations. Russell and Russell1138 report that about 75 per cent of the induced mutations at the seven loci are recessive lethals. This proportion varies among loci, according to present reports, from about 50 to 100 per cent of the number of observed mutants. The locus with the highest gonial mutation rate, the piebald locus, also happens to have produced only lethal mutations. These data pertain to those mutants derived from the single dose tests with the dose delivered at high intensity. Whether or not the same ratio of lethal to viable mutants will occur among those induced by low-intensity continuous exposure remains to be seen. Presumably, there should be no qualitative difference between mutations induced at different rates of radiation dosage, even though the mutation rates them- selves may vary (see below). However, the recovery process, acting upon premuta- tional damage to reduce the mutation rate under continuous exposure as compared to single-dose exposure, could conceivably act in a selective manner at the molecular level. On the assumption that variation in genie action is associated with variation in the molecular structure of the gene, it is then conceivable that some forms of genetic damage may be more amenable to spontaneous recovery. Whether or not lethal mutations are selectively acted against cannot be stated, but this would seem to be a potentially important point to have clarified in a mammalian test system. An additional method that has been employed to determine the recessive lethal mutation rate for autosomal genes can be designated as the linked-lethal procedure. In its simplest form, a single recessive marker gene is carried homozygously in the irradiated parent; the parent is outcrossed and the heterozygous progeny are inbred to produce the normal 3 : 1 ratio in the F2. The absence of the marker in the segregating generation is accepted as prima facie evidence of the induction of a lethal closely linked to the marker. The linked-lethal procedure was tried by Snell,1250 but without success. His test entailed the screening for aberrant segregation ratios in F3 progeny produced by backcrossing an F2 mouse heterozygous for a marker with its Fx parent. An obvious deficiency of the method is in the limited length of the chromosome under test. Crossing over will naturally occur with greater frequency as the map distance between the marker and the lethal increases. Minor aberrations in the segregation ratio would require extensive testing for proof of presence of a lethal mutant, which could go far beyond its economic value in terms of information yield. MAMMALIAN RADIATION GENETICS 139 The linked-lethal method can be increased in its efficiency through the use of a number of independent marker genes.517 The statistical complications of the method have been worked out by Haldane. These include the question of allowance for chance fluctuations in the F2 segregation ratio and the length of the chromosome scanned on either side of the marker gene. The total swept length will naturally increase with the number of markers employed and with the number of F2 progeny raised. The method requires three generations; the first is from crossing the homozygous marker stock inter se, one parent of which has been irradiated. The second generation is an outcross of the marker stock which may now carry a lethal in the heterozygous form to a wild strain. The use of what might appear to be an extra generation of breeding, that is, the carrying of the homozygous marker stock one generation beyond the irradiated generation, eliminates sibmating the progeny of the irradiated parents and the accompanying chance of introducing a lethal from both parents independently induced. In other words, the second generation progeny trace back to only a single irradiated gamete in the original parents. The third generation is produced by sibmating the second generation progeny and significant deviations from the expected 3 : 1 ratios are sought. Carter158 tested the system of Haldane with a stock carrying seven recessive, visible genes. These are the same as those noted earlier with the exception that pink-eye (p) had been replaced by waved-l (wa-\). The results suggested that the dose required to induce one autosomal recessive lethal per gamete in spermatogonia is probably no less than about 800 r of X rays delivered as a single dose. Carter con- cluded that the method is far less efficient than the specific-locus method for the detection of lethal mutations. An elaboration of the linked lethal procedure has also been described by Carter.170 This involves the use of linked marker genes and the detection of lethals located be- tween the markers. On a theoretical basis, Carter could not conclude that it would be more efficient than the procedure employing independent markers. Apparently, the method has not been subject to experimental test. Haldane, in an appendix to Carter's paper, noted that the use of linked markers could be reasonably efficient as a means of detecting sublethal recessive mutants, at least for sublethals with a viability between about 5 and 50 per cent. Further exploration of these techniques in the laboratory would appear desirable. Still another procedure available for the detection of recessive lethals has been described and tested by Carter.165 This test uses the reduction in litter size produced by the sons of irradiated mice, when these sons are crossed to their daughters. Thus, this is an application of the backcross procedure previously described that can also be used for detection of visible mutations. The probability of homozygous expression of a recessive lethal in the backcross progeny is again one in eight, which now is detected as a one-eighth reduction in litter size in comparison to the control. The regression of litter size on radiation dose was employed for the analysis and led to the conclusion that a dose of about 300 r produced one recessive lethal per gamete in postmeiotic male 740 RADIATION GENETICS germ cells. The exposure in this experiment was protracted over a 5-week period at rates of 1.64, 8.0, and 33.3 r per week. No exposures of single dose with high dose rate were reported, so no comparison can be made for possible effects of dose rate. This procedure would seem sufficiently straightforward to warrant the development of data for analysis of effects of dose rate on the induction of recessive lethals. Definitive study of the mutation rate for sex-linked lethals still remains to be done. One attempt was made in Charles' experiment, with equivocal results. The breeding procedure is simple. Irradiated males are outcrossed, and their daughters, which may now carry a sex-linked recessive lethal as a heterozygote, are mated to unrelated males. If a lethal segregates, the litters will show a 2: 1 sex ratio. Due to the normal fluctua- tions in sex ratios, the test system is inefficient. In recent years, several sex-linked genes have become recorded in the mouse. Recombination percentages vary from 4 to 16 among the known loci,492 and this is close enough to permit the suggestion that Carter's technique of detecting lethals between linked markers would be worth exploring. Even the use of a single marker, such as tortoise (To), which is dominant for its effect on coat color and recessive for its lethal effect, would provide some useful data. If an induced lethal is closely linked to tortoise, test matings would have few or no male offspring, thus simplifying the test system. Dominant visible mutations. — Data on the induction of dominant visible mutations are exceedingly unsatisfactory and for practical purposes may be considered as virtually nonexistent. This is not the fault of those investigators who have made valiant attempts to detect these mutations. The problem is that there is no simple quantitative method available for their detection. Basically, all that can be done is to screen the offspring of irradiated parents for detectable anomalies and mate these animals to determine the heritability of the trait. A total gametic rate is therefore observed. Since there is almost no limit to the range of dominant morphologic and physiologic variants that can be measured, it is difficult to determine what the true mutation rate is. The single greatest effort yet reported was made by Charles et a/.198 The male offspring of irradiated male parents were sacrificed, dissected, and inspected for morphologic variants. Obviously, no heritability tests could follow. The female off- spring were mated to unrelated stock; thus externally visible mutants could be studied for transmissibility. One hundred and twenty possibilities were tested, and fourteen proved to be bona fide mutations. The data provided an estimated mutation rate of 5.4 x 10 "6 per roentgen per gamete. These mutations were observed among the offspring of males subject to daily X irradiations that were permitted to mate con- tinuously during the course of the experiment. The mutation rate thus cannot be applied to any single type of germ cell but probably includes contributions from all stages of gametogenesis. Russell1134 reported the detection of five X-ray-induced, dominant, visible mutants affecting the coat color, the ears, and the tail. These were the only morphologic traits screened for dominant changes in the course of early tests on the induction of MAMMALIAN RADIATION GENETICS 141 recessive visibles. Although no mutation rate could be calculated, Russell noted that 3 of the 5 new mutants affected a trait that was also controlled by one of the specific recessive loci in the main test. Since 32 recessive visibles were uncovered in the same group of animals, he therefore concluded that the mutation rate to dominant visibles is probably significantly lower per roentgen than the rate for recessive visibles. Recessive visible mutations. — The determination of the mutation rate for recessive visibles induced by irradiation at specific loci in mice has been one of the major research efforts in radiation genetics by the Atomic Energy Commission for the past decade. The results of this effort, along with the many ancillary findings, now constitute a major contribution to our knowledge of genetics. This subject has been the topic of so many discussions that the reader should properly address himself to the original reports. The studies do provide some interesting sidelights on the problems of methodology, however. The basic procedures for the specific-locus test (described above) enjoy the economy of requiring only one generation of breeding to bring the progeny to test. The spermatogonial mutation rate induced by single doses of X rays delivered at a rate of90r per minute is approximately 25 x 10_8/r/genefor total doses up to 600 r.1140, 1142 At 1,000 r, the rate falls to about one-half the above figure, which is interpreted to be due to selection against a more radiosensitive class of spermatogonia.1111, 1131 The most significant finding, and the one which has received worldwide attention, concerns the effect of the dose rate of radiation on the observed mutation rate. If the exposure is delivered, essentially continuously, at rates of either 10 r/week or 90 r/week (from 1 to 10 milliroentgens/minute), the mutation rate falls to about one-fourth that observed following the single dose exposure.1140, 1142 The difference is significant and has also been confirmed for the female.1126, 1139, 1143 The effect had also been noted by Carter,164 but, unfortunately, his data and experimental conditions could not clearly substantiate the interpretation that an effect of dose rate existed, although it was suggested. He was forced to compare the mutation rate following single dose exposure in males with the rate induced by continuous exposure in females without the benefit of the reciprocal comparison. The interesting side effect of this finding in mice was that it produced a great deal of concern and some criticism among geneticists, all of whom had learned as a basic principle that the mutagenic effect of radiation always acted additively, regardless of the manner in which the radiation was delivered. The original data that suggested an effect of dose rate did contain a few uncertainties to warrant some of the criticism. The protracted exposures were provided by the radioisotope cesium- 137, which emits a 0.7 Mev gamma ray. The ionization density is less than that for 250 kev X rays, and an RBE of 0.8 or 0.9 might be required to make the physical parameters constant. The standard errors of the initial data were quite large and point-by-point significance testing gave no real assurance of a difference between high- and low-intensity exposure except at the level of the 600 r dose. It should be recalled, however, that the original concept of dose-rate independence was derived from data obtained on irradiated Drosophila sperm. Analogous data derived from the exposure of mature germ cells 742 RADIATION GENETICS of the mouse do not challenge the basic Drosophila data. The dose-rate effect is definitely limited to spermatogonia and oocytes.1140, 1142 It should also be noted that a significant dose-rate effect has now been demonstrated for Drosophila oogonia exposed to Co60 gamma radiation.972 The flies were exposed to a total dose of 4,000 r over either a period of two weeks or in 31 seconds. The percentage of sex-linked lethals was 1.3 + 0.5 and 3.4 + 0.7 for the continuous and single-dose exposures, respectively. The difference is significant. Studies on microorganisms indicate that some portion of the induced genetic damage falls into a category now labelled as premutational damage. A recovery process, requiring active protein synthesis, acts to prevent the fixation of part of the premutational damage, thus reducing the mutational yield.514, 708 The reduced mutation rate in mouse gonia and oocytes following low intensity irradiation is inter- preted to be the result of a process of metabolic recovery, which may itself be quite radiosensitive and therefore fail under high-intensity irradiation. The study of mutation at the molecular level has therefore become of increased importance, and new insight into the mechanisms of radiation protection may also result. Important unanswered questions now concern the effects of fractionated ex- posures in all their complexities of interminable variables. Is there a limiting low dose rate, above which the mutation rate jumps to the higher level? Will there be all gradations between the high and the low ? What will be the effect of intermittent exposure at high intensities but to very small doses? If the answer to this previous question is that the low mutation rate prevails, will it do so only under specific condi- tions of dose rate, dose per fraction, and fractionation interval ? These questions are important for both industrial and medical situations where there is often no regular pattern of exposure. In addition, the dose-rate effect requires careful evaluation with exposure to the densely ionizing radiations, such as alpha particles and fast neutrons. Because of their greater efficiency in inducing genetic and general cellular damage, is it possible that exposure to neutrons, for example, may produce more concurrent injury to the pre- sumed recovery mechanism and thus a lesser reduction in mutation rate with declining dose rate ? The neutron may be of particular value in these genetic studies because of its known peculiar behavior with regard to somatic lethal effects. Let me refer to two studies as an example. Sproul,1262 using the traditional paired-dose technique to estimate recovery from acute radiation injury, found that the rate of recovery is essen- tially the same following initial exposure of the whole body to a single dose at high intensity of either Co60 gamma rays or 14 Mev neutrons. On the other hand, Upton et a/.1336 have indicated that the RBE for shortening of life induced by neutrons pro- duced by a Po-Be source (~4 Mev) progressively increases as the dose rate declines. The data suggest that the long-term, lethal effects of neutrons are considerably less dependent on dose rates than are those following Co60 gamma irradiation. It is entirely conceivable that genie mutational damage induced by fast neutrons may also be less dependent on dose rates. MAMMALIAN RADIATION GENETICS 143 Cytogenetic analysis. — Two subsequent chapters by Drs. Klein and Yerganian amply cover the methodologies of cytology and cytogenetics. The application of tissue-culture techniques to genetic analysis, radiation and otherwise, is also covered by Drs. Ford and Puck in the previous volume in this series, Methodology in Human Genetics. The use of mammalian cells in tissue culture for radiation-genetic analysis will assuredly be an area of increasing scientific effort. The opportunity has become available to make elaborate comparative quantitative analyses of radiogenetic sensitivi- ties among all important domestic and laboratory animal species. Besides interspecific comparisons, strain differences in radiosensitivity within species can be explored at the cellular level. To date, the recognized genetic differences in radiosensitivity in mice have been attributed to physiologic genetic factors, rather than to basic differences in the resistance or sensitivity to cellular genetic damage.473, 474, 475 This point can easily be checked in tissue culture. Strains may differ in their sensitivity to mutagens, at least for chromosomal aberration and restitution rates. If so, new techniques would be provided to study the cellular mechanisms of somatic injury and recovery. QUANTITATIVE GENETIC EFFECTS OF RADIATION Although geneticists have been estimating spontaneous and induced mutation rates in a number of species of plants and animals for many years, virtually all of our present estimates of the potential genetic hazards of radiation to man have been made independently of this accumulated experience. There are several reasons for this. First, while fairly good data for spontaneous mutation rate are available for man, the radiation-induced rate is a complete unknown. Second, the bulk of data on detri- mental genetic factors in man are in the form of morbidity and mortality statistics. Although many clear-cut mutant phenotypes that severely affect viability are recog- nized, their frequencies are generally masked by the normal rates of infant and child- hood mortality in human populations. Thus, more concern is expressed by geneticists about the total mortality than about the individual syndromes of disease con- stituting it. An excellent summary of several hundred detrimental genetic qualities in man has been prepared from experience in Northern Ireland.1282 Mortality rates at birth and among adults are given, although the temporal patterns of mortality for the various characteristics have not been derived. In addition, the genetic basis for many traits is known to be uncertain and to involve considerable environmental interaction. At present, best approximations of the normal load of detrimental genetic factors in man have been derived from the analysis of the progeny of consanguineous marriages. The analytic technique requires the maximum likelihood estimate of the regression of early mortality on the inbreeding coefficient.901 The procedure has also been em- ployed by Schull1169 and Slatis1216 with considerable success. The basic biological data in nearly all instances include some or all of the following : fetal deaths, neonatal and infant deaths, childhood and juvenile deaths. Such data are comparatively easy 144 RADIATION GENETICS to obtain with a high degree of accuracy among most Western nations. Unfortunately, comparable data for laboratory animal populations are not generally available, even though many of the laboratory animals enjoy a standard of living far superior to that of the civilization that maintains them. The importance to be placed upon these data is emphasized by the fact that the most widely accepted and quoted baseline predictions of the radiation hazard to man are those provided by Crow,231 involving an expansion of the analysis of Morton, Crow, and Muller, referred to above. These calculations, although ingenious, require the mixed application of parameters from Drosophila, mice, and men. As they stand, they are in need of updating and revision, but, basically, the calculations require careful evaluation for populations of laboratory mammals. In addition, the genetic analysis of the progeny of the survivors in Hiroshima and Nagasaki was largely restricted to data on quantitative anthropometry, morbidity, and mortality.939 In general, the basic techniques of breeding employed for the detection of qualita- tive changes can be used for the study of the more subtle quantitative expressions of damage. In many instances, the two types of data can be obtained concurrently. The analytic procedures are more complex, however, and thoughtful statistical design and analysis are required. The key to success is no longer a matter of overcoming the issue of events of low probability by sheer weight of numbers, but rather overcoming the problems of ordinary random fluctuations. Sex ratio. — Prior to a discussion of the evaluation of mortality statistics, brief mention should be made of studies of sex ratio. This is a semiquantitative trait that does not require any specialized approach. This parameter has become a con- troversial issue since Schull and Neel1170 reported a significant shift in sex ratio among the progeny of the survivors at Hiroshima and Nagasaki. Shifts in sex ratio have not been uniformly seen among the progeny of irradiated mice.1128 A very recent report729 also indicates that the sex ratio of mice remains substantially unaltered following single and fractionated exposure of the male parent. An earlier preliminary report by Kalmus et al.676 did indicate a deficiency of female offspring from irradiated male mice for litters sired within 40 days of the exposure. The shift in sex ratio was in the direc- tion of genetic expectation, but this study has apparently not been confirmed. It can be suggested that man may be more sensitive to the induction of sex-linked lethals by radiation. Since the X chromosome of man carries more recognizable detrimental genes than have been noted in the mouse, man may have a qualitatively different genetic potential for induced genetic damage. Viability. — The general term viability is chosen to cover the full range of mortality periods: stillbirths, neonatal deaths, infant or preweaning deaths, and adult mortality. Data on these traits are scattered and incomplete. Russell,1128 in his review of the early literature, has indicated that an increase in stillbirth rate and mortality to the time of sexual maturity has been observed among the progeny of irradiated male mice for litters sired in both the pre- and poststerile periods. Similar observations have been made with guinea pigs.1289 MAMMALIAN RADIATION GENETICS 145 More recently, additional attempts have been made to detect an over-all effect on viability. Boche et a/.113 reported on the life expectancy of the progeny of irradiated male and female mice, as measured from the time of weaning. Their controls con- sisted of litters sired prior to irradiation and the experimentals were produced at three intervals of time after irradiation: 10 days, 120 days, and 180 days. The study was done in two replications; the first indicated a shorter life expectancy among the experi- mentals, the second was negative. The use of controls of a different parity than any of the experimental groups is not to be recommended. Litter size and maternal nutritional factors will be confounded with any induced genetic effects of the radiation. Russell1133 reported a significant shortening of life among progeny of irradiated males for mice conceived during the presterile period. The offspring had their mean life expectancy from weaning reduced by about 60 days per 100 rep exposure of the male parent. This is generally considered to be a maximum effect, since the parents were subject to a prompt neutron exposure from a nuclear weapon detonation and the progeny were conceived from cells in postgonial stages at irradiation. The mutation rate for these stages is twice that for spermatogonia and the n:x RBE may lie any- where between 2 and 6. Russell and Russell1137 have indicated that follow-up studies employing X irradiation of spermatogonia confirm the earlier report on neutron effects but the expression is less readily detected. Preweaning mortality in mice has been measured as a reduction of litter size at three weeks of age.1137 Litters produced from germ cells exposed in the spermatogonial stage show a 3 to 4 per cent reduction from control values following a 300 r, X-ray exposure. No information on the time of death of the animals during the three-week period was given. Total infant mortality, including stillbirths, has recently been studied in rats with the suggestion that some part of the mortality is genetically caused rather than a random function of maternal and litter-size factors.824 The technique consisted of a comparison of control and experimental litters of similar size for differences in postnatal mortality, rather than a comparison of litter sizes themselves, since progeny were produced dui^ig the presterile period and the litter sizes reflected dominant lethal effects occurring prenatally. Of the studies reported to date, none has really obtained the data required to provide an accurate and quantitative assessment of the full expression of viability mutations. It is imperative that studies be done with the view of encompassing the whole life table; populations must be studied from birth through death as a unit experiment. It is especially important that careful attention be given to the period of neonatal and infant mortality because of the significance of such data for comparison to man. It is further suggested that the analysis of data for viability be done by standard methods of actuarial statistics. These are amply described by Pearl.995 Surprisingly, complete life tables have apparently never been obtained for populations of laboratory mammals. The author is presently endeavoring to accumulate a sufficient number of mouse days of experience to generate the tables for about a half-dozen standard 146 RADIATION GENETICS inbred strains and hybrids. These are: A/He, A/ Tax, BALB/c, C3Hf/He, C57BL/6, and Fi and F2 hybrids from the BALB/c x C57BL/6 cross. Rather large numbers of mouse days of experience are required for adequate analysis of the young adulthood portion of the life table. For example, the BALB/c x C57BL/6 Fx hybrid at weaning age has a death rate approaching a low of 1 in 10,000 per day. Prior to this time and beyond 150 to 200 days of age for most mice, the daily rates of death are high enough to permit reasonable data to be derived from samples of only 100 to 200 mice when 50- to 100-day intervals are used. The greatest advantage of using life tables lies in their more direct comparability to human experience. The full form of the life table is remarkably similar for most mammalian species. This similarity has been employed to extrapolate the life-shortening effects of radiation from mice to man.333- 1144 All analyses to date have concerned only adult populations, however. Figure 19 shows a comparison of murine and human populations in the neonatal, infant, and childhood periods. The U.S. population data were obtained from the published vital statistics of the United States available from the National Office of Vital Statistics, U.S. Public Health Service. The data for mice are some of those of the writer's. The F2 was derived from the reciprocal crosses of the BALB/c and C57BL/6 inbred strains, and the referred inbred in figure 19 is the BALB/c parental line. Although these data are for the combined sexes, routinely sex is determined at birth and litters are checked daily for deaths. For the sake of ease in final recording and coding of IBM punch cards, the data are assembled in the intervals : 0-5 days, 6-15 days, and 16 days to weaning, which provides a mean of approximately 30 days of age. In time, the data will be analyzed by sex and strain for maternal age, parity, and effects of litter size. Preliminary analysis of the BALB/c strain, for example, shows that litters of 1 to 3 mice have a 2- to 3-fold greater mortality rate during the full preweaning period than do litters of 4 or more. The point of interest in figure 19 is the nearly complete superposition of the human and mouse infant mortality data. The ratio of time scales is approximately 120:1. This ratio is 2 to 4 times greater than that customarily noted in the comparison of adult populations and undoubtedly reflects the rather attenuated prepubertal develop- mental period of man. Physiologically, the first 30 days of life for the mouse and the first 10 years of life for man are generally comparable. For example, it is recognized that a small proportion of the females of both species will reach sexual maturity by the end of the indicated age intervals. Many of the major hereditary defects in man express themselves during this early period of life. The same is undoubtedly true for mice and other mammals. The basic similarity in the temporal course of spontaneous mortality for the two species certainly encourages more complete and quantitative study of laboratory animals in order to evaluate the general adherence of the expression of genes for viability to genetic theory and expectation. Of particular note here are the data of Russell and Russell1138 indicating that most of the recessive lethals detected in the specific-locus tests induce death in the neonatal and preweaning period rather than in prenatal life. MAMMALIAN RADIATION GENETICS 147 Fig. 19. Neonatal, infant, and childhood mortality rates for inbred and outbred POPULATIONS OF MICE COMPARED TO UNITED STATES POPULATION. _# U S POPULATION 1956 _j}. Fj MOUSE POPULATION (All O Q INBRED MOUSE POPULATION (L.it... ol 7. 8. & 9) MOUSE DAYS 0 MAN YEARS 0 (Age scales adjusted to maximize comparability.) Growth and maturation. — Although there are no special genetic techniques required for studies in this category, a deficiency of information still remains. Russell1134 has reported that mutants at the piebald locus nearly always show a poor rate of growth and an associated higher mortality. The data of Boche et al.113 did not indicate a growth decrement, however. Possibly most effects on these traits will be due to a few major 148 RADIATION GENETICS mutants and thus show up in only a few animals per generation, the detection of which would be statistically difficult. Reproductive performance. — Here, too, there appears to be a shortage of data, with the exception, of course, of the studies on dominant partial sterility discussed earlier. As noted by Russell and Russell,1138 there will be a small percentage of the progeny of irradiated parents that will be completely sterile. Minor reductions in reproductive performance most certainly will be difficult to detect and will require good sampling statistics. The author has a small quantity of data for lifetime performance. The experimental group, the progeny of C57BL/6 males subject to 5 r/day of Co60 gamma radiation for 120 days, shows a 7.5 per cent drop in average size of litter and a slightly more rapid senile decline in productivity. Since only ten breeding females were sampled in each of the two groups, no real significance of the data can be claimed. The study was done on a pilot basis in the period 1956-1958 to test the effects of protracted irradiation on the induction of mutations affecting viability and reproduc- tion and to explore the attendant statistical problems (which are many). It can be noted that the careful initial pairing of the breeding stock by age, for experimentals and controls, will automatically lead to a high degree of comparability of the data, litter by litter, throughout the reproductive lifetime. At the same time, changes in the interval between litters will become apparent, if this is to be one of the manifestations of genetic damage. Behavioral traits. — There are no data on radiation-induced mutations that affect behavioral characteristics, although attempts to detect genetic damage of this type are being made by Green.481 PROTECTION AGAINST INDUCED GENETIC DAMAGE Basically, there are two principal methods of approaching radiation protection : preventive therapy and supportive therapy. The latter is typified by the successful use of postirradiation injections of bone marrow to improve survival. Bone-marrow therapy replenishes the animal's hematopoietic tissues during a critical period in the acute syndrome. Postirradiation supportive therapy appears to offer nothing to the geneticist interested in reducing the genetic hazards of radiation. Preventive therapy does hold promise. In this case, treatment is required in the immediate preirradiation period or even during exposure. Various chemicals have been tested along with modifications of oxygen tension. The theory behind the use of chemotherapeutics is that they act to reduce oxygen availability, quench active radical transport, or generally act as a competing target system for the initial events of energy absorption. While much effort has been spent on radiation-genetic protection studies with microorganisms,591 very little has been done with mammals. Hypoxia was tried by Russell et al.1136 as a means of reducing the induction of dominant lethals in X-rayed male mice. The animals were exposed to 800 r while in a chamber flushed with MAMMALIAN RADIATION GENETICS 149 5 per cent 02 and 95 per cent He. No evidence for protection was noted. A similar study with female mice did suggest some minor degree of protection.1116 Mature sperm may innately be hypoxic and thus no oxygen effect would be expected.1140, 1142 Kaplan and Lyon686 tried the compound mercaptoethylamine which, like hypoxia, is effective against general somatic damage. A 4 mg. dose given intra- peritoneally about 5 minutes before exposure did not protect against dominant lethal damage in males subject to 500 r of X rays. The same drug was used in a 10 mg. dosage with rats exposed to 300 r of X rays.839 Testicular weight loss was the end point and no effect was noted. Eldjarn et al.320 point out, however, that only very small amounts of the drugs cysteamine or cystamine are localized in the testes (only 1 to 1 0 per cent of that observed in other organs) . The same may be true for the compound used by both Kaplan and Maisin, which would suggest that considerably larger dosages would be required. Rugh and Wolff1086 succeeded in reducing damage to the ovary of the mouse with a 3 mg. intraperitoneal dosage of either cysteamine or cystamine. The measures here were the number of litters per mouse-week and the average number of fertile weeks. Animals protected with cysteamine, for example, produced 0.084 litters per week as compared to only 0.030 per week for controls after 50 r of whole-body X irradiation. Protected females were fertile an average of 18 weeks, compared to 6 weeks for the controls. Recently, Mandl846, 847 has reported success with male and female rats protected with B-mercaptoethylamine and cysteamine, respectively. She employed a histologic measure of effectiveness : primordial-oocyte survival and spermatogonial- cell survival. Intraperitoneal injections of 20 to 30 mg. were used, and a dose- reduction factor of 1.5 to 3.0 was achieved over the dose range of 100 to 400 r. ,No attempts have been made to study protection against specific mutational damage. As Mandl has pointed out, the earlier failures may not have reflected an inefficacy of the therapeutics but rather the wrong choice of dosage of both drug and radiation, the former too low and the latter too high. Her success in increasing survival of gonia and oocytes may encourage a more determined effort to search for an agent that will reduce the yield of genie mutations. Selection of mammalian species and systemis) of mating. — To a degree, there is really little room for choice when one wishes to carry out studies in mammalian radiation genetics. The mouse has been and certainly will continue to be the favored species. Some consideration, however, should be given the rat, at least for studies on growth and maturation, since it may offer the opportunity to do more sensitive studies on these traits. The unfortunate disadvantage for both mice and rats, and to some degree for all litter-bearing animals, is the partialy uncontrollable incidence of neonatal mortality. Since most litters are born during the early morning hours, the stillbirth and im- mediate neonatal, or perinatal, mortality can be obscured by the cannibalistic ten- dencies of the dams. In the writer's experience, it has sometimes been impossible to determine the number born, although positive evidence of stillbirth, or livebirth, or both, in the form of a few scraps of progeny partly eaten, may be available. An 150 RADIATION GENETICS eight- to ten-hour shift in the diurnal cycle could possibly overcome this problem and would seem worth a try. Since most modern animal quarters are windowless and equipped with resettable automatic light switches, no technical difficulties need be anticipated. For the most part, other species either have too few young per cycle or are too expensive to maintain. Nevertheless, a major Atomic Energy Commission-supported program with swine is under way at the Iowa State University, under the direction of Drs. J. L. Lush and L. N. Hazel, which may provide the controlled early mortality data that are required. However, swine after bearing young also tend to trample and crush part of the litter. Once the species is selected, the next question concerns the genetic composition of the chosen animals. Should they be inbred, single-cross hybrids, or should they be random-bred or mixed hybrids, such as double crosses? This is not an easy question and the answer any particular investigator gives may not be entirely unbiased. Answering the question raises additional ones on the significance of polymorphism and heterosis in mammalian species. Will inbred animals tend to express heterosis for induced mutation more readily than crossbred animals, or will this expression occur to any significant degree at all? Will genetically heterogenous material have a greater probability of masking the minor detrimental mutations because of an innately greater viability and genetic flexibility ? There may be a way out, however, as in the study Green481 describes, in which four different levels of inbreeding are employed on two different populations — one genetically homozygous and the other genetically heterozygous. An additional procedure is that used by Chapman.196 In his study, recurrent incrossing and out- crossing is compared with continuous incrossing and outcrossing. In both studies, the animals are irradiated every generation in order to check for cumulative genetic injury but under the different selection pressures induced by the varying degree of inbreeding. The only opinion the writer offers is that the outbred system may be the choice for the sake of its greater comparability to man if the investigators cannot afford to check both inbred and outbred genetic systems. RADIATION AS A TOOL FOR GENETIC RESEARCH It is an understatement to say that radiation has become an extremely useful tool in experimental biology and medicine. A glance through the contents of this volume should substantiate the fact that many aspects of mammalian genetics have profited as well. No attempt will be made to describe all applications. However, particular contributions to mammalian genetics have been provided through the use of radiation techniques in the areas of physiologic genetics, immunogenetics, developmental genetics, the genetics of disease resistance, the genetics of viability, genetics of cancer, and somatic-cell genetics. Pertinent specific references will be found throughout this volume, and the reader should consult the studies of E. S. Russell, H. Chase, R. D. Owen, MAMMALIAN RADIATION GENETICS 151 D. Uphoff, L. B. Russell, J. W. Gowen, D. Grahn, W. E. Heston, K. Atwood, S. Scheinberg, and certainly many others. Techniques developed by mammalian geneticists, originally for genetic purposes, are now finding usefulness in other areas of radiobiology. For example, a group in the Medical Department of the Brookhaven National Laboratory is making extensive use of measurements of survival of spermatogonial cells for detailed analysis of the relative effectiveness of different energy levels of neutron irradiation.60' 1075 This judicious mixture of genetic techniques with those of the medical and radiologic physicist promises to offer much valuable data to the science of radiobiology. The geneticist's adherence to quantitative methodology and to sound concepts of the cellular basis of radiation injury have generally assisted in establishing a high standard of scientific accomplishment in the comparatively young and fast-growing field of radiation biology. Geneticists can take pride in their total contribution, direct and indirect, to this scientifically and politically important field of endeavor. Perhaps many remaining problems in radiobiology will ultimately be solved either by geneticists or through genetic techniques and interpretation. DISCUSSION Dr. Degenhardt: I am very pleased to open the discussion of this excellent paper presented by Dr. Grahn. We are all impressed by the recent advances in this specialized field, but we also recognize that our knowledge is fragmentary in certain areas. On the whole, we have advanced little beyond the first steps into the field of radiation genetics, in which methodologic problems still prevent accurate, rapid, and economic progress. The few well-equipped teams of scientists, who are investigating problems in mammalian radiation genetics, are not sufficient even though their individual efforts are excellent. I should emphasize that it is absolutely necessary (1) to encourage international cooperation and international exchanges of ideas and information in the field of mammalian genetics and (2) to encourage cooperation and exchange of ideas between scientists working with experimental mammals and those working with human beings. This would enable us to collect all information in this field within a reasonable space of time and to estimate better than before the hazard of nuclear damage to human beings. After these general comments I should like to direct attention to a certain problem, mentioned by Dr. Grahn: the induction of dominant lethals in germ cells. This is a critical means for examining the areas of physiologic and developmental genetics in early embryonic stages. W. L. Russell, L. B. Russell, and E. F. Oakberg1142 have demonstrated that the induction of dominant lethals in male germ cells leads to a high incidence of lost conceptus prior to days 10-11 of gestation. Pre-implantation losses rise to about 7 per cent and post-implantation losses to about 25 per cent of the number of corpora lutea at doses below 100 r. 152 RADIATION GENETICS In female germ cells, dominant lethal incidence was shown to be strikingly depend- ent on the stage of oogenesis during irradiation;950, 951, 1123 a dose of 70 r will induce about 50 per cent dominant lethality in oocytes in the first meiotic metaphase. When Bateman57, 59 computed theoretically the relationship beween the postulated number of chromosomal breaks and time of death of the conceptus due to dominant lethals in the female germ cells, he found that single lethals per egg permit survival to implantation but multiple lethals usually cause loss of the egg before implantation. These facts should encourage cooperation among geneticists, radiologists, embryologists, cytogeneticists, and biochemists to focus their attention on the direct relationship between chromosomal breaks and abnormal embryonic development. Regarding post-implantation losses, I wish to draw special attention to seven-day- old embryos in mice. We have information, gained from direct irradiation effects (130 r) upon embryonic differentiation of somatic tissue in successive stages of gestation, that day seven seems to be most critical in early morphogenesis (figure 20). In comparison to embryonic development in human beings, this would be the third week of gestation (day 17 to day 21), a stage of development in which most mothers are not aware of their pregnancy. This conclusion agrees with those given by Russell and Russell,1121, 1122 and they recommend that, whenever possible, pelvic irradiation of women of child-bearing age should be restricted to the two weeks following the first day of menstruation. Radiation hazards to the embryo are being avoided to a large degree by following this advice. Dr. Burdette: Fairly recently we have been able to demonstrate in Drosophila, although we have not tried it in mice, that actinomycin D, which is often used in con- junction with irradiation, will reduce the frequency of mutations induced by X irradia- tion by approximately 50 per cent, although we do not have sufficient data to demonstrate the effect on natural rates of mutation. I am wondering whether you will comment on possible pressures in nature which may reduce the numbers of muta- tions. Photoreactivation is an example of the type of phenomenon I had in mind. Dr. Grahn : For mammalian systems or in man, for example, I do not know what particular pressures there would be to reduce the hazard of radiation. Such things as photoreactivation are probably not effective in mammalian systems. Generally it is very much easier to find all the different environmental stresses that will increase the mutation rate rather than to find anything that will act to reduce the potential hazard. Dr. Burdette: More attention directed toward reversal and inhibitory effects may be very profitable in view of the current disproportion between this type of study and those dealing with enhancement. Dr. Crow, do you have a comment ? Dr. Crow: There was a suggestion a few years ago by Carter and Haldane 170, 517 that one might study the over-all mutation rate in mice by finding lethal mutants linked with known visibles. I wonder how widely this is being applied ? Dr. Grahn: Essentially not at all. Both the use of independent markers and linked markers in the location of a lethal in between the markers should be mentioned. Haldane517 has worked out most of the statistical problems and has even been able to MAMMALIAN RADIATION GENETICS 153 demonstrate that this procedure can be used to look for sublethals. Carter158 tried the linked-lethal procedure and came to the conclusion that it has no improvement in efficiency over the specific-locus, test procedure. However, the procedure possibly has not been exploited as far as it could be. Fig. 20. Congenital deformities produced by acute X irradiation of pregnant mice of INBRED STRAIN C57/BL/K.S AT successive stages of gestation. STAGE IRRADIATED ( DAY « HOUR POST COITUM ) pTa fz*;o|gi|l*« |x*-2o| ACEPHALY CRANIOSCHISIS CYCLOPIA ENCEPHALOCOELE OTOCEPHALY EYE DEFECTS BASE of CRANIUM ABNORMAL 8ASISPHENOID HOLE CLEFT PRESPHENOID DEFECTIVE SCOLIOSIS of CRANIAL BASE FACIAL PORTION of CRANIUM ABNORMAL CLEFT PALATE CLEFT PALATE- JAW- LIP NASAL BONES ABNORMAL BOTH JAWS UPPER JAWS CLEFT " FUSED LOWER JAWS REDUCE 0 FUSED UPPER INCISORS ABNORMAL " " FUSED LOWER " ABNORMAL FUSED MALFORMED VERTEBRAL COLUMN VERTEBRAL CENTRA ABNORMAL NEURAL ARCHES " RIBS • STERN EBRAE LUMBARIZATION and SACRALIZATION TAIL ABNORMAL POLYDACTYLY, HIND FOOT SUSCEPTIBLE STA«E peak or tusccmwLiTY Dr. Burdette: Would you comment on Dr. Degenhardt's first point? Is there an effort to correlate research efforts on irradiation globally by the World Health Organization and others? A summary of these activities should be of general interest. 164 RADIATION GENETICS Dr. Grahn: The United Nations, in conjunction with the World Health Organiza- tion, is interested in the problem of effects of low-level radiation in man.1411 The United National Scientific Committee on the Effects of Atomic Radiation is also interested in improving the collection of vital statistical data so that it will be of some value in human or genetic studies. I know of no specific program or plan to bring together the diverse efforts of different countries other than those agreed upon in an informal way, such as might be arranged between the United States and the United Kingdom and Canada. Dr. Gowen: In discussing rates of mutation, you placed quite heavy emphasis on the seven loci in mice. Knowing what has been learned about mutations in Drosophila and realizing the variations possible when one considers that crossing over occurs within the gene, so to speak, would you put a premium on the seven loci, or would you prefer to have a much more general survey of the mutation frequencies for as many loci as one can keep under reasonable control ? Dr. Grahn: What do you mean when you speak of premium, Dr. Gowen? Dr. Gowen: Premium in the sense that it would be strange indeed if such a group of loci were representative or even suggested the range of mutation rates or the types of changes which may be expected to occur in, say, mammals or man. Dr. Grahn : Certainly data on more loci are desirable if for no other reason than to give assurance that these seven loci and the mean or median rates of mutation derived therefrom are reasonable descriptions of the average mutation rate for the total genome. This is neither cheap nor easy, so the solution of the problem is not obvious. Total gametic methods are not too efficient either. In other tester stocks that have been or can be synthesized, their reduced viability, reduced fertility, over-lapping phenotypes, etc., make the problem additionally very difficult to solve. Dr. Yerganian: Dr. Grahn's remarks on reverse-lighting patterns of illumination in animal-breeding rooms should be supported wholeheartedly for the benefit of the investigator who wishes to initiate convenient and controlled matings.1462 Dr. Lederberg : It seems to me there has been such a frenzy of interest in radiation- induced mutation that we are very seriously neglecting the other environmental factors which, from a practical point of view, may be much more important to man. I am talking about chemically induced mutation and antimutation effects. As far as I can tell, there has been a studious neglect of the very well-documented observations that mutation rates in bacteria, and very much more recently in Drosophila, can be markedly influenced by the presence of various purines in the diet. Furthermore, the compound adenosine will go so far as to reduce the mutation rate in bacteria by the factor of a half. Now if you are concerned about the incidence of mutation in the human popula- tion, I think it is reasonably plain that you could exert on a global basis a much more significant effect by alteration of diets which would have some effect on the spontaneous mutation rate than we are now likely to accomplish by small changes in the environ- mental radiation hazard. I am not saying we are not justified in making strenuous efforts to reduce the radiation hazards to manageable levels; but I feel now that there MAMMALIAN RADIATION GENETICS 155 is a tremendous distortion of interest at the present time in radiation biologic genetics and away from the chemical aspects of mutations, and I think more could be done about this, f | Subsequent to this symposium, the problem has been reviewed : Genetics, Proc. of the Second Conference, 1960 (Mutation), Josiah Macy, Jr., Foundation, 1962. See especially the discussion by Dr. A. Goldstein. PHYSIOLOGIC GENETICS Sewall Wright, Sc.D. GENIC INTERACTION Genie interaction is a subject of great importance in two of the major branches of genetics. It is obviously fundamental in physiologic genetics and is almost as funda- mental in population genetics, including the genetic aspects of the theory of evolution. I have been interested in genie interaction for both reasons and in about equal measure since about 1915. In trying to keep up with both, it has sometimes seemed as if I were trying to ride two wild horses bent on going in different directions. I shall try to justify the attempt to keep them together. INTERACTION IN EARLY GENETIC RESEARCH The unit characters of the early geneticists probably seemed to most other bio- logists merely another revival of the ancient doctrine of preformation, discredited by all who, like Aristotle, Harvey, Wolff, and von Baer, had attempted to trace the step-by- step elaboration of complexity in the process of development. The early geneticists did relatively little to disavow this interpretation. They were too busy working out the laws of transmission of these units and the patterns of organzation in the germ cells to have much time for theoretical discussion. In pursuing this task, they were interested in characters mainly as markers for genes. They were interested, for the most part, only in good genes, consistently associated with easily classifiable characters. The catchwords used for convenience in naming genes often seemed to others to imply that geneticists thought of the organism as a mosaic of unit characters. The extent to which even the earliest Mendelians actually were preformationists can easily be exaggerated. Interaction effects, such as those responsible for the familiar 159 /ffO PHYSIOLOGIC GENETICS modifications of the 9 : 3 : 3 : 1 F2 ratio (9 : 3 : 4, 9 : 7, etc.) , were recognized very early61 • 232 and interpreted as due to epigenetic chains of reactions. ELEMENTARY GENIC ACTION It is generally accepted now that the physiologic action of genes is wholly by imposition of specific patterns on macromolecules1450 and even this is thought to be by steps (DNA-RNA-polypeptide, etc.). The classes of characters that have seemed most Fig. 2 1 . Diagram of relations of genome and external environment to observed CHARACTERS AT VARIOUS LEVELS OF ORGANIZATION. Behavior External Environment Histogenesis Extraorqanic Structure Homeostasis vs. Disease Organic Structure. Cell Constitution Morphogenesis Genome Gene Duplication / Cell Product Metabolism Genome / V / Macromolecular Pattern X Enzyme Antigen favorable for study of the first links in the control of characters have thus been antigenic and enzymatic specificities and the more elementary metabolic processes. One of the most significant results from immunogenetics has been the usual occurrence of a one-factor relation between any particular antigenic reaction and a particular gene (or group of alleles) irrespective of the situation at other loci. Even so, the demonstration of hybrid substances by Irwin shows that there may be interactions among loci even at this level.641 Another significant result is the occurrence of a very large number of alleles at many of the loci with antigenic effects. While attempts at subdivision of the loci according to the pattern of responses to multiple test sera have been demonstrated GENIC INTERACTION 161 to be unsound,985 the extraordinary number of alleles in some cases (more than 250 at the B locus in cattle) and the curious intragenic, interaction effects indicate great complexity of genie pattern and the existence of much primary pleiotropy. The association of blood-group genes in man with fetal hemolytic disease and with various diseases of adults demonstrate much secondary pleiotropy. There is no good reason to suppose that multiple allelism and pleiotropy are any less frequent with loci for which such delicate methods of demonstrating differences are unavailable. Fig. 22. Diagram of factor interactions in the determination of coat color of the GUINEA PIG. Predisposition (epidermis) Absence of Melanocyte WHITE Differentiation (melanocytes) Enzyme system (granules) Pigment (from tyrosine) Dinginess (I) Spotting S,s(+) I (Ms) df,?(+) Dam's age(+) Region Z(Db) "> W(+),w Androgen(+) — •Spotting v. Region Chance J Chance /\l^ ■ \Spotting j Region /p» i Chance . Silvering Si,si(0 S(Msi) Region Chance (3) Diminution^ Dm,dm(+) (4) Grizzling Eumel. predisp Phaeomel./i predisp. (10) f \y f f-(26) <( (25) AgeH 2 (Leu) b^\ i L-{27) - (28)- a p) \Mp,mp(-)| (22)x Spotting ^"-"v AgeH J >f\ Gr,gr(+) I (Mgr) Age (+) Region Chance Sootiness I (So) Region Temp.(-) Agouti AMXa Z(MA) Region \—-^ /_(„)_ -JJc^r^L *-SEPIA v»-BR0WN >YELL0W The study of the genetics of metabolic processes in such favorable organisms as Neurospora, Aspergillus, and Escherichia coli has been characterized by the demonstration of long, branching, reaction chains in which each link is controlled by a particular locus. Nonadditive interaction effects are the rule.1353 Such processes are merely the first steps leading to the variations observed at the morphologic level in higher ani- mals. Figure 21 is intended to represent the hierarchy of levels at which inter- action may occur.1413, 1424, 1433, 1434, 1441, 1450 The interaction patterns found among the genes concerned with a number of such characters of the guinea pig will be reviewed here. 162 PHYSIOLOGIC GENETICS COAT COLOR OF THE GUINEA PIG Figure 22 is an interpretation of the interaction pattern with respect to quality, intensity, and pattern of the coat color of the guinea pig. Color depends on the pres- ence of pigment granules (sepia, brown, or yellow) or their absence (white). The pig- ment granules are produced in special cells (melanocytes) in the hair follicles, basal layer of the epidermis, chorioid coat of the eye and certain other tissues, and in retinal cells. All but the last migrate from the neural crest. The melanocytes may pass the pigment granules to epidermal cells. Absence of pigment may be due to failure of the melanocytes to reach the normal site,1177a' 1352a to death in the site, probably in silvering and grizzling (at 1, 2, and 4, respectively, in figure 22), or to failure of melanogenesis without death of the cell (albinism caca).e54- 1204- 1315 The primary differentiation in quality is between cells that produce eumelanin (sepia, brown) and phaeomelanin (yellow). The most important factor in relation to this differentiation in the guinea pig is the e locus (E typically self-eumelanic, ee typicaly self-phaeomelanic, epep, epe a mosaic (tortoise-shell) of the colors found with E and e). The ^-alleles cannot be supposed to determine differentiation of melanocytes directly but merely to produce an effect that predisposes toward one or the other type of differentiation (at 5 and 6 in figure 22) . Thus with EA each hair follicle produces (at 8 in figure 22) the same sort of yellow as with ee, during a brief phase in the growth cycle of each hair, in spite of the presence of E. This results in a subterminal yellow band in otherwise eumelanic hair. A locus with similar action in the mouse acts on the melanocytes from adjacent epidermal cells.1203 On the other hand, pigment cells in animals with pure yellow coats at birth because of ee may produce much black or brown pigment later (sootiness at 10 in figure 22) in the presence of favorable un- analyzed heredity 2 {So) and low temperature.1401. Gene ep is definitely not mutable in the germ line in inbred strains. The nature of the all-or-none process that occurs early in development in tortoise-shells to distinguish different cell lineages (perhaps in the epidermis) is not known. Cells with phaeomelanic differentiation produce only phaeomelanin but those with eumelanic differentiation produce granules that, while largely eumelanic, may contain a small amount of phaeomelanin, which is revealed under conditions that differ- entially reduce the eumelanic constituent. Visual grades, based on standard squares of skin chosen so that each grade is barely distinguished from the preceding, have been assigned each animal at birth and often later. The corresponding amounts of pigment, relative to intense black (grade 21) or intense yellow (grade 1 1 ) have been estimated by extraction from weighed samples of hair and colorimetry.542' 1096- 1453 In the later years,1415 reflectionmeter readings (R) were taken of many genotypes using amber, green, and blue filters. Indices closely paralleling the visual grades were obtained by the equation Ix= \0{\ogRw-logRx) GENIC INTERACTION 163 in which log Rw is the average logarithm of readings of white and Rx in the reading in question. A final intensity index was obtained from the sum of the indices from the three filters. Relative quantities of pigment were estimated from the colorimetric determination by way of the relations to the visual grades. Figures 23 and 24 are based on such estimates for various genotypes; figure 25 is based on the unweighted average of estimates from the indices and earlier estimates from visual grades. An index of quality (Q) was obtained1438 from the ratio of the index from the blue filter to the total index (/). Average quality (Q) is plotted against average intensity (/) for various genotypes in figure 26. The melanoproteins of the granules consist of melanoid derived by oxidation of Fig. 23. EUMELANIN AND PHAEOMELANIN IN GUINEA PIGS. cac° crca crcr cdca cdcr cdcd ckca ckcr ckcd ckck C 0 i caca crca crcr cdca cdcr cdcd ckaa ckcr ckcd ckck c Estimates of amounts of eumelanin (100-intense black), above, and of phaeomelanin (100-intense yellow), below, in guinea pigs with combinations of is, e; B, b; C, ck, cd, cT, c°; P, p and F,f. The eumelanic estimates are based on animals with three or four plus factors at the loci Si, si; Dm, dm. The phaeomelanic estimates are based on those with three plus factors. The estimates are transformed from reflectionmeter readings. 76'4 PHYSIOLOGIC GENETICS tyrosine, under control of the enzyme tyrosinase, firmly linked with protein.1192 The specificity of the enzyme seems to depend primarily on the c series of alleles which is interpreted as acting pleiotropically on the phaeomelanic and eumelanic processes (13 and 19 respectively in figure 22). The complete or very nearly complete domi- nance of C in all combinations indicates that its product is in excess in both reactions Fig. 24. EUMELANIN AND PHAEOMELANIN IN GUINEA PIGS. c°c° crc° cV cdc° cV cdcd ckc° ckcr ckcd ckck C (sisi-n-) ca ca crca crcr cdca cd cr cdcd ck ca ck cr cKcd CK ck C Estimates of amounts of eumelanin (above) in guinea pigs of genotype EBP and of phaeomelanin (below) in ones of genotype eeFF, associated in both cases with the various c compounds and with 0 to 4 plus factors at the loci Si, si; Dm, dm. Comparable with figure 23. while the intermediacy of heterozygotes among the other alleles indicate that their products are limiting factors in both. The evidence as a whole indicates the order in amount of product to be C > ck > cd > cr > ca but the actual orders of intensity vary from case to case (figure 23). The lower alleles are interpreted as competing (20 in figure 22) for processes 13 and 19 with different degrees of efficiency in order to GENIC INTERACTION 1 65 account for the qualitative differences shown in figure 26. Thus cr and ca are inter- preted as unable to produce any yellow whatever, even in cells with phaeomelanic differentiation, cd is interpreted as relatively efficient in producing yellow, but less Fig. 25. EUMELANIN IN GUINEA PIGS AT BIRTH AND SIX MONTHS LATER. cV crca crcr cdca cdcr cdcd ckca ckcr ckcd ckck C 4Eppff caca crca crcr cdca cdcr cdcd ckca ckcr ckcd ckck Estimates of amounts of eumelanin (above) in guinea pigs at birth (broken lines B, dotted lines bb) and at about six months of age (solid lines). Similar estimates for phaeomelanin (below) in guinea pigs at birth (broken) and at about six hours (solid). These are based on unweighted averages of estimates from transformed visual grades (back as whole) and transformed reflectionmeter readings (darkest spot near midline of back). efficient than cr in producing eumelanin because of this competition. It is more efficient in the eyes (cdcd, cdcr, cdca black eyed, cV dark red, crca light red) in which there is no production of yellow and thus no competition. Gene ck is interpreted as somewhat less efficient than cd in producing yellow but much more efficient in producing eumelanin. 166 PHYSIOLOGIC GENETICS The top lines in the upper and lower parts of figure 23 compare the various c compounds in the most favorable combinations with other loci. Returning to figure 22, the intensity of phaeomelanin is represented as affected by F (strong) and/ (weak) at 14 and 15 in figure 22 following reaction 13 and as subject to a threshold (at 1 7) . What is left is represented as interacting (at 18) with a limiting factor 2 (Lph) to give yellow pigment. The averages in figure 23 show a slight reduction brought about by replacing FF by Ff and great reduction by replacing it by ff. The absence of yellow pigment in the heterozygotes represented collectively by ckdcraff, in contrast with the small amounts in ckdckdff, is one of the evidences for a threshold. Great differences in the intensity of reds, eeCF, among inbred strains (about twice as much pigment in strain 32 as in strain 2) indicate a variable ceiling. Crosses indicated multiple factors, 2 (Lph). Turning to the eumelanic colors, P (or an allele pr described by Iljin626 as similar in effect to P except for lighter eye color) is represented as necessary for the production of Fig. 26. Mean quality index (Q) plotted against mean intensity index (/) for VARIOUS GENOTYPES. Q 50 48 46 44 42 40 38 36 34 32 Cream (ee) cdca- cdcdffcdcd^xckcd v\ Eppff > CFf « CFF Red M White (upper quartile • Pale brown cdcMcr (EbbppF) .drd_x^_ rd rd Dark brown cc Very pale sepia dn + , Jc<«u,;f j Dark sepia (EBP) EbbP cickcfcrcr Black 0 10 20 30 40 Both indices are derived from reflectionmeter readings. 50 I GENIC INTERACTION 167 the full amount possible with each c compound. Compounds FF, Ff or ff make no detectable difference in the intensities of any of these. The replacement of P in EBPF by pp (figure 23) results in a great reduction in the amount of pigment in both coat and eyes (pink). There is disproportionately great reduction in the lower c compounds, especially crcr and crca. With crca the color is often indistinguishable from white at birth, indicating a low threshold (at 24 in figure 22). The fact that even with crcr there is less pigment than with cdca, in contrast with the situation in the presence of P, seems to require a carrying through of the effect of the specificity difference between cr and cd to the reaction at 22 (in figure 22). The replacement of F in EBppF by ff reduces pale sepia to a very much paler brownish cream, sometimes indistinguishable from the pure pale yellow of eecdcdff. This color is interpreted as due largely to the uncovering of a feeble underlying yellow (at 1 5) in the absence of both P and F. The trace of eumelanin, usually present, is attributable to feeble action of/ (at 23) . The fact that replacement of F by/f makes no recognizable difference if P is present, but almost complete absence of eumelanin with pp is interpreted as meaning that P, without F, is sufficiently efficient to produce as much eumelanin as the c compound permits but that in the absence of P, F acts as a feeble substitute, and if this also fails, only a trace at most can be produced by/. It may be noted from figure 23 that with any lower c compound and Eppff the color is pure white, indicating that the eumelanic and phaeomelanic processes fall below the threshold in these cases. The replacement of B in EBP by bb gives brown in coat, skin, and eyes in place of sepia and approximately halves the apparent intensity by colorimetric determinations of these closely similar pigments. There is parallelism between sepia and brown among the c compounds (figure 23) to the extent that caca is white and cdca and crca definitely more dilute than the others (except certain combinations involvoing C), but the differences among all the other c compounds are much less in browns not only absolutely but on a percentage basis. This can be interpreted on the hypothesis that bb imposes a ceiling on the possible amount of pigment at about half that imposed by B and that this ceiling is approached to such an extent even with crca and cdca that there is no possibility of much further increase with higher compounds. If P in EbbPF is replaced by pp, there is marked reduction in intensity but not as much proportionately as in the sepias. The eyes are again pink. There is a marked qualitative difference from the pale sepias (figure 26) in contrast with the slight apparent difference between dark browns and dark sepias of the same intensity. Pale browns with C have much more than half as much pigment as the corresponding pale sepias and among the lower c compounds there are no consistent differences in quantity in the somewhat unsatisfactory data on this point. The smallness of the quantitative differ- ence in comparison with that in the P eumelanics may be interpreted as due to remote- ness from the ceiling. Ebbppff is wholly indistinguishable from EBppff The most remarkable interaction effect is one that affects only browns of genotype EbbCP.li31- 1439 There is an optimum genotype with respect to the c, p, and /loci, 168 PHYSIOLOGIC GENETICS probably CcxPpff in which x is any lower c allele. Thus in the presence of Ebb, cxczppjy is pure white, Cppff has a trace of color, cxcxppF is very pale brown, CppF pale brown, cxcaPff slightly dilute brown, ckckPff intense brown, CPpff possibly slightly more intense, but CPpFF slightly dilute (dingy on the head), CPPff slightly more dilute and CCPPFF often with less than half as much pigment as the intense browns. Re- placement of CC by Ccx in these has a slight darkening effect demonstrated only in adults. The type of dilution in the higher combinations is of a peculiar sort (dinginess) which ranges from a mere sprinkling of dark tipped light hairs on the cheeks and nape to uniform dilution of all hairs, except at the extreme tips, to a color as pale as pale sepia (only in EbbCCPPFF with favorable modifiers). This type of dilution could not be produced at all in the presence of ckck or oi pp. This effect is represented in figure 22 as the result of a destructive action of CPF product in excess of that necessary to saturate the limited bb product. The failure to observe any such effect in blacks in this colony may be interpreted as due to the higher ceiling provided by the B product. Ibsen and Goertzen624 described an incompletely dominant modifier of dinginess [W) which completely inhibits brown pigment in a subterminal band in EbbCCPPFF. They found a slight effect in blacks in the presence of WW. While W was clearly absent from my colony, dinginess was much affected by other modifiers. It was possible to bring even EbbCCPPFF to full intensity in a few cases by selection. We will go back to consider modifiers of some of the other processes here. The most important genetic ones are Dm, dm and Si, si (figure 24). 1415, 1432, 1440 The most conspicuous effect of sisi in otherwise intense animals is to cause a sprinkling of white hairs (silvering) in the coat which does not progress after birth in contrast with the effect of grgr (grizzling)7*5- 1431 with no effect at birth but progressive whitening later. We are here concerned with dilution effects of si on colored hairs. Replacement of Dm by dm (diminution) has no recognizable effect on intense (C) blacks or yellows but causes dilution in lower c compounds, most conspicuously in cdca (both sepia and yellow) and in crca sepias. Factors si and dm dilute both colors cumulatively (figure 24). The combination sisidmdm is pure white except for occasional pale spots on the head. Eye color is slightly reduced. There are other effects which suggest that the effect of these genes is on the vitality and metabolic efficiency of certain types of cells rather than on the pigment process in as specific a way as seems to be the case with many of the other loci. Animals with sisidmdm suffer a high mortality after birth and an anemia in which the red blood count is reduced by a third. In males, testicular size is .reduced to 25 per cent of normal and there is complete absence of spermatogenesis and thus sterility. About half the females tested have been sterile and the remainder rather low in productivity. Assuming that the effect of these genes on color is on meta- bolic efficiency of the pigment cells (if not actually lethal to these as in silvered hairs) it appears that among the specific color factors only the efficiency of the c alleles is affected. The effects of si and dm have been studied intensively only in dark sepias EBP and yellows eeFF, but as far as determined they have proportional effects in browns (EbbP), pale sepias (EBppF), and pale yellows (eeff). GENIC INTERACTION 169 There are at least two pairs of modifiers, Mpx, mpy, Mp2, mp2, that profoundly affect the intensity of pale sepias (EBppF), pale browns (EbbppF), and probably the trace of eumelanin in pale brownish creams (Eppff) but have no recognizable effect in dark sepias {EBP), dark browns (EbbP), or yellows (V (Leu)] as well as those already referred to that affect the ceiling for yellow [T (Lph)]. ENVIRONMENTAL EFFECTS ON COAT COLOR There are interaction effects with temperature and age1401, 1421 (figure 25). Most of the processes become weaker as the animals grow older, independently of temperature. Thus pale sepias fall off some 50 to 60 per cent in intensity by a half year of age (attributable to process 22 in figure 22). In dark browns that are not dingy there is a reduction of about 12 per cent, probably from lowering of the ceiling (27 in figure 22). However, the dingy females darken, indicating a weakening of dingy modifiers (28 in figure 22), but the males become even lighter. Experimental evidence indicated that this lightening was an effect of androgens.1400 Pale browns diminish in intensity but probably somewhat less than pale sepias. Intense yellows (FF) decrease some 20-30 per cent (14 in figure 22), while fading yellows (ff) show a much greater reduction, some 50-60 per cent (15 in figure 22). Low temperature has already been referred to as a condition for sootiness of yellow (10 in figure 22). The most striking effects of temperature, however, are those on the lower c alleles, the products of which seem to be markedly thermolabile (12 in figure 22). This effect is not recognizable in the case of C, the product of which is presumably present in great excess. In the case of ck product, the effect of tempera- ture, if any, does not compensate for the reduction with ageing. Thus yellows of genotypes ckckF, ckcrF, and ckcaF become significantly paler after birth when production of pigment occurs at a lower temperature than before birth. This is also true of sepias carrying ckP. On the other hand, yellows carrying cdF all become more intense. In these, the decreased loss of cd product with lower temperature more than compensates for the effect of ageing. This is also true of dark sepias with cdcd, cdcr, crcr and especially cdca and crca. Albinos (caca) are pure white at birth, although pigment cells are present.1204 Soon after birth, those with EBcacaP and EbbcacaP develop much black or brown pigment, respectively, in the skin and in hairs on feet, nose, and ears and traces of eumelanin on the back. In dark browns, the lowering of the ceiling with age and the tendency to darkening, especially of crca and cdca, from lowered temperature almost remove all differences among c compounds except for caca and CPPFF males. SPOTTING We have not yet considered spotting and its remarkable interaction effects.199, 1439> 1454 Spotting (colored spots on a white ground) depends primarily 7 70 PHYSIOLOGIC GENETICS on an incompletely recessive gene s. There are unanalyzed modifiers, 2 (Ms), that can shift the median percentage of white in inbred strains with ss from about 10 per cent to 98 per cent. In all cases females average a little whiter than males. The same female produces whiter young on the average as she becomes older. The pattern has some orderliness — with the strongest tendency to color near eyes and ears, strongest tendency to white on feet, nose, and midline of belly; but there is always an enormous amount of variability that can only be attributed to accidents of development. An inbred strain may range from a trace of white to black-eyed self white and yet show by the absence of correlation between parent and offspring that there is no genetic varia- bility. In such a strain there is indeed no correlation between points one third of the length of the animal apart with respect to presence or absence of white. Gene s is not mutable germinally in inbred strains. Spotting is probably due to interaction of two modifiable patterns: migration of melanocytes from the neural crest into the skin, and differentiation of the skin.117a- 1352a The points of most interest here are interaction effects with certain other processes indicating that the spotting process is not merely one which leads to presence or absence of pigment cells. It is a process in which the pertinent cells in the areas where color is present fall into different states in a spotting pattern which affects certain color processes but not others. Those affected most conspicuously are the tortoise-shell pattern due to ep (5, 6 in figure 22) and the pattern of dinginess in browns of genotype EbbCCPPFF (28 in figure 22). The pattern of silvering due to sisi (2 in figure 22) and the intensity of pale sepia or pale brown (22 in figure 22) are affected similarly but much less frequently. There are very rare cases of mosaics with respect to other loci which suggest somatic mutation but which are usually related to white spotting in a way that suggests that gene s has something to do with them.1456 Tortoise shells of genotype SSevep are predominantly eumelanic but usually show scattered yellow hairs and less frequently more or less yellow in blotches. With ssepep or even Ssepep, the amount of yellow is increased and there is a strong tendency to segregation of yellow and eumelanin into a few large areas each often with scattering admixture of the other color. 199a' 627, 1419 These areas are often separated in whole or part by white streaks. Sometimes a streak between eumelanic areas is white at one end, yellow at the other, indicating that the determination of yellow is related to the process that leads in more extreme cases to white by absence of the pigment cells. Similarly the orderly pattern of dinginess, found in the presence of SS, is broken up in the presence of ss into a coarse mosaic of light and dark dingy areas, often separated in whole or part by white streaks. In this case, it seems to be the determination of the darker areas that is most closely related to the determination of white.1439 COLOR FACTORS AND ACTIVITY OF TYROSINASE There has been considerable research on differences in enzymatic activity in pigment cells of different genotypes. There are definite differences which may be GENIC INTERACTION 171 attributed to color factors (E, e; A, a; C, ck, cd, cr, ca; P, p; F, f), but the correlation with the intensity of pigment production is far from perfect. The dopa reaction has been studied in frozen sections of the skin at birth735' 113° and in colorless extracts from the skin at birth.433 Foster394 has studied both the oxygen consumption curves and amount of darkening in the Warburg apparatus on adding tyrosine or dopa to homogenates of fetal skin. There are some apparent inconsistencies in the results, but these seem to trace largely to the stage of the reaction on which observation was focussed. Foster's tech- nique was designed to distinguish differences in moderate to strong reactions and gave less certain results in distinguishing very feeble reactions from the background oxygen consumption of controls. The earlier studies of the dopa reaction distinguished different end results of very feeble reactions from that in controls but gave such saturated results from only moderately strong reactions that these were not always distinguished from very strong ones. The dopa reactions in frozen sections were moreover very seriously obscured where there was much natural pigment (dark sepias, dark browns). It may be noted first that tyrosinase and dopa oxidase activities were very greatly reduced by replacing E by ^.394 Foster found similar reduction in the yellow phase of agouti follicles (EA) in comparison with the black phase (Eaa) . Much black melanin was, however, ultimately produced from dopa in frozen sections735- 1130 and extracts433 from intense yellow skin although both Russell and Ginsburg found significantly less than from even pale sepia. Foster was unable to demonstrate any tyrosinase or dopa-oxidase activity from lower c compounds including EBckcTPPFF which is intense black. Russell found considerable ultimate darkening of dopa in frozen sections from pale sepias (EBppFF) and yellows (eeFF) carrying ckck, ckcd or cdcd though much less than with C, and even some darkening in the corresponding heterozygotes ckdcra. Russell, Ginsburg, and Foster all found no evidence of a reaction from the near blacks of genotype crcr. It would appear that the enzymes produced by the c compounds tend to be lost, after forming the natural pigments, almost as rapidly as they are produced unless produced in great excess by C. We have already noted the thermolability of the product of the lower c alleles which would especially affect Foster's studies of homogenates from fetal skin. Foster found that homogenates from pale sepias (EBCppF) and pale browns (EbbCppF) oxidized tyrosine or dopa significantly less rapidly than homogenates from intense blacks or browns (P in place of pp) but the reduction was far from proportional to the great reduction in amount of natural pigment (in contrast with his results from lower c compounds) . This agreed with Kroning's results in hair735 and Russell's results at least in the epidermis. Replacement of F by /caused no reduction in tyrosine or dopa oxidase activity in intense blacks or browns. With the same replacement in the presence of pp, the moderately strong reactions were reduced to zero (tyrosinase) or very weak (dopa oxidase) according to Foster. These parallel the striking interaction effect in vivo {EPF and EPff, equally intense; EppF, pale; Eppff, very pale). The dopa 172 PHYSIOLOGIC GENETICS reaction was, however, moderately strong in frozen sections and indistinguishable in pale cream and yellow areas of 14 tortoise shells of genotype epepCppjf (W. L. Russell, unpublished results of F. Appel and of L. B. Russell). These reactions were, however, significantly less than in pale sepias (ECppF) and reds (eeCF). It appears that in the eumelanic series F(in ppF) and even /(in ppff) substitute for P somewhat more effectively in oxidising added tyrosine or dopa than in producing natural melanin. Replacement of B by bb causes no reduction in tyrosinase or dopa oxidase activity by any test. There is, indeed, considerable evidence that the reactions are stronger with bb, even in the extreme dingy browns. It appears that B, b takes no direct part in the enzyme system responsible for oxidising added tyrosine or dopa. Perhaps again greater utilization in blacks than in browns is associated with greater loss. CONCLUSIONS ON COLOR INTERACTIONS It may be seen that the products of the color factors enter into a rather compli- cated pattern of interactions before formation of the final products: sepia, brown, or yellow pigment granules. The pattern resembles somewhat the branching chains of reactions worked out for gene-controlled metabolic reactions in microorganisms. Some of the steps may indeed be of similar nature, since most of the factors probably act in the nuclei of the pigment cells themselves as demonstrated in mice by Reed and Henderson.1044 Others, however, probably act from adjacent epidermal cells as al- ready noted. The difference between adult male and female dingy browns is probably under endocrine control. Some seem to be concerned directly with the pigment process but others undoubtedly affect pigment merely because of effects on the vitality or metabolic efficiency of the pigment cells. HAIR DIRECTION We will consider next a morphologic character, hair direction (figure 27) . In wild cavies, the hair is directed away from the snout on the body, with minor qualifications, and towards the toes on the legs. On introducing the dominant gene R into a genotype that is otherwise like that of the wild species (rrMMrerestst) there is more or less reversal on the feet, especially the hind feet (grade E) and occasionally a slight crest along the back.1428' 1429 Those with RMm are highly variable, but most of them have either a strong dorsal crest (grade D) or a single pair of rosettes, halfway along the back (grade C). Some of those called grade C had only a single dorsal rosette. This was almost always (96 per cent) on the right side, a curious example of regular asymmetry. A few were called grade E and many in certain strains were called grade B, or better, CA, with two pairs of dorsal rosettes (as in grade A) but no rosettes on head or belly (as in grade C) . With Rmm, there are typically two strong pairs of dorsal rosettes, anterior and posterior, radiation from the ears, a strong forehead rosette, eye, check, and groin rosettes, and feathering along the midline of the belly (grade A) . The GENIC INTERACTION 173 number of dorsal rosettes varied in both directions. It might be at least doubled (grade A++) or it might be reduced to two (grade AC). In cases of asymmetry, rosettes again tended to occur more on the right than on the left side. Genes M, m are specific modifiers of R with no detectable effect in its absence. Other specific modifiers were clearly present. As indicated above, high grade RMm (grade CA) differed markedly from low grade Rmm (grade AC). It appears that the number of dorsal rosettes was especially subject to modifiers, 2 [MRd), in both of these genotypes. The effects of these modifiers were, however, confused by a very Fig. 27. Diagram of factor interactions in the determination of hair direction and PLEIOTROPIC EFFECT ON WHITE FOREHEAD SPOT IN THE GUINEA PIG. StSt>Sts1 St St RR-Rr R r / — - / i i MM (I) / Mm (1,2) u /— mm (1,2,3) M m -X / Growth pattern n skin ReRe>Rere Re rj £(MRe C Melanoblast / \V//' Forehead--' Process //A ~v6] /// /\ -\.(2)-^Back^[*Mid R " [3LAnterior Process (3) Posterior Hind feet Fore feet White spot considerable amount of nongenetic variability. Thus the regression of offspring on midparent in matings of Rmm x Rmm, including an excess of the extremes AC and A++, was only 0.48, indicating that only about half of the variability was genetic. Three generations of selection did not suffice to fix grade AC.1428, 1429 As indicated above, eye rosettes ordinarily appear only in roughs of high grade (Rmm). Strong eye rosettes were, however, recorded in four individuals that were otherwise of grade E and of genotype RMM, from separate matings that were only remotely related and had no record of the character in their ancestry (other than in grade A). A considerable number (171 born alive, 21 born dead) were derived from one of these lines but a complete analysis of the genetics was not made. There are, however, a number of points of interest with respect to interaction in the unpublished data. The character proved to be a very troublesome one because of intergradation with the condition in normal, smooth-furred animals (rr) which themselves show a slight divergence of hair direction about the eyes. Among those considered to have the 174 PHYSIOLOGIC GENETICS anomalous eye rosette and born alive, 96 were recorded as having a strong pattern and 75 a weak one. There was rather frequent irregular asymmetry, including a few cases in which there was a strong rosette about one eye and none about the other. This indicates that developmental accident played a considerable role. In matings in which Mm and MM were segregating, there was no significant difference in incidence of eye rosettes among grades E, D, and C contrary to expectation in view of their invariable occurrence in grades AC and A, genotype Rmm. There was also no relation to presence or absence of a forehead rosette in matings segregating in Stst and stst, discussed later. On the other hand, segregation of R and rr made a great deal of difference. Thus a group of matings that produced 16 per cent with eye rosettes among 208 RM young produced only 2 per cent among the 217 rr young. A group that produced 55 per cent among 88 RM young, produced only 20 per cent among the 46 rr segregants. Only liveborn are considered here because of somewhat greater uncertainty in classification of stillborn. Thus R greatly favors manifestation of the character but is not necessary. Strong eye rosettes were present in eleven other- wise completely smooth animals (rr). The highly sporadic occurrence at first, including such records as 1 in 15, 16, or 17 RM young from scattered matings between normals, of 0 in 27 and 2 in 29 from matings of strong x normal and of 3 in 15 from a mating of strong x strong, suggested either low penetrance or a recombination effect of two or more genes. After all young were examined carefully for weak eye rosettes at both birth and weaning (when they are often more conspicuous), it became apparent that penetrance at this level could be high at least in RM young. The pattern of occurrence indicates a single essential gene, Re (rough eye) . The results of outcrosses indicated dominance in some degree but with penetrance far from perfect in heterozygotes carrying RM and low but not zero in ones carrying rr. There was an approach to 100 per cent penetrance but not expressivity of RMReRe, but only moderate penetrance in rrReRe. Another aspect of the full rough pattern of Rmm, the forehead rosette, can also appear in the absence of R.li29, 1430 All animals in the colony of this type traced to three obtained from Mr. I. J. Wachtel. This character, Star (St), behaved in a remarkably different way from rough eye (Re) in two respects. First, it showed no intergradation with normal and behaved as a simple dominant in eight successive backcrosses to lines in which it had never been present and there was no difference between rrStSt and rrStst.li29- 1430 Second, instead of showing enhancement in the presence of R there was a tendency toward reciprocal inhibition.1430 In rrSt-, the forehead rosette is almost invariably (99.6 per cent) single and very flat. In about 19 per cent of RMMSt it is replaced by two weak rosettes. This forehead roughness is combined with typical grades D or E but the proportion that are grade D is reduced. In RMmSt-, this reduction of the rough pattern goes farther. Most show roughness of grades D or even E combined with a forehead rosette that is doubled in 64 per cent. In the 1 7 per cent called grade C the rosettes were usually weak and lateral. The forehead rosette was weakened in 78 per cent. Finally in RmmSt in which an exception- GENIC INTERACTION 175 ally strong forehead rosette might have been expected by combination of the effect of Rmm and of St, there was actually only a slight irregularity of hair direction on the forehead. The anterior dorsal rosettes of grade A were weak and lateral in position, leaving a broad smooth shield anteriorly on the back. This last feature refers to RmmStSt. In this case only, St ceased to be completely dominant and RmmStst showed a great variety of intermediate conditions of the dorsal rosettes. It is interesting that RMM and RMm tend to inhibit the effect of St in a part of the coat, the forehead, in which they have no visible effect in the presence of stst and that St in turn inhibits the effects of RMm and Rmm in a region, the anterior back, in which it has no visible effect in the absence of R. In view of these effects, it ceases to be surprising that Rmm and St interact to prevent development of the forehead rosette which each determines by itself. Gene St has an interesting pleiotropic effect in a tendency toward a white spot in front of the center of the forehead rosette. It was shown by Bock114 that this does not require even heterozygosis in the spotting factor and that it tends to be inhibited by R, especially Rmm. Table 42 gives a condensed summary of his results for young with no Table 42 Percentage of white forehead spot in guinea pigs with no white outside of the forehead Grade stst St- (excluding Typical Per cent Per cent forehead) genotype No. spot No. spot Smooth rr — 294 2.4 530 46.6 Rough E R-MM 27 3.7 116 42.2 Rough C, D R-Mm 75 8.0 181 37.0 Rough A, B R-mm 59 5.1 164 15.2 Total 495 3.7 According to grades of roughness due to Rr, Mm and to presence (St) or absence (stst) of forehead rosette from matings that involved St. white outside of the forehead and from matings that involved St. The results indicate that the white forehead spot is a secondary consequence of the effect of St on the skin of the forehead, manifested in the forehead rosette. The neutralizing effect of R on the latter, especially in the combination R-mm, also tends to neutralize the white spot- ting. Another element of the full rough pattern of Rmm, the feathering on the belly, has also been observed independently of R in a certain somewhat inbred strain.1416 It followed an irregular course of inheritance, neither a simple dominant nor a simple recessive. Dorsal irregularity of hair direction has also occurred independently of R but so sporadically that mere developmental accident seemed indicated. The most important general result of this study of the genetics of hair direction seems to be another revelation of the extraordinary diversity in the relation of genes to characters, and in 7 76" PHYSIOLOGIC GENETICS particular in kinds of interaction, that is to be found in this case in even a rather limited genetic system. POLYDACTYLY Attention is now directed to a different sort of morphologic character, the occur- rence of atavistic digits. The guinea pig, like all species oiCaviidae, lacks the thumb, great toe, and small toe. A fourth toe, exactly like the small toe of related rodents, is not, however, uncommon. Among 22 inbred strains there were 1 1 that were invariably 3-toed on the hind feet (including strains 13 and 32), the small toe appeared sporadic- ally in five (including strain 2, the major branch of which, however, was entirely free of the trait) and in six the incidence was fairly high (including strain 35). 1445, 1446' 1447 A strain, D, with 100 per cent occurrence of a well-developed small toe was produced by Castle181 by selection. Crosses were made between strain D (4-toed) and the 3-toed strains 2, 13, and 32 and between D and strain 35 with 31 per cent in the branch des- cended from a single mating in the 12th generation.1451 The results from 2 x D and 32 x D gave complete dominance of 3-toed in Fl5 except for one individual in 26 in the latter, and passable 3 : 1 and 1 : 1 ratios in F2 and the backcross to strain D respectively. These results suggest segregation of a single, essential, recessive factor for the small toe but tests of the supposed segregants from (2 x D) x D gave results that completely vitiated this hypothesis. The 3-toed segregants produced only 23 per cent 3-toed (in 186), whereas the 4-toed segregants gave nearly the same result (16 per cent 3-toed in 119). The most plausible hypothesis seemed to be cumulative action of multiple factors on the underlying physiology and two thresholds: one for any development of the small toe, below which there is homeo- static control of the normal 3-toed foot and, slightly higher, a threshold (or better, a ceiling) above which development of the small toe is controlled homeostatically, or canalized in Waddington's1352 terminology. The cross 13 x D gave 67 per cent 3-toed and 33 per cent 4-toed in Fx. Tests of the two Fx types gave results that did not differ significantly either in F2 or in the backcross to D. There was no simulation of one- factor heredity, but the results fit well with the interpretation of multiple factors and two thresholds. Most of the young from 35 x D were 4-toed. The results in F2 and the backcross to 35 again fit the above hypothesis. The results from these crosses in F1 and F2 are represented in figure 28 according to this threshold hypothesis. There was considerable variability in strain 35 in relation to age of mother (high percentage of 4-toed from immature mothers) and season (excess of 4-toed in winter and early spring) ,1414 The standard deviation on the physio- logic scale can be determined as that of the normal curve that yields the observed trichotomy, taking the interval between the thresholds as the unit. It is approximately 0.80. The same standard deviation was assumed to apply to the other inbred strains and to the three Fj/s. The mean of D was put at 2.5a (2 units) above the upper threshold as a value which might be attained by selection but at which overlap of the GENIC INTERACTION 7 77 threshold would be rare. Because one 4- toed had been obtained in Fj (32 x D), a cross which gave results very close to those 2 x D in F2 and the backcross to D, the mean of (2 x D) was put only 2.5a below the lower threshold. The means of the pure strains 2 and 13 were located on the hypothesis that Fx in each case was exactly half way between the parental strains on the physiologic scale. Fig. 28. Factors determining development of the small toe. 3-toe' 2 13 35 poor I good 4-toe1 4-toe (2*D)2 03xD)2(35xD)2 -9 - Theoretical distribution of factors determining development of the small toe on an underlying physiologic scale in relation to thresholds for any small toe and for perfect development, in four inbred strains, in Fx and F2 of crosses of one (D) with the other three. The relations of variability in F2 (2 x D) to the interval between the means of these two most extreme strains permit a minimum estimate of the number of gene differences, assuming equal effects and no dominance. The number is approximately four. The hypothesis of one major factor, responsible for half the difference and practically all of the variance plus a great many minor factors, fits equally well. So 178 PHYSIOLOGIC GENETICS does the hypothesis of a great many factors with contributions in geometric series (40 per cent: 24 percent: 14.4 per cent: 8.6 percent, and so forth).1448 Results in third backcrosses best fit an intermediate hypothesis somewhat similar to the last. It is assumed that the effects are additive on the physiologic scale. However, the factors were not isolated. It may be suspected that, if they had been isolated, a more complex situation would have been found with specific interactions as in the cases of color and hair direction. As indicated above, the hypothesis of two thresholds permits considerable elasticity with respect to the effects on the underlying scale. Similar small toes have been restored in a wholly different way, in this case often associated with thumbs and great toes resembling those of other rodents.1413 A single mutant individual showed those characters imperfectly developed. From thousands of descendants it appears that a dominant gene (Px) with variable penetrance was respon- sible. In the stock of origin, about 82 per cent showed one or more atavistic digits. After certain crosses, penetrance fell to 20 per cent or less. Crosses with strain D revealed interesting interaction effects. It was not surprising that penetrance of the small toe in Pxpx rose from 62 to 100 per cent by combining these two heredities which favor it. More interesting is the fact that penetrance of the thumb rose from 74 to 100 per cent, although thumbs were wholly absent in strain D. Similarly, the pene- trance of the great toe rose from 2 to 18 per cent after one backcross to D and to 55 per cent after two backcrosses. The homozygote PxPx turned out to be lethal and grossly abnormal. It was found by Scott1184 that about 92 per cent died and were absorbed at about the twenty- eighth day of gestation. Those that reached birth 1185 had short legs, rotated hind legs without tibiae, and broad paddle-shaped feet with 7 to 12 digits each. The animals were always microphthalmic and either hydrocephalic or with bi'ains protruding from the skull, usually harelipped, and grossly abnormal in most of their internal anatomy. The defect was manifest at about 18 days of gestation in limb buds of double width and overgrown midbrains and hindbrains. This constitutes an extreme case of pleiotropy. The atavistic return of digits, lost in the evolution of the whole family Caviidae, raises a question on the nature of homology. Homology is usually treated as an all-or- none matter: particular organs of two species either are or are not homologous. The language used in discussing evolution of organs often seems to imply preformation of an extreme sort, a heredity evolving separately for each part. It is obvious, however, from the genetics of morphologic characters in all organisms studied, that replicative homologs develop to a large extent under the same systems of genes. Thus genotype RRMM has similar effects on hair direction and the underlying pattern of growth of the skin, not only on right and left hind feet but on hind feet and fore feet (with some difference in threshold) and on the separate digits on these feet (a pattern of partings along the sides and across the upper side of these digits shaped like a letter H). The situation is similar with the genes that tend to restore the pentadactyl foot. It is supposed that all of the genes under which any part of the ances- tral pentadactyl foot developed were so deeply involved in the development of other GENIC INTERACTION 1 79 parts of the foot and the organism as a whole that most of the genetic system concerned with thumbs, great toes, and small toes is necessarily still present. The loss of these digits is presumably due to some simple, superimposed, inhibitory process that stops formation of the normal number of lobes on the developing limb buds. Any genetic or environmental effect that inhibits this inhibitory process releases the substrate for action of the whole array of genes that shaped these missing digits in the remote ancestors. Homology, whether replicative in the same individual or phylogenetic, must be con- sidered to be a matter of degree, the result of calling into play more or less similar systems of genetic reactions by more or less similar developmental processes.1434 The multifactorial Polydactyly may conceivably involve ancestral genes that have been carried at low frequencies throughout the history of the Caviidae or have been brought back by reverse mutation, but this is hardly likely for Px. Yet if Pxpx acts merely by inhibiting a relatively simple inhibition acquired in evolution, the thumb, small toe, and great toe that develop in this genotype under the released ancestral heredity may be considered as essentially homologous to these digits in the mammals that have never lost them, in spite of the monstrous characteristics of the foot in PxPx. Thresholds have been shown to play a major role in other morphologic deviants of the guinea pig of which otocephaly has been most intensively studied.1433, 1455, 1458 It may be added that Griineberg501 reports that similar "quasicontinuous" variation interrupted by thresholds is the commonest situation in the mouse in the case of morphologic deviants. TYPES OF INTERACTION The effects of factor replacement under varied genetic or environmental conditions may be put into four categories. Constant effects. — There is usually constancy of effect in cases in which the replace- ment in question is associated with variations in a character that seems obviously un- related; for example, R, rr (rough and smooth fur respectively) and any pair of colors. This also often holds for widely different aspects of a character that is single only in a broad sense; for example, A, aa {agouti and nonagouti respectively) on a black (B) or brown (bb) background. With respect to grades of a single quantitative character, constancy of effect requires somewhat arbitrary definition. One may, for example, choose to treat either a consistent additive effect or a consistent multiplicative effect as a constant effect. Strict examples of either of these types of constancy are, however, probably uncommon. They were not found in the quantitative studies of intensity of the colors of the guinea Pig- Cumulative but not constant effects. — There are many examples of this among the guinea pig colors. Thus on assigning 100 as the measure of quantity of yellow pigment with CF, that with cdcdF was 38, with Cff 33 and with cdcdff 5. Any two-factor case such as this could be made additive arbitrarily by a suitable transformation of scale, I HO PHYSIOLOGIC GENETICS just as multiplicative constancy can be converted into additive constancy by the use of logarithms. Such transformations are often useful but usually break down more or less in more complicated systems. No effect in some combinations. — The frequent reduction of the F2 ratio 9:3:3:1 to 9 : 3 : 4 or to 12:3:1 indicates the frequency of specific modifiers of the effect of a domi- nant or of a recessive gene, respectively. Thus in guinea pigs carrying EBF, CP is black, Cpp is pale sepia, but either cacaP or cacapp is white. In those carrying EBC, either PF or Pffis black, ppF is pale sepia, and ppff is pale brownish cream. The effect of M, m in rough (R) and smooth (rr) guinea pigs illustrates the case of a specific modifier of a dominant that itself lacks dominance (Rmm, full rough; RMm, inter- mediate rough; RMM, rough only on the toes; but rrMM, rrMm, and rrMM all equally smooth). There may be mutual instead of one-sided dependence as in the cases of comple- mentary dominants (F2 ratio 9:7), complementary recessives (F2 ratio 1 5 : 1 ) , or comple- mentary dominant and recessive (F2 ratio 13:3). In guinea pigs, CP gives dark eyes, but Cpp, cacaP, and cacapp are all pink-eyed (although Cpp can often be distinguished by traces of color). With sisidmdm, guinea pigs are anemic, spermatogenesis is absent in the diminutive testes of males, and fecundity is low in females, whereas with either Si or Dm or both, these are normal. There are, however, cumulative effects of Si, si, Dm, dm on intensity of color. All such two-factor cases can be treated as multiplicative by assigning zero to the effect of one member of one or both pairs of alleles and choice of a suitable scale, but this sort of constancy of effect cannot as a rule be extended to all combinations. Threshold and ceiling effects of a more complex sort may also be included in this category. All of the colors of the guinea pig are subject to threshold effects. A ceiling effect is most conspicuous in the case of the dark-eyed browns, especially when adult. The irregular penetrance that is characteristic of most mutational effects on morphologic characters implies a threshold. The occurrence and variations in degree of develop- ment of the small toe give an example of a case in which there is homeostatic control of development both below a threshold and above a ceiling. Opposite directions of effect in different combinations. — In guinea pigs with CPff, those with E (black) have much more pigment than those with ee (dilute yellow) whereas with Cppff, those with E (pale brownish cream) have much less pigment than those with ee (same dilute yellow as in the preceding case). The intensifying effects of C, P, and F in brown (Ebb) up to a certain point and their dilution effect beyond this point is another example. In this case, Pp always shows overdominance, a phenomenon in which the alleles necessarily reverse their order of effect in different compounds. The increase in intensity after birth of cd sepias and yellows, in contrast with the decrease in intensity after birth of ck sepias and yellows, illustrates reversal of effect of a non- genetic condition in different genotypes. In this case ck sepia is darker than cd sepia at both ages, and ck and cd yellows hardly differ at birth. In the case of comparison of intense browns (EbbCPp) and sepias of genotype GENIC INTERACTION 181 EBcdcaP at birth and as adults there is reciprocal reversal of order of effect, the order of intensity being adult sepia (highest), newborn brown, newborn sepia, and adult brown. The case of rrSt and Rstst, both with strong forehead rosettes, RSt with mere irregularity on the forehead, and rrstst, smooth, {mm present in all) represents an example in which reciprocal inhibition by two dominants causes reversal of direction of effect in two pairs of loci. SELECTIVE VALUE Interaction effects of this fourth type are undoubtedly less common among simple characters than are those of the second or third type. There is, however, a complex character in which such interaction effect must be almost the rule. This is selective value, which, in relation to the total environment of a given population, is the character that is all important in the evolution of the latter, with the qualification that an array of genotypes may be even more important than any one genotype. The reasons for the prevalence of this sort of interaction are partly indicated in the following quotation from an earlier paper1422 which is cited because a recent account of the history of population genetics860 attributes to it the assumption of absolute selective values for each gene and thus of no interaction. The actual assumptions in this respect (and in most others) were the opposite of those stated. "Selection, whether in mortality, mating or fecundity, applies to the organism as a whole and thus to the effects of the entire genie system rather than to single genes. A gene which is more favorable than its allelomorph in one combination may be less favorable in another. Even in the case of cumulative effects, there is generally an optimum grade of development of the character and a given gene will be favorably selected in combinations below the optimum but selected against in combinations above the optimum." The sort of case referred to last is illustrated in the upper part of figure 29 in which it is assumed that a character varying quantitatively is determined by four pairs of alleles with equivalent effects and no dominance. It is supposed that selective value falls off from the optimum at the midgrade in proportion to the square of the deviation. There are six optimal genotypes in this case, each distinguishable from each of the others by at least two homozygous replacements. With more loci and multiple alleles at each locus, the number of optimal genotypes in this sort of case may be enormous.1423 It is assumed, for simplicity, that the effects of the genes are additive on the under- lying physiologic character. It is probable that, in any real case, the situation would be complicated by specific interactions as in the case of coat and hair direction. The case of coat color may, indeed, be cited as such an example. The cream agouti color of the wild ancestor of the guinea pig (Cavia cutleri) is presumably the optimum in nature. It is intermediate in intensity and can be simulated more or less in at least four independent ways, none of which are similar to it genetically.1427 The quotation above does not refer to a point that was taken up later in the 1 93 1 182 PHYSIOLOGIC GENETICS paper, the probable universality of pleiotropy and its consequences. Pleiotropy makes it unlikely that any allele is the most favorable in all respects even in a given combination with other genes. High selective value in a genotype requires a great deal of compensatory interaction among the genes. The consequence is a tendency toward overdominance and interaction among loci of the fourth type. Fig. 29. Selective values of alleles. _W 1.0000 0.9375 0.7500 0.4375 abed _W 0.2500 0.1250 0 Abed ABcd ABCd aBcd AbCd ABcD abCd AbcD AbCD abcD aBCd aBcD abCD aBCD ABCD aabbAabbAAbbAABbAABB oaBbAaBbAaBB. aaBB Above: Selective values assigned homozygous combinations of four pairs of alleles, assumed to have equivalent effects on a quantitative character, but intermediate optimum. Below : Selective values assigned to additive pleiotropic effects of two of the pairs of alleles. In the case of an intermediate optimum illustrated in the upper part of figure 29, the six optimal genotypes all have the same value of W so that it would be a matter of indifference which one is established in evolution. This ceases to be the case if there are small pleiotropic effects. It is assumed in the lower part of figure 29 that genes A and B have additive pleiotropic effects while C and D do not. Figure 30 shows the selective values ( W) of all homozygous genotypes except for those at the extremes of the quantitative character {abed with W = 0 and ABCD with W = 0.25). The six peak genotypes are now at three levels: one low, four intermediate, and one high. In the case of a character that is dependent on cumulative action of multiple factors above a threshold or below a ceiling, even selection directed toward one of these GENIC INTERACTION 183 extremes is likely to lead to multiple peak genotypes in the neighborhood of the threshold or ceiling on the underlying scale, on the hypothesis that the pleiotropic effects of the favored genes are largely deleterious.1427 Fig. 30. Mean selective values of 14 homallelic populations. 1 1.25 1.00 abCD A\ 0.75 Based on the selective values of figure 29. and abed (W = 0) are omitted. Population homallelic in ABCD ( W = 0.25) The effects on the underlying scale need not be uniform. Figure 31 illustrates a case in which a mutation (M') has an effect that offers the promise of a major step in advance but has other effects that make it highly deleterious. By introducing modifiers A', B', C", D', E', F' that neutralize these other effects of M' and have only very slightly deleterious effects otherwise, the direction of effect of M' may be reversed in a combination that is far superior to the initial one. THE IMPORTANCE OF INTERACTION IN EVOLUTION There may be some genes that have such unequivocally injurious effects that these cannot be reversed by any combination with other genes. In general, however, it seems safe to conclude of any pair of alleles that one is more favorable in some combina- tions, the other in others. The primary condition for an effective evolutionary process would seem to be that selection operate to increase the frequencies of favorable multi- factorial genotypes (or even systems of genotypes) rather than of single genes because of their favorable net effect in the population in question. The least effective evolutionary process is the one that is still, perhaps, most widely accepted : evolution as a succession of rare favorable mutations, each gradually displacing its type allele in what is at each stage essentially a single type genotype. 184 PHYSIOLOGIC GENETICS Morgan896 stated this viewpoint as follows: "If we had the complete ancestry of any one animal or plant living today, we should expect to find a series of forms differing at each step by a single mutant change in one or another of the genes, and each a better-adapted or a differently-adapted form from the preceding." Under this theory Fig. 3 1 . Mean selective values of populations homallelic with respect to a MAJOR PAIR OF ALLELES (M, M') AND SIX MODIFIERS. Initial Selective Peak M M M M ABCDEF A'BCDEF A'B'CDEF A'B'C'DEF A'B'C'D'E'F Later Selective Peak IvV a'b'c'd'e'f' etc. etc. etc. M M M a'bic'd'ef A^'c'rJE'F a'b'c'd'e'f' etc etc. there is no recombination, and interaction effects are significant in evolution only in a restrictive sense. The other two theories to be considered here are based on the concept that the population is something more than a lot of individuals of the same genotype. Under them a population is characterized genetically by an array of genie frequencies. The elementary evolutionary process becomes a change of genie frequency.373, 516, 1422 The observations which led to this concept were indeed largely on such obviously variable populations as those of man, cultivated plants, domestic animals, and laboratory rodents rather than on wild species, although some support could be drawn from such GENIC INTERACTION 185 studies as those of Sumner1302, 1303 on the genetics of variability within and between subspecies of the deermouse Peromyscus maniculatus. More recently this general view- point has received increasingly massive support in species of Drosophila under the leadership of Dobzhansky, 283 and we have an enormously richer concept of what the genetics of such wild species is actually like. We consider first the case of a species (or subspecies) that is essentially homo- geneous throughout its range. Because of the conditions of balance that maintain the array of alleles at each locus, this would consist largely of isoalleles. The virtually infinite array of possible recombinations would insure the genetic uniqueness of every individual even though there would be little conspicuous variability. Since, however, the recombinant genotypes are broken up in each generation by the reduction division (with qualifications that are unimportant unless linkage is nearly complete and selection very strong), selection is based only on the net effects of the genes. If the absolute selective values of genotypes are independent of the genie frequencies, Fisher's373 fundamental theorem of natural selection holds: "The rate of increase of fitness of any organism at any time is equal to its genetic variance in fitness at that time." Genetic variance was here defined as merely the additive component. Thus favorable interaction effects of genes with unfavorable net effects cannot be utilized any more than under the first theory. There is no way by which a species can work its way from a lower to a higher selective peak with respect to mean selective value. Once the population has arrived at a selective peak, further evolution can occur only by a change of conditions or a wholly favorable mutation, both of which change the whole system of selective values. If, however, such a change occurs, the heterallelic character of the population permits an extensive readjustment on the basis of interaction effects until a new peak is arrived at, a process that cannot occur under the first theory. In the third theory, it is assumed that there is sufficient isolation of small local populations (denies) in at least some part of the range of the species to permit significant genetic differentiation, but not so much that a successful deme cannot modify the genetic composition of less successful neighboring demes by emigration and crossbreeding.1422 The introduction of immigration pressure into the conditions of balance at each locus makes for much more strongly heterallelic arrays, locally as well as in the species as a whole, although these conditions are still such that these arrays consist largely of isoalleles and minor modifiers. In such a species, population is continually welling up in some places, falling off in others, but the sites of the population sources and sinks may be changing from time to time. This process of interdemic selection supplements the continuous process of intrademic change of the sort considered in the second theory. With a suitable balance between selection and immigration on the one hand, and between the resulting tendency toward and away from equilibrium from the cumulative effects of random processes on the other, the array of genie frequencies may occasionally pass from control by one selective peak to control by another. Figure 32 shows the paths which the array of genie frequencies tends to take under selection alone in two 186 PHYSIOLOGIC GENETICS small parts of the four-dimensional field of mean selective values {W) expected from the selective values in figure 30. In addition to selective peaks and pits in the corners, there are shallow selective cols between the lowest peak (abCD) and the intermediate one that is shown (aBCd), and between the latter and the highest peak (ABcd). Figure 33 shows the mean selective values along the most favorable path connecting the lowest peak with the intermediate and this with the highest (as well as a less favorable direct path from lowest to highest through a four-dimensional col not shown in figure 32). The mean selective values of the gene-frequency systems of figures 32 and 33 were calculated from the selective values in figures 29 and 30, using formulas 16 and 19 in a 1935 paper 1444 with respect to interaction effects. Fig. 32. Trajectories of gene-frequency systems. ABCd ABcd 1.000 — *~-r* »*l.250 aBCD 0.875 t£99C^ v^ t/ 1.000 abCD 0.875 aBcd -0.750 abCd Trajectories of gene-frequency systems on surfaces of selective values on two faces of the four-dimensional field defined in figures 29 and 30. Random processes need shift mean selective value only 8 per cent as much counter to selection as would be involved in fixation of gene B. This figure brings out the reason why I have held that fixation from unbalanced random drift (the Hageodorn effect) is of no importance in progressive evolution. As this is the point on which my position in the 1931 paper has been misinterpreted most frequently, I will give another quota- tion from it.1422 The reference is to an extremely small, completely isolated popula- tion. "In too small a population, there is nearly complete fixation, little variation, little effect of selection and thus a static condition, modified occasionally by chance fixation of a new mutation, leading inevitably to degeneration and extinction." It should be said that the conditions of balance are such that passage directly from one selective peak to another is not likely to occur except from arrays of genes with GENIC INTERACTION 1H7 Fig. 33. Mean selective values of gene-frequency systems. Selection h- — I Selection Random drift Random drift aBCd (1.125) Random drift ABcd .250 abCD (1.000) abed (0.875) abCd (0.750) Mean selective values of gene-frequency arrays along the path of least depression from the lowest peak abCD through an intermediate peak aBCd to the highest peak ABcd, of figures 30 and 32, and along the direct path from the lowest to the highest peak through the four-dimensional col. only slight differences in their net selective values. But as can be seen from figure 3 1 , the process can apply indirectly to a pair of alleles (M, M' ) with major differential effects. Random drift may lead to sufficient frequencies of several of the very slightly deleterious modifiers A', B', C, D', E', and F' at some time in some deme to reverse the selection against M' which, with its associated modifiers, will then start to spread through the species. Table 43 makes a comparison between the selective process in a homogeneous species373 and the double process (intrademic and interdemic) in a finely subdivided species.1422 The conditions of balance among net selection coefficients, immigration, and random drift may not have held anywhere at any time in the ranges of some species. They almost certainly have held in others (for example in primitive man and many other mammals) . Where they have not held at all, the evolutionary process has been restricted to that of the second theory. The first theory is merely the limiting form of the second under conditions in which all loci are only very weakly heterallelic. The three are really complementary. 1HH PHYSIOLOGIC GENETICS Table 43 Comparison of evolutionary processes in homogeneous and subdivided populations Homogenous population Population subdivided into numerous partially isolated demes Entity selected Source of varia- tion Process (1) Conditions static (2) Conditions change Gene, differing from alleles in net selective value Gene mutation Selection among individuals Progress restricted to exten- sion of currently control- ling peak Progress up most available peak in new surface of mean selective values Set of genie frequencies, characterized by harmonious genie effects, con- trolled by a selective peak Shift in controlling peak in a deme (by random drift and selection toward new peak) Selection among demes (by differen- tial growth of population and migration) Continual shifts in prevailing selective peak Interdemic selection relative to all available selective peaks in the new surface of mean selective values A major evolutionary step under the third theory, as discussed above, simulates fixation of a favorable mutation in the first or second, but in this case is a byproduct of a continuous process of intrademic and interdemic selection that is occurring below a superficial appearance of near-uniformity in the species. The theory helps account not only for occasional major steps in evolution less miraculously than in the other theories but also for the extraordinary perfection of fine details that we find in the pro- ducts of evolution. The essential difference is between a theory in which selection can operate in the virtually infinite field of interaction effects of recombinants instead of almost exclusively on the net effects of each separate gene. DISCUSSION Dr. Burdette: Thank you, Dr. Wright. The discussion of Dr. Wright's paper will be opened by Dr. Herman Chase. Dr. Chase : This is an excellent and exhaustive paper, leaving nothing to be taken away and very little to add. Compared with the guinea-pig work of Dr. Wright, there has been relatively little done with mice in the way of genie interaction. Since this is a symposium supposedly dealing with methodology, I would like to point out what I consider to be the four main approaches or general methods in physiologic genetics. They are: first, genie substitution; second, genie interaction; third, comparison of genetic constitutions; and fourth, teratogens. Simple genie substitution with resulting analysis of the varying phenotype is certainly used the most and is basic in work with GENIC INTERACTION 189 Neurospora, bacteria, maize, mice, and so forth. The comparison of genetic constitu- tions, as when nutritional requirements, sensitivities to drugs, and the like of inbred strains are compared, is another approach often used. The effect of teratogens, the production of phenocopies, is a method which has some merit when cautiously inter- preted. Genie interactions are fundamental in the complex of interrelationships involved in the realm of physiologic genetics, the genetics of expression; but genie interaction (the second approach) as a deliberate method of investigation has been used all too little, except for Dr. Wright's long-term studies on guinea-pig coloration and hair direction. Relating directly to the details of this paper, pink-eyed dilute (p) in combination with Light (Blt) prevents basal dilution in the mouse, a situation somewhat analogous to one mentioned by Wright. Recently we have discovered a possible interrelationship between murine Polydactyly and anophthalmia. In no case, however, have we made a systematic attempt to combine our mutants for study in the Wrightian manner. Murine genetics during the past 30 years has placed more than enough emphasis on the development and maintenance of inbred strains and on single-gene differences; perhaps now more should be done to explore genie interactions. After all, as Dr. Wright said at the beginning, "genie interaction ... is obviously fundamental in physiologic genetics and is almost as fundamental in population genetics, including the genetic aspects of the theory of evolution." Whereas he said in his introduction that he was riding the two wild horses of physiologic genetics and population genetics, I would like to make the observation that the one horse of physiologic genetics, tamed by Dr. Wright, has been enough for me. Dr. Popp: Frank Moyer at Johns Hopkins University has been interested in differences among pigmented granules of murine melanoblasts as regards possible differences in tyrosinase in such cells;914 information on structural differences among tyrosinases of the mouse may be forthcoming in a year or two. Dr. Russell: Dr. Douglas Coleman at the Jackson Laboratory also has under way a splendid program analyzing genetically determined differences in tyrosinases in mouse skin.213 Dr. Yerganian: With respect to outlining the genes involved in diabetes mellitus of the Chinese hamster, Cricetulus griseus, we have conducted appropriate hybridizations involving symptomatic animals representing four different strains. To our surprise, instead of the anticipated retention of the high incidence of diabetic animals among the Fx of single- and double-hybrid crosses, the frequency of symptomatic hamsters was virtually nil. The syndrome865, 866 reappeared after three and four generations of brother-sister matings. We have estimated the number of genes to be two, in addition to an unknown number of modifiers that control the degree of penetrance and age of onset. These observations have been repeated on numerous occasions, but the role of modifiers remains to be fully disclosed. Since diabetes failed to occur following the hybridization of symptomatic animals, the role of modifiers is most perplexing. An interaction among the genes as well as 190 PHYSIOLOGIC GENETICS the environment is strongly implicated. An example of the former or genie role is the fact that diabetics have a sixfold increase in alpha-2-serum proteins whereas hybrids, stemming from two or four diabetic parents from different inbred families, exhibit low or normal levels of this diagnostic protein.494 Dr. Degenhardt: Is there any information about a preferred time for genie interaction in early embryonic development ? I am thinking only of Polydactyly. Dr. Wright: Dr. Scott worked out the embryology of the polydactylous monster. About the seventeenth or eighteenth day he found broad, paddle-shaped limb buds and that the hindbrain was growing too much for the rest of the body, causing it to bulge out through the top of the head. Probably action of the gene occurred much earlier than that. Dr. Degenhardt: We found in our investigations on the effect of irradiation early in embryonic development that there is a critical stage for inducing Polydactyly in the inbred strain C57BL/KsJ, in which polydactyly normally appeared at a 2.2 per cent rate, between day seven to day ten of gestation (table 44) . The table illustrates Table 44 Incidence of polydactylism in controls and after X irradiation (130 r) at SUCCESSIVE STAGES OF EARLY GESTATION IN MICE OF THE INBRED STRAIN C57BL/KsJ Controls Time of X-irrad. Bir th data Polydactylism Summary in pregnancy Li NB 0 right left both sides n Per cent ofENB Day hour 39 269 6.9 3 3 — 6 2.2 269 VI 8 PM 17 108 6.4 4 _ 4 3.7 107 VII 8 AM 25 106 4.2 9 — — 2 1.9 103 VII 8 PM 21 112 5.3 17(3)T 5(l)t 22 19.6 112 VIII 8 AM 22 112 5.1 13(2)t 2 1 16 14.3 112 VIII 8 PM 17 115 6.8 12 4 1 17 14.9 114 IX 8 AM 20 114 5.7 22(2)f 7(l)f 2 31 27.2 114 IX 8 PM 17 112 6.6 11 4 — 15 13.5 111 X 8 AM 16 105 6.6 13 3 — 16 15.4 104 X 8 PM 18 126 7.0 20 4J(l)t 1 25 20.0 125 XI 8 AM 15 95 6.3 1 1 — 2 2.1 94 115 30 5 150 18.2 1096 Li = Litter NB = Newborn 0 = Average litter size ENB = Examined newborn t = Incidence of 7 toes X = One case of postaxial polydactylism the incidence rate up to 27.2 per cent, and it may be possible that this is the threshold of genie interaction. But there seems to be another critical phase. This work has been done by Dr. Charles P. Dagg.236 He gave 5-fluorouracil at a certain stage of GENIC INTERACTION 191 embryonic development in the strain 129/J, and he found a second critical phase during the eleventh or twelth day of inducing Polydactyly. Is that the second critical stage for genie interaction ? Dr. Wright: These critical stages probably have very little to do with the gene under study. There are certain weaknesses in the developmental process leading to the development of Polydactyly, microphthalmia, or anencephaly according to the time of development. Anything environmental or genetic which produces a sufficient disturbance at that critical time would bring out that particular type of defect. The specificity is not so much in the gene, although the timing of genie action may be re- garded as specific ; the character of the effect depends on the developmental background. Also the same gene may bring out other different developmental effects if it comes into play at different times. Dr. Steinberg : I hope that in working with mice, investigators will not go through the same series of mistakes that happened when flies were used to detect the time of genie action. When it was found that there is a specific period during development when temperature would affect the size of the eye, it was labelled the temperature- effective period, and it was concluded that this was the time when the gene was acting. However, when the embryology was pursued, it was found that a detectable effect of the gene on the development of the eye occurred some 24 to 36 hours before temperature could affect it.1273 Furthermore, it was found that oxygen could affect the develop- ment of the eye two or three days after temperature could affect the development of the eye.849 It became, as more and more agents were used, a little more ludicrous to say that the gene was acting at this time, that time, or the other time. The effect of the environment on the development of the organism may be related to the gene, but usually it is not and certainly it need not be. Cause and effect are not so easily related. Dr. Burdette: I regret that we do not have more time to devote to the dissection of these complexities. Dr. Wright, perhaps you wish to make a few concluding remarks at this time, particularly with relation to evolution. Dr. Wright : I will not attempt to summarize in any detail what I said in my paper. I wish merely to emphasize again that because of the prevalence of intermediate optima in quantitative variability, and also of pleiotropy, multiple selective peaks are. to be expected in any species in the virtually infinite field of possible sets of genie frequencies. Each of these peaks tends to be closely related to an equilibrium point determined in part by the pressures of recurrent mutation and immigration but primarily by mean selective value. These equilibrium points have the property to which Lerner has applied the term genetic homeostasis. After any slight shift, the system tends to return to the same equilibrium. The selective peaks may be expected to differ greatly in adaptive value. The evolutionary problem is the mechanism by which the species may work its way from lower to higher peaks, necessarily somewhat against the pressure of selection in the first stage. In a sexually reproducing species under given environmental conditions, the only known mechanism is that in which some sort of random drift brings about 192 PHYSIOLOGIC GENETICS differences among local populations which can provide material for interdemic selection (by differential population growth and dispersion). The relation of random drift to selection among demes is precisely analogous to that of random mutation to mass selection within demes, but whereas the latter deals only with the momentary net effects of single gene differences, the former deals with genetic systems as entities. As I have always emphasized, neither random drift nor random mutation is of appreciable evolutionary significance except in conjunction with its appropriate kind of selection. It is because of the necessary prevalence of types of genie interaction that lead to multiple selective peaks in the genetic system that I have always considered interaction to be as important in evolutionary theory as in physiologic genetics. D. S. Falconer, Ph.D. QUANTITATIVE INHERITANCE A quantitative character is any attribute for which individual differences do not divide the individuals into qualitatively distinct classes. Antigenic differences, for example, separate individuals into qualitatively distinct classes and are therefore not quantitative characters. But an antibody titer, which varies continuously from one individual to another, is a quantitative character. The inheritance of quantitative characters is generally under the control of many genes (that is, it is polygenic), but this is not necessarily so. The essential feature is that the segregation of the genes, whether few or many, is not manifest in phenotypic discontinuity. This feature of quantitative characters precludes the application of the ordinary methods of genetic analysis used for the study of single-gene effects. Special methods are therefore re- quired, and at the same time the questions that can be answered are different in nature. In general, the genetic properties of a quantitative character that can be investigated are those arising from the simultaneous action of many genes. It will not be possible to explain fully the theoretic nature of these properties here; nor will it be possible to describe all the methods available for their study. So this discussion will be confined to a description of the simpler methods which may be of use to those for whom genetics is a secondary rather than a primary interest. Explanations of many of the points which cannot be fully explained here will be found in the treatise by Falconer.335 The first and simplest question to be asked about any quantitative character is: are there any genetic differences ? To answer this question it is only necessary to com- pare different strains maintained in the same place and under the same conditions. If strains differ in their mean values of the character this proves the existence of genetic differences. So many characters are known to exhibit strain differences that the exist- ence of genetic differences in any character can almost be taken for granted. The 193 194 PHYSIOLOGIC GENETICS demonstration of strain differences is therefore no more than the starting point of the investigation. There are, broadly speaking, four sorts of genetic investigations of a quantitative character that may be made. The first two — estimation of the degree of genetic determination and of the heritability — are concerned with the relative importance of genetic and environmental factors as determinants of an individual's phenotypic value of the character. The third is a description of the effect of selection applied to the character. This may be of practical interest, but the additional genetic conclusions that can be drawn are rather limited. The fourth is a description of the effect of inbreeding or of crossing inbred lines, and again the conclusions that can be drawn are rather limited. None of these investigations can be made without a noninbred, or genetically heterogeneous, strain. The most satisfactory strain for this purpose is one that has been maintained by random mating among a large number of parents over many generations, so that its genetic properties have had time to become stabilized. But to advocate the use of such a strain is obviously a counsel of perfection. In the absence of a random- bred strain the most convenient form of genetically heterogeneous strain is an F2 of a cross between two inbred lines, or the third generation of a 4-way cross of four inbred lines. These synthetic strains have the advantage over a random-bred strain that they can be reconstituted at will from the original inbreds. Their disadvantages are that the conclusions about their genetic properties have less generality and that their genetic properties are in some ways necessarily unnatural. The lack of generality can be expressed through the inbreeding coefficient. Nobody would claim generality for any biologic property discovered from the study of a single highly inbred line. The F2 of a 2-way cross is 50 per cent inbred, and the third generation of a 4-way cross is 25 per cent inbred; in other words, a strain derived from a 2-way cross is equivalent to the progeny of a single self-fertilized individual, and a strain derived from a 4-way cross is equivalent to the progeny of a single pair of full sibs. To this extent, therefore, synthetic heterogeneous strains resemble inbred lines in lack of generality. The un- natural features of the genetic properties of synthetic strains are the restricted range of genie frequencies, nonrandom linkage associations, and the absence of lethal and severely deleterious genes. The disadvantages of synthetic strains, however, are only relative. Complete generality is an unattainable ideal; no random-bred strain of mice, however carefully constructed and maintained, is truly representative of all mice, just as no laboratory mammal is truly representative of all mammals. DEGREE OF GENETIC DETERMINATION If there are genetic differences, then how important are they? This is the nature- nurture question: what is the relative importance of heredity and environment in determining the value of the character in an individual ? (For the sake of convenience all nongenetic differences are referred to as environmental, so that the genotype and the QUANTITATIVE INHERITANCE 195 environment of an individual are the only determinants of its phenotypic value.) The question has precise meaning only when framed in terms of the variation between inidividuals: how much of the variation is caused by genetic differences between individuals and how much by nongenetic differences? To answer the question it is necessary to eliminate one cause of variation and to measure the variation remaining, which will be due to the other cause only. If the amount of variation is measured as the variance, then the total variance when both causes are operating is the sum of the variances when each cause is operating separately. It is not possible in practice to eliminate nongenetic causes of variation, because, however careful the experimenter may be in insuring uniformity of external conditions, there always remains a substantial amount of nongenetic variation from intangible causes. The genetic variation, however, can be eliminated by inbreeding, although this is practicable only with fast-breeding laboratory animals. A strain that has been inbred by brother-sister mating for upwards of 20 generations is, for practical purposes, free of genetic variability. The variation within the strain, therefore, is purely environmental in origin. There is, however, a difficulty here, because inbred animals have often been found to be more susceptible than noninbred animals to the normal range of environmental differences. An inbred line therefore does not always provide a reliable measure of the environmental variation for comparison with a noninbred strain. The difficulty can be overcome by taking a cross between two inbred lines. If both lines are highly inbred, then the first genera- tion of the cross (that is, the Fx) is equally free of genetic variation and provides a more reliable estimate of the environmental variation. Better still, however, is to make several different crosses between highly inbred lines and base the estimate of environ- mental variance on the mean variance within the crosses. This overcomes the possible objection that one cross provides only one particular genotype whose sensitivity to environmental differences may not be typical. The observed variance of the individuals of a genetically variable strain is the sum of the genetic variance in that strain and the environmental variance. Subtraction of the environmental variance, estimated from the cross-bred individuals, therefore gives an estimate of the genetic variance. In this way the total, or phenotypic, variance in the variable strain may be partitioned into two components, genetic and environmental. This partitioning expresses the degree of genetic determination, as the percentage of the total variation that is attribu- table to genetic differences among individuals of the strain. An example of the estimation of the degree of genetic determination is provided by the following data on the age of vaginal opening in female mice, from Yoon.1465 Two inbred strains, BALB/c and C57BL/10, were crossed (Yoon's cross 2), and the age of vaginal opening was recorded from 192 F1 animals and 163 F2 animals. The variance among the Fx animals was 19.1 and the variance among the F2 animals was 34.9. The difference, which estimates the genetic variance among the F2 individuals, was 15.8. The degree of genetic determination in the F2 of this particular cross was therefore 15.8/34.9 = 45 + 8 per cent. (The standard error is calculated from equation 4, as explained below.) 196 PHYSIOLOGIC GENETICS Experimental design. — The only technical problem that needs consideration in connection with the degree of genetic determination is the scale of experiment required to estimate it with a given degree of precision. The standard error to be expected when a given number of animals has been measured can be deduced in the following way. Let V be the estimate of the variance in the genetically variable group, based on Nv degrees of freedom; and let U be the estimate of the variance in the genetically uniform group, based on Nu degrees of freedom. For the purposes of planning the degrees of freedom may be taken to be the number of animals measured. The sampling variances of these two estimates of variance are given by a\ = 2V2INv and o\ = 2U2/NU, (1) where a2 is the sampling variance. The standard error of each estimate is the square root of its sampling variance. The ratio of the standard error to the variance itself is av/V= V'Wv and av\U = V2[N~U. (2) This ratio is plotted in curve (a) of figure 34. The degree of genetic determination, g, is estimated as g=(V- U)\V= 1 - U/V (3) The sampling variance of the degree of genetic determination (a2g) can be deduced from the general properties of variances as follows : n 2 — n2 '->g — ° (1- UIV) — n2 = Y*(v2(y2v+ u2°'' u2 v2 Since UjV = 1 — g, from (3), the standard error of the estimate reduces to °^^-^HhI) (4) Now, the total number of animals that can be measured is fixed by the amount of space available, or of effort that can be expended. The formula deduced above for the standard error (4) shows that with a fixed total (that is, Nu + Nv), the standard error will be minimal if Nu = Nv. Therefore the best design for the experiment is to have equal numbers of animals measured from the genetically variable and from the genetically uniform groups. If this number is N (so that a total of 2N animals are measured), then we obtain the relationship -*- = — ■ (5) This ratio is plotted in curve (b) of figure 34. QUANTITATIVE INHERITANCE 197 The relationship in equation (4) shows that the precision of an estimate of the degree of genetic determination depends on the value ofg itself. The higher the value, the greater the precision with a given number of animals measured. To plan an experiment it is therefore necessary to guess what the degree of genetic determination may be. This is not very satisfactory, but it is better than working completely in the Fig. 34. Graphs of y — \/2/x and y = 2/Vx. ■10 (a) Graph of y — V2/x; (b) y = 2/Vx. Two scales are given: the left hand scale of y is to be read against the bottom scale of x, and the right hand against the top. Uses 1. Standard error of estimate of a variance, V, (graph a) : av — yV, when x is the number of degrees of freedom. 2. Standard error of estimate of the degree of genetic determination, g, by comparison of variances of genetically uniform and genetically heterogeneous groups with equal numbers in each group (graph b) : ag = y{\ — g), when x is the number of degrees of freedom in each group. 3. Standard error of estimate of heritability by regression of offspring on parents, with one offspring from each parent or pair of parents, y is the standard error when x is the number of parents or pairs of parents. Graph a refers to regression on the mean of both parents, graph b to the regression on one parent. The inset graphs show the effect of in- creasing the number of offspring when the number of parents remains the same. The vertical scale gives the factor by which y (from graphs a and b) is to be multiplied to give the standard error. The four inset graphs refer to different characters with phenotypic correla- tions (t) between offspring of the same parents as shown. 198 PHYSIOLOGIC GENETICS dark. Suppose, for example, that the degree of genetic determination is thought to be 40 per cent, which is a reasonable figure to guess for many quantitative characters; how many animals must be measured to obtain an estimate with a standard error of, say, 10 per cent? Herecrg = 0.1, and 1 — g = 0.6. Entering these values in equation (5) gives N = 144. Therefore about 144 animals must be measured in each group, or 288 altogether, to obtain a result that would read g = 40 + 10 per cent. To take another example showing how the relationship might be used the other way around, suppose a character is difficult to measure and it is decided that the greatest number of animals that could be measured would be 40, twenty of each group. Would it be worth while to try to estimate the degree of genetic determination? Reading graph (b) in figure 34 from the upper and right-hand margins, crg/(l — g) is approximately 0.44. So, if g were 40 per cent, its standard error would be 0.44 x 0.6 = 0.26. Thus the expected result of the experiment would be g = 40 + 26 per cent. The estimate would not be significantly different from zero, and therefore even demonstrating the existence of any genetic variation at all could not be expected. From all this it will be evident that the degree of genetic determination cannot be precisely estimated without a very considerable expenditure of effort, especially if the character is only weakly heritable. But an estimate with a standard error even as high as 20 per cent would not be entirely without interest, and, if suitable data could be accumulated from routine measurements made for the other purposes, information of great interest could be obtained. HERITABILITY The degree of genetic determination, although of great intrinsic interest, is of little practical use. It cannot be used to predict the speed of progress to be expected if selection were applied to the character. For this prediction it is necessary to deter- mine the heritability of the character. The determination of the heritability involves the further partitioning of the genetic component of variation into two parts, although the method of partitioning is now entirely different. The need for the additional partitioning arises from the mode of hereditary transmission — from the fact that gametes are haploid and that genes, and not genotypes, are transmitted from parents to offspring. The idea of heritability is therefore of fundamental genetic importance. The genetic variation is of two sorts. Part of it may be thought of as arising from the genes con- sidered singly, instead of paired in the diploid genotype. This component of the genetic variation is called the additive variance. The heritability is the ratio of the additive variance to the total phenotypic variance ; or, in other words, the fraction of the total variance made up by additive variance. The other part is the additional variation that arises from the genes coming together in pairs to form genotypes. This component is called the nonadditive variance. The cause of nonadditive variance, in terms of the properties of individual genes, is dominance and interaction (epistasis) between different loci. If there is no dominance and no epistasis there can be no nonaddititive variance; QUANTITATIVE INHERITANCE 199 and, conversely, if no nonadditive variance is found, it can be concluded that there is no dominance or epistasis: the genes are then said to act additively. Methods are available for separating the different sorts of nonadditive variance.538 Because the amount of additive variance reflects the variation that is transmitted from parent to offspring it is responsible for the resemblance between relatives, and its estimation depends on the measurement of the degree of resemblance between relatives. The most commonly encountered sorts of relationship are between offspring and their parents, and between full or half sibs. It can be shown from theoretical considerations that the degree of resemblance, measured as a regression or correlation coefficient, in these relationships is related to the heritability as follows (the symbol h2 stands for the heritability) : Regression of: offspring on one parent = \h2 offspring on mean of two parents = h2 Correlation (intraclass) of: half sibs = ih2 full sibs > \h2. Thus, for example, to estimate the heritability from the resemblance between half sibs it is only necessary to compute the intraclass correlation and multiply this by four; or, from offspring and parents, to compute the regression of the offspring on the mean of their two parents which itself estimates the heritability. A graphic illustration of the resemblance between offspring and parents is shown in figure 35, with the regression which estimates the heritability. There are, however, two complications which have an important bearing on the choice of the relationship from which to estimate the heritability. The first concerns only full sibs. The genetic cause of resemblance between full sibs is not confined to the additive variance, and the correlation is augmented by part of the non-additive variance if any is present. Therefore an estimate of the heritability from full sibs is, strictly speaking, valid only in the absence of nonadditive variance. The error is, however, seldom likely to be serious, and an estimate from full sibs is by no means to be rejected as valueless on these grounds alone. The second complication is much more important. It arises from the fact that there are often nongenetic causes for the resemblance between certain sorts of relative, which may increase the regression or correlation and lead to a serious overestimation of the heritability. These non- genetic causes of resemblance are particularly prevalent in mammals. They arise from maternal effects and circumstances generally referred to as common environment. For example, large mothers tend to give more milk to their young than small mothers. Therefore the offspring of large mothers tend to be larger than the offspring of small mothers, and so the offspring tend to resemble their mothers in body size for purely nongenetic reasons. Also, for the same nongenetic reason, offspring of the same mother tend to resemble each other in body size. It is in the full-sib relationship that 200 PHYSIOLOGIC GENETICS resemblance tends most often to be augmented by nongenetic causes. Full sibs always have the same mother and they tend, especially if they are also littermates, to be subjected to similar environmental circumstances. For these reasons the correlation between full sibs is to be avoided as a means of estimating the heritability. Fig. 35. Heritability of the number of urethan-induced pulmonary tumors in mice of a random-bred strain (JC), by regression of offspring on the mean of both parents. MID-PARENT Based on preliminary data from a current experiment by Miss J. L. Bloom, a total of 299 offspring from 46 pairs of parents are represented. Each point shows the mean of all offspring from parents with the given mean tumor-number, the approximate number of off- spring in each point being indicated by the size of the dots: small = fewer than 10, medium = 10-20, large = more than 20. The straight line is the computed regression line, each point being weighted by the number of offspring contributing to it. The regres- sion coefficient, which estimates the heritability, is 0.173 ± 0.043. The absence of nongenetic causes of resemblance in a relationship is the most important criterion in choosing a method of determining the heritability. After that, the choice of method is a matter of deciding which gives the most precise estimate with a given expenditure of effort. In general, low heritabilities are more efficiently esti- mated from the half-sib correlation, and heritabilities higher than about 20 per cent are more efficiently estimated by offspring-parent regressions. The offspring- parent regression is usually the more convenient method for work with laboratory mammals. It is usually also the more efficient because the total number of measure- ments made is not usually the limiting factor, but rather the number that are made at one time. By the measurement of parents and offspring, the work is spread over two generations. The rest of this section is concerned with the technical problems that arise in the estimation of heritability, first from the offspring-parent relationship and then from sibs. QUANTITATIVE INHERITANCE 201 Offspring-parent regression. — The chief problem of experimental design concerns the number of offspring from each parent or pair of parents that should be measured. When the most efficient design has been decided, the number of animals that must be measured to attain a given degree of precision can be determined. The size of an experiment is limited either by the amount of breeding or rearing space available or by the number of individuals that can be measured. The problem is to decide how to divide the space or effort among the offspring of different parents. Either few offspring from each of many parents or many offspring from each of few parents can be reared and measured. The solution of this problem comes from a consideration of the expected standard error of the estimate of heritability that will be obtained. Approximate formulae for the variance of the estimate are as follows:335 By regression : „ . \ + (n — l)t ,r. on one parent, op = 4 ^ (b) on mean of both parents ofe = 2 ^^ (7) In these formulae n is the number of offspring measured per parent (equation 6) or pair of parents (equation 7), TV is the number of parents or pairs of parents, and / is the pheno- typic correlation between the offspring of the same parent or pair of parents. In work with laboratory animals the offspring are likely to be full sibs. In this case the phenotypic correlation, t, will be approximately equal to half the heritability, or greater than this if there are also nongenetic causes of resemblance between the offspring of the same parents. The solution of the problem of design depends on the circum- stances that limit the size of the experiment. The simplest solution is when the limiting factor is the total number of offspring that can be reared or measured. The total number of offspring is nN, and, if this is fixed, then the denominators of both the above formulae are fixed. The variance of the estimate is then, in both cases, minimal when n = 1 . This means that the most efficient design has only one offspring measured from each parent or pair of parents. The standard error of the estimate of the herita- bility then becomes: By regression : on one parent op — V2/N (8) on mean of both parents ah^—2jvN. (9) These relationships are also shown by the graphs in figure 34. Graph (b) refers to the regression on one parent and graph (a) to the regression on the mean of both parents. The horizontal axis shows the total number of offspring measured (that is, N) and the vertical axis the corresponding standard error expected. It is again evident that precise estimates of the heritability cannot be obtained without the measurement of large numbers of individuals. For example, to attain a standard error of 0.10 it is 202 PHYSIOLOGIC GENETICS necessary to measure 200 offspring if both parents are measured and 400 offspring if only one parent is measured. The situation discussed above is likely to apply when the labor of measurement is the limiting factor, rather than the amount of breeding or rearing space available. When labor of measurement is not the limiting factor, then it is often possible to rear and measure more than one offspring from each parent or pair of parents without any reduction of the number of parents that can be used. The limiting factor is then the number of parents. If this is fixed, it follows that any increase in the number of off- spring measured will improve the precision of the estimate. The question then is how many offspring are worth measuring. The reduction in the expected standard error effected by increasing the number of offspring from 1 to 8 is shown by the inset graphs in figure 34. The figures on the vertical scale give the factors by which the standard error obtained from equations (8) and (9), or from the main graphs in figure 34, are to be multiplied. The improvement in precision depends on the phenotypic corre- lation between offspring of the same parents, and graphs for four correlations, ranging from 0.05 to 0.5, are given. The cause of the correlation is immaterial in this connection ; it may be genetic or nongenetic, or both. When the correlation is high there is little to be gained by increasing the number of offspring measured, but when the correlation is low a substantial improvement of precision results from even a small increase in the number of offspring. If the value of the correlation is not known beforehand, it would seem worth while, as a general rule, to aim at measuring about four offspring from each parent or pair of parents. Planning on paper is easy, but executing the plan by obtaining and measuring the animals exactly as required is often impossible. Even if it is planned to measure only a few offspring from each parent, some parents will inevitably fail to produce the required number of offspring, and there may be other losses later, so that the plan cannot be strictly followed. This raises two problems. The first is at the stage of the collec- tion of data : should the experimenter discard all families that have failed to provide the right number; and if he does not discard them, should he supplement the total number of offspring by measuring more than was planned from some other families ? My answer is that he should include every animal measured and measure any additional animals that he can, because every additional offspring measured adds something to the precision of the estimate. The second problem then arises with the computation of the offspring-parent regression from the data, because the number of offspring will not be the same for all parents. How is the computation to be made? (In what follows I shall use the word family to mean the offspring of one parent or pair of parents, and the word parent to mean equally the single, measured parent or the mean of both parents, according to which regression is to be computed.) There are two simple courses of action, neither of which makes the best use of the data. One is to take each offspring separately and count each parent as many times as it has offspring. This gives too much weight to the larger families but is reasonably satisfactory when the heritability is low and there is very little resemblance between QUANTITATIVE INHERITANCE 203 the offspring of the same parent. The second simple course of action is to take the mean of each family and to regress this on the parental value. This gives too little weight to the larger families, but is reasonably satisfactory when there is a strong resemblance between offspring of the same parents, either for genetic or for nongenetic reasons. A slightly less simple, but much more satisfactory, procedure is as follows. The families are divided into groups according to the number in the family, and the regression of family means on parental values is computed separately within each group. The variance of each estimate is computed in the usual way, the degrees of freedom being based on the number of families in the group. A weighted average of the regression coefficients is then taken, the weight being the reciprocal of the variance of each estimate. The variance of this average regression is the reciprocal of the sum of the weights. The chief disadvantage of this method is that one degree of freedom is lost for each subdivision of the data, a sacrifice to simplicity that may not willingly be made when much effort has gone into the collection of the data. It may therefore seem worthwhile to use a fully satisfactory, but more complicated, method of combining the data from families of different size. The method that makes the best use of all the data depends on combining the sums of squares and products from families of different size according to a weighting factor appropriate to the family size. The derivation of the weighting factors is explained by Kempthorne and Tandon702 and Reeve.1046 The principle is that families of different size are weighted in proportion to the reciprocal of the variance of the estimate of re- gression that would be obtained from families all of that particular size. It has already been pointed out, in connection with planning, that the proportionate effect of the number in the families on the precision of the estimate is governed by the phenotypic correlation, t, between members of families. But it also depends, to a lesser extent, on the value of the regression being estimated, and, although this factor can be omitted in connection with planning, it must be brought in if the best use is to be made of the data. The first step in computing the weighting factors is therefore to determine the phenotypic correlation, /, between members of families, from an analysis of variance of the offspring, within and between families. Then one must make a rough estimate of the regression, b, which is to be estimated. This need not be very precise, and it can be obtained from a graphical representation of the data in a scatter diagram of the values of parents and the means of their offspring. Approximate values of the corre- lation, t, and the regression, b, having been obtained, the weighting factors can most easily be calculated in two steps. First compute the quantity T, as follows : when the regression is to be made on a single parent T = (t - b*)l(\ - t), and when the regression is to be made on the mean of both parents T=(t-±b*)l(l -t). Then the weighting factor, w n, appropriate to families of n offspring is wn = (n + nT)l(\ + nT). 204 PHYSIOLOGIC GENETICS Table 45 Some weighting factors to be applied to the means of full-sib families of different sizes, in regressions of offspring on the mean of both parents Listed according to the phenotypic correlation (t) between full sibs, and the heritability (h2) of the character t = 0.05 0.1 0.2 0.4 0.7 h2 = 0.1 0.1 0.2 0.2 0.4 0.2 0.6 0.2 0.6 T = 0.047 0.106 0.089 0.225 0.150 0.633 0.367 2.267 1.733 n 1 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 2 1.91 1.83 1.85 1.69 1.77 1.44 1.58 1.18 1.22 3 2.75 2.52 2.58 2.19 2.38 1.69 1.95 1.26 1.32 4 3.52 3.11 3.21 2.58 2.88 1.85 2.22 1.30 1.38 5 4.23 3.62 3.77 2.88 3.29 1.96 2.41 1.32 1.41 6 4.89 4.06 4.26 3.13 3.63 2.04 2.56 1.34 1.44 7 5.51 4.45 4.70 3.33 3.93 2.10 2.68 1.36 1.46 8 6.08 4.80 5.09 3.50 4.18 2.15 2.78 1.37 1.47 Some examples of weighting factors are given in table 45, the value of T under which the weights are listed being appropriate to the heritabilities shown at the heads of the columns when the regression is to be made on the mean of both parents. The meaning of the weighting factor, wn, is that the mean of each family of size n, and the correspond- ing parental value, are to be counted wn times in the computation of the sums of squares and products. [It should be pointed out that the weighting factor given above differs slightly from that of Kempthorne and Tandon in that it gives unit weight to families of n — 1, which theirs does not do. The weight given here is equal to theirs divided by 1/(1 -f T), which is their weight for n = 1 .] The computation of the regression coefficient, b, may be summarized as follows. Let X be any parental value, Y the mean value of the corresponding family of offspring, and w the weighting factor appropriate to the number in that family. Then the regression of offspring on parents is computed thus, 2 indicating that the quantities are to be summed over all families: where and b = 1 (wxy) > w z w Also needed will be 2 ( w QUANTITATIVE INHERITANCE 205 The variance of the estimate of the regression coefficient should, in my opinion, be computed in the usual way but from the weighted sum of squares. Thus °* I w - 2 12 (wx2) b\ The error variance on which this is based is the weighted mean of the squared deviations of family means from the regression line. Kempthorne and Tandon,702 however, say that the appropriate error variance is the variance (of Y) within families. Sib analyses. — Although the correlation between sibs is usually less efficient than the offspring-parent regression as a method of estimating heritability in laboratory mammals, there are occasions when it has to be used. If the measurement of the character requires the death of the animals before they can breed, then the regression method is obviously inapplicable, and the correlation between sibs is the only method that can be used. The correlation of full sibs does not provide a reliable estimate of heritability because it may be augmented by nonadditive genetic variance and often also by non- genetic causes of resemblance. A sib analysis designed to estimate heritability should preferably, therefore, be based on the correlation between paternal half sibs. To provide half-sib data, each of a number of males is mated to several females and the progeny of each male constitutes a half-sib family. Ideally, if the estimation of heritability is the sole object, only one offspring of each female should be measured, so that there are no full sibs within the half-sib families. The problem of design is then a fairly simple one. The total number of progeny that can be measured will be fixed by the amount of space available or by the amount of effort that can be expended on the measuring. The question then is : how large should the families be ? Where does the optimum lie between the extremes of having many small families and few large families ? It can be demonstrated 1061 that the sampling variance of the correlation coefficient will be mini- mal when n = \jt approximately, n being the number of offspring per family and t the phenotypic correlation. So, again, it is found that the optimal design cannot be determined precisely without prior knowledge of the correlation to be estimated. But, by guessing whether the heritability of the character is likely to be high or low, a useful guide to the design can be obtained. Since the correlation between half sibs will be one quarter of the heritability (/ = \h2), the optimal design will have n — 4jhz. Therefore if the heritability is 20 per cent, 20 offspring per family should be planned, and if it is 40 per cent, 10 offspring per family should be planned. The precision of the estimate falls off much more rapidly when the families are smaller than the optimum than when they are larger. Therefore it is better to err on the side of having too many offspring per family than too few, and, in the absence of any knowledge of what the heritability is likely to be, it would seem reasonable to plan on having families of about 20 half sibs. To mate each male to 20 females and to measure only one offspring from each female, as the optimal design requires, is, however, not a convenient design to carry 206 PHYSIOLOGIC GENETICS out with laboratory mammals. And, furthermore, the estimation of heritability is not the only interest in a sib analysis. The correlation between full sibs is also of interest, because, when compared with the half-sib correlation, it shows how important nongenetic causes of resemblance between progeny of the same mother are. It would therefore seem desirable to sacrifice some precision in the estimate of heritability in order to have a more convenient design and to obtain the additional information about full sibs. To include full sibs within the half-sib families it is only necessary to measure more than one offspring from each mother. The optimal design is then more complicated and must necessarily be a compromise.1061 If it is desired to estimate the full-sib and the half-sib correlations with equal precision, and if the full-sib correlation is not augmented by maternal effects, then each male should be mated to three or four females, and between 5 and 10 offspring from each female should be measured. If, however, the full-sib correlation is augmented by maternal effects, then it is better to have more females mated to each male and fewer offspring from each female. The computation of the heritability from data obtained in this way needs some explanation. The computation consists of an analysis of variance leading to the estimation of three components of variance, attributable to sires, to dams, and to individuals. The correlations are estimated from these components. Sums of squares are computed in the usual way for the following sources of variation : between sires (that is, between half-sib families) ; between dams within sires (that is, between full-sib families within half-sib families) ; within dams (that is, among individuals within full-sib families). The computation of the three components corresponding to these sources of variation is straightforward if all dams have the same number of offspring and all sires are mated to the same number of dams.1230 But equality of numbers is seldom achieved and a modification of the procedure is required. The procedure will be described without ex- plaining the reasons for it. A full explanation is given by King and Henderson.710 The sums of squares are composed of the three components in certain proportions which de- pend on the distribution of numbers within the classes and subclasses. These expected compositions of the sums of squares are given in table 46. It will be seen that each com- ponent appears with a certain coefficient which is some function of the numbers. By computing these coefficients and equating the expected composition to the observed value of each sum of squares, three equations containing three unknowns are obtained. (The sum of squares for the total is entered in the table only for the sake of complete- ness; it is not required.) The coefficient of the within-dam component, W, in each sum of squares is equal to the number of degrees of freedom corresponding to that source of variation. The first step in the solution of the equations is therefore to divide each sum of squares, both expected and observed, by the appropriate degrees of free- dom, and so obtain the expected and observed mean squares. The solution of the equation is then straightforward. QUANTITATIVE INHERITANCE 207 Table 46 Estimation of components of variance from a sib analysis with unequal numbers in the families Source of variation Degrees of freedom Expected composition of sums of squares Total Between sires Between dams (within sires) Within dams N - 1 m — \ N - n (N - \)W + (N - KJD + (N - K2)S (m - \)W + (K3 - KX)D + (N - K2)S (n - m)W + (N - K3)D (N - n)W S = Component of variance attributable to sires. D = Component of variance attributable to dams. W = Component of variance attributable to individuals within full-sib families. N = Total number of offspring. n = Total number of dams (full-sib families). m = Number of sires (half-sib families). kd = Number of offspring of any one dam. ks — Number of offspring of any one sire. I k\ v _ I k\ r_V2 *2<*t A-i - Ko *3=2 TV "* N "° £- ks f This means: compute 2 k?djks for each sire group and then sum over all sire groups. Table 47 Numerical example of the computation of the coefficients of the components of variance in a sib analysis with unequal numbers in the families Number of offspring pei dam per sire Sire Dam kd Ks IW Ki 77 + 34+ 120 _ 53?2 A a 6 b c 4 5 15 11 K, - 152 + % + 20= - 16.023 B d e 3 5 8 34 77 34 120 *■ - is + i + f - 15-383 C I 8 g 2 20 120 h 4 i 6 231 m = 3 n = 9 N = 43 N = 43 Expected composition of: Source d.f. sums of squares mean squares Total 42 42 W + 37.63 D + 26.98 S W + 0.90 D + 0.64 S Sires 2 1W + 10.01 D + 26.98 S W + 5.01 D + 13.49 S Dams 6 6W + 27.62 D W + 4.60 D Within dams 34 34 W W 208 PHYSIOLOGIC GENETICS Table 48 Components of variance of litter size in mice, estimated from a sib analysis (Data of J. C. Bowman, unpublished) Mean square Source d.f. Expected Observed W + 4.16 D + 9.75 S = 17.10 W + 3.48 D = 10.79 W = 2.19 Components: W = 2.19 10 79 - 2 19 D _ 10.79^2.19 = 247 s = 17.10 - 2.19 - (4.16 x 2.47) _ g Ag Sires 70 Dams 118 Within dams 527 S + D + W = 5.14 Correlation of half sibs = 0.48/5.14 - 0.093 ± 0.064 Correlation of full sibs =2.95/5.14 =0.57 ± 0.036 Heritability: h2 = 4 x 0.093 = 0.37 ± 0.258 The computation of the quantities Kx, K2, and K3, which appear in the co- efficients of the components in the sums of squares, need not be as troublesome as the formulae given at the foot of table 46 might suggest. The computation, which is illustrated numerically in table 47, may be done as follows. Tabulate the sires and the dams to which each sire is mated. Against each dam tabulate the number of its off- spring that were measured (kd). Add these and tabulate the number of offspring measured from each sire (ks) . Now square each kd and enter the sum of these squares obtained from each sire group as shown under 2 kd2 m tne table. This gives every- thing needed for the computation of the three K's ; the computations are shown at the right-hand side of table 47. The coefficients of the components in the sums of squares and mean squares can now be entered, and these are shown at the foot of table 47. An example to show the solution of the equations with real data is given in table 48. The sib correlations are obtained from the components of variance as follows : S Correlation of half sibs = Correlation of full sibs = S + D + W S + D S + D + W The estimate of the heritability is four times the correlation of half sibs. If the component of variance between dams (D) is not greater than the component between sires (S), then there is no evidence that the full-sib correlation has been augmented by nonadditive variance or by nongenetic causes of resemblance. If this is the case, then the full-sib correlation provides the more reliable estimate of the heritability. The heritability is twice the full-sib correlation. QUANTITATIVE INHERITANCE 209 The standard error of the heritability estimated from a sib correlation is rather laborious to compute. The following method, based on that of Osborne and Pater- son,971 was devised by Dr. B. Woolf, to whom I am much indebted for explaining it to me and for permission to describe it here. It simplifies the computation considerably and at the same time makes allowance for unequal numbers in the classes. The procedure for computation is set out in table 49 and is illustrated numerically from the Table 49 Procedure for computing the standard error of the heritability estimated from a sib analysis, illustrated numerically from the data of table 48 I. Symbols used for the mean squares an d the coefficients of the variance components in the mean squares. Mean squares Degrees of In terms of Source freedom Observed components Sires m — 1 A W + aD + bS Dams n — m B W + cD Within dams N — n C W II. Sampling variances of the observed mean squares. Sires: VA = 2A2jm - 1 - 2 x 17.102/70 = 8.35 Dams: VB = 2B2/n - m = 2 x 10.792/118 = 1.97 Within dams: Vc = 2C2/N - n = 2 x 2.192/527 = 0.018 III. Coefficients of components in mean squares, and other quantities required. a = 4.16 be = 33.93 b = 9.75 x = b - a = 5.59 c = 3.48 y = be - c - x = 24.86 T = bc(S + D + W) = 33.93 x 5.14 = 174.40 c Half-sib correlation : P = s — = 0.093 S + D + W S + D Full-sib correlation: R = -= — = 0.574 S + D + W IV. Sampling variances of the correlation coefficients. Half-sib correlation: T2aP2 = (c - cP)2VA + (a + xP)2VB + (c - a + yP)2Vc 9.96 x 8.35 + 21.90 x 1.97 + 2.66 x 0.018 - 126.36 Full-sib correlation: T2aR2 = (c - cR)2VA + (* - xR)2VB + (x - c + yR)2Vc 2.20 x 8.35 + 5.67 x 1.97 + 544.76 x 0.018 = 39.35 V. Standard errors of correlation coefficients and heritability. Half sibs: aP = Vl^'36= 0.064 ah2 = 4aP = 0.258 Full sibs: oR = ji;35 = 0.036 ah2 = 2aR = 0.072 210 PHYSIOLOGIC GENETICS data of table 48. The computation is divided into five steps in the table and it needs little further explanation. The observed mean squares are denoted by A, B, and C, and the coefficients of the variance components in the mean squares by a, b, and c, as listed in section I of the table, which represents the analysis of variance as already explained and exemplified in table 48. Next (section II), the sampling variances of the observed mean squares are computed; these are twice the square of the mean square, divided by the corresponding degrees of freedom. Under III the values of the co- efficients (a, b, and c) of the components are listed and three quantities denoted by x, y, and T are computed for later use. The sib correlations, denoted here by P and R, will have already been obtained from the analysis of variance. Now the sampling variances of the two correlation coefficients (half sibs and full sibs) can be computed fairly simply from the formulae given in section IV. Finally (V), the standard errors of the correlation coefficients are the square roots of their variances. Since the half-sib correlation must be multiplied by four to give an estimate of the heritability, the standard error of the correlation must also be multiplied by four to give the standard error of the heritability. If the heritability is estimated from the full-sib correlation (which, however, is not justified in the data presented), its standard error is twice the standard error of the full-sib correlation. The heritability estimated from the data of table 48 has a very large standard error. The poor precision of this experiment was due partly to its design. There were 2.6 females per male and 3.7 offspring per female. It would have been better, in view of the high full-sib correlation, to have had more females per male and fewer offspring per female. SELECTION Artificial selection, as a method in quantitative genetics, may be used for the utilitarian purpose of producing a strain with a higher, or lower, mean expression of a character ; or, alternatively, as a means of investigating the genetics of the character. In both cases the interest centers on the connection between the heritability of the character and the response to selection. The response to be expected can be predicted from a knowledge of the heritability ; or, alternatively, the response observed can be used for the estimation of the heritability. However, the words "in principle" should be added to both these statements because experiments with laboratory animals have shown that there is a complication, not yet fully understood, which interferes with the simple theoretical relationship between heritability and response. This complication will be mentioned later; meanwhile it will be ignored. In principle, the response expected (R) is equal to the product of the heritability (A2) and the selection differential (S) : R = h2S, QUANTITATIVE INHERITANCE 21 1 and so the heritability may, in principle, be estimated from the ratio of the response obtained to the selection differential applied : h2 = R/S. The selection differential, S, is the difference between the mean value of the selected animals and the mean of all the measured animals, including themselves, out of which they were selected. If the individual values of the character are normally distributed and the selected individuals are all those with measurements exceeding a certain value, then the selection differential depends only on the proportion selected and the standard deviation. For example, if the best 25 per cent of individuals are selected their mean superiority, which is the selection differential, will be about 1.3 standard deviations; and if the best 50 per cent are selected the selection differential will be about 0.8 standard deviations. The proportion selected depends, in turn, on the number of offspring produced by the parents. On the average two offspring per pair of parents must be selected if the strain is to maintain itself. So, if each pair produce 8 young, 2 must on the average be selected, and the maximum possible selection differential will be 1.3(7 corresponding to 25 per cent selection. The selection differential can be increased by waiting for the parents to produce more young. With laboratory mammals the production of more young means waiting for second or third litters, and this takes time and adds to the interval between generations. The highest rate of progress in time is therefore not necessarily achieved by the highest possible selection differential. The best procedure depends on the number of young per litter and the interval between litters. An examination of the procedure for selection applied to mice,335 shows that it is best to raise only one litter unless the number per litter is as low as 4, when it becomes worth while to wait for second litters ; third litters are worth waiting for only if the number per litter is as low as 2. (The number per litter means, of course, the number of offspring measured and available for selection.) At the risk of being rash, I shall make the following generalization about how rapidly an investigator may reasonably hope to improve a strain of laboratory mammals, such as mice, rats, and perhaps rabbits, by selection. The selection differential achieved will be about one standard deviation, and the heritabilities of most characters will lie between about 20 and 50 per cent. The rate of improvement will therefore be between about one-fifth and one-half of a standard deviation per generation. If 5 generations can be covered in a year, which is just possible with mice, the investigator can reasonably hope for between 1 and 2.5 standard deviations of improvement in a year. The prediction of a response to selection is theoretically valid only for one genera- tion, because the selection must be expected to change the genetic properties on which the response depends. But experiments have shown that, in practice, the initial rate of response can be expected to continue for 5 or perhaps even 10 generations. Eventu- ally, selection applied to a closed population leads to a limit beyond which there is no further response. The time required to reach the limit, and the final level reached, 212 PHYSIOLOGIC GENETICS cannot be predicted a priori. However, experiments suggest, although they are really too few to justify a generalization, that the response may be expected to continue for about 20 generations and that the total improvement one might reasonably hope to achieve by selection is probably of the order of 4 to 10 standard deviations. These con- clusions, however, rest on a very tenuous factual basis. The complicating factor which lays any prediction of the response from a know- ledge of the heritability open to doubt is this: several experiments in which selection Fig. 36. Two-way selection for six-week weight of mice The generation means are plotted against the cumulated selection differentials, and linear regression lines are fitted to the points. The slopes of these lines, which estimate the realized heritabilities, are: upwards, 0.175 + 0.016; downwards, 0.518 + 0.023.341 was made in both directions, that is, for an increase and also for a decrease of the character, have shown that the rate of response in the two directions was not equal. One such experiment is illustrated in figure 36. The heritability estimated from the response, which may be called the realized heritability to distinguish it from estimates based on the resemblance between relatives, was 17 per cent in the upward direction and 52 per cent in the downward direction. The heritability, if it had been estimated from the resemblance between relatives, would presumably have been about 35 per cent, and, if a prediction had been made for upward selection, the response achieved would have fallen short of expectation. Since the reasons for these asymmetrical responses are not yet fully known, there is no means of predicting when they are likely to occur. Consequently it would be unwise to put much faith in any prediction of a QUANTITATIVE INHERITANCE 213 response to selection in one direction. And, for the same reasons, if selection is to be used as a means of estimating the heritability, it is essential that selection should be made in both directions and the heritability estimated from the divergence between the two selected lines. There are two details of procedure connected with the estimation of heritability from the response to selection that should be briefly mentioned. The first concerns the selection differential. The selected parents may contribute unequally to the off- spring from which the response is measured. It is therefore necessary to compute the selection differential as the weighted mean of the superiority of each parent, the weight being the number of offspring of that parent that were measured. It is of interest to compare the weighted with the unweighted selection differential, because if the weighted is less than the unweighted this gives evidence of natural selection opposing the arti- ficial selection. It shows, in other words, that the best parents have produced fewer offspring than the less good. The second point of procedure concerns the averaging of the response from successive generations. The best way to do this is to plot the mean of each successive generation against the cumulated selection differential. In other words, plot each generation mean against the sum of the previous selection differentials up to that point. This will give a graph such as that shown in figure 36. The slope of the line up to any point is the ratio of the response to the selection differential, and this ratio (RjS) provides the estimate of the realized heritability. The slope may remain constant over many generations, in which case the average slope may be estimated from a linear regression line fitted to the points, as illustrated in figure 36. Or the slope may change, in which case the fitting of a linear regression over the whole experiment would not be justified. There is always a disconcerting amount of erratic variation of the mean values from one generation to the next. One cannot, in conse- quence, hope to assess the rate of response with any degree of precision until at least 5, or preferably even 10, generations have been obtained. Selection is therefore a time-consuming method of study. Although selection takes a long time before reliable conclusions can be drawn, it does not require a great deal of cage space at any one time. The amount of space required is inversely related to the rate of inbreeding that can be tolerated. If the aim is to produce a useful strain, then it is important to keep the rate of inbreeding low, because there is no opportunity for selection between lines and whatever inbreeding depression occurs must be tolerated. I suggest that for a program intended to last for 10 or 20 generations, the selected individuals should be drawn from not fewer than 10 families. This can be done with the greatest economy of space by mating 10 pairs in each generation and selecting the best two offspring from each family. Selection within families in this way, however, reduces the selection differential because the variation between families is not utilized. If, on the other hand, selection is made purely on individual merit, which should usually give a better rate of progress, then it is necessary to mate substantially more than 10 pairs in order that 10 families will be represented among the selected individuals. 214 PHYSIOLOGIC GENETICS INBREEDING AND CROSSING The effects of inbreeding or of crossing lines on the mean expression of a quantita- tive character provide some information about the dominance of the genes that affect the character, and may perhaps allow us to draw tentative conclusions about the role of the character as a determinant of natural fitness. The effects of inbreeding and of crossing are the same but opposite in direction and they are both the outcome of the same properties of the genes, namely dominance. If inbreeding produces a change in one direction, then the genes on the average should show dominance of one allele over the other and the recessive alleles should affect the character in the direction of the change on inbreeding. To be specific, if the character declines on inbreeding, then the alleles that reduce the character tend to be recessive to their alleles that increase it. If the character does not change on inbreeding it cannot, however, be concluded that the genes do not have dominance. It can only be concluded that they do not have directional dominance; there could be dominance at every locus if some genes were dominant in one direction and some in the other. The presence or absence of directional dominance is the only thing to be learned about the genetic properties of the character from the presence or absence of inbreeding depression or heterosis. An empiric conclusion based on much evidence is that characters obviously connected with natural fitness exhibit inbreeding depression and heterosis. (By characters obviously connected with natural fitness is meant characters reflecting some aspect of fertility or general viability.) This gives some grounds, therefore, for suppos- ing that if a character shows inbreeding depression and heterosis, then it probably has a close connection with fitness, and natural selection in the past has favored individuals with high rather than low or intermediate values. The presence of heterosis is to be judged, in this connection, as any deviation of the Fx from the mid-parental value. THE NUMBER OF GENES The question of how many genes are concerned in the genetic determination of a quantitative character is one of great interest. But unfortunately the answers that can be obtained have not much meaning, and the question itself is not very meaningful. The number of genes cannot be stated meaningfully without specifying the magnitude of their effects. It is not impossible that all genes segregating in a strain affect every character in some slight degree. However, if the suppositions are made that genes either affect the character or do not and that all those that affect it do so by roughly the same amount, then an estimate of the number that may have some meaning can be obtained. The procedure for estimating the number of genes is to cross two strains that differ in the mean value of the character and to measure the variance in the F2 generation and in the F2 generation. The difference of variance between the F1 and F2 gives QUANTITATIVE INHERITANCE 215 an estimate of the total genetic variance in this particular F2 generation. The estimate of the number of genes that cause this genetic variation then comes from a comparison of the amount of genetic variance with the difference of mean between the parental lines that were crossed. It is easy to see that if there are very few genes segregating — say one or two — then the parental values will be easily recovered in the F2 ; but if there are any more than a few genes the parental values will not be recovered. Therefore the greater the variance in relation to the parental difference, the fewer are the genes segregating. The relationship that estimates the number of genes is 1 R2 n = •= where R is the difference between the parental strains, and V(F2) and V(Fl) are the variances of the F2 and Fx generations respectively. This relationship, however, is based on a number of assumptions, and if these do not hold it is not valid. The conditions for its validity are : 1 . All the + genes are concentrated in one of the parental strains and all the — genes in the other. 2. The parental strains are highly inbred (or long selected), so that the genes segregating in the F2 are all at frequencies of one half. 3. There is no dominance. 4. There is no linkage. 5. All the genes have effects of equal magnitude on the character. The first of these conditions will hardly ever be met unless the parental strains have previously been selected to their limits in opposite directions. Inbred lines that have not been selected for the character under study are therefore of very little use for this purpose. The second condition is easily satisfied, and will be automatically satisfied if the first condition is met. The third condition is probably not very important, because even if all the genes were fully dominant, the error would not be very great compared with the other sources of error. The fourth condition (absence of linkage) is unlikely to be fulfilled unless there are very few genes. Linkage sets an upper limit to the estimate that it is possible to obtain for the number of genes. There cannot be more genes estimated than there are independently segregating segments of chromo- some. The last condition has already been discussed. It is not so much a condition as a definition of the meaning of the number of genes. The consequence of any of these four conditions not being fulfilled is that the estimate obtained will be lower than the estimate that would have been obtained if the condition had been fulfilled. This is the only comforting feature of the situation because it means that we can be sure that the real number of genes is greater than our estimate. 216 PHYSIOLOGIC GENETICS SUMMARY The two properties of primary interest in the genetics of any quantitative character are the degree of genetic determination and the heritability. The degree of genetic determination is the proportion of the total phenotypic variance that is attributable to genetic differences between individuals. It is estimated from a comparison of the variances of a genetically uniform group, such as an Fx of two inbred lines, and a genetically heterogeneous group, such as an F2 or a random bred strain. The number of animals that must be measured in order to attain a given degree of precision in the estimate can be determined from formulae and graphs presented. The heritability is the proportion of the total phenotypic variance that is attribu- table to additive genetic variance. It is estimated from the degree of resemblance between relatives. The regression of offspring on parents is usually the best method for laboratory mammals. The design of the experiment to give maximal precision is discussed. In general it is best to have as many parents as possible at the cost of fewer offspring per parent. The method of computing the regression when the families contain unequal numbers of offspring is explained. The correlation of half sibs is another method of estimating the heritability. The most efficient design is very inconvenient with laboratory mammals, and as a compromise it is suggested that as many males as possible should each be mated to 3 or 4 females and between 5 and 10 offspring from each female should be measured. The structure of the sib analysis with full and half sibs is described ; and the method of computing the components of variance, and from them the correlations when there are unequal subclass numbers, is explained. The genetic information that can be obtained from artificial selection and from inbreeding or crossing inbred lines is briefly explained. Finally, the estimation of the number of genes contributing to the variation is discussed. Only in very restricted circumstances does the estimate have any useful meaning. Elizabeth S. Russell, Ph.D, PROBLEMS and POTENTIALITIES in the STUDY of GENIC ACTION in the MOUSE * Studies of the physiologic genetics of laboratory mammals have a special role in the biomedical sciences in that they can and must be the integrating link between basic information from lower organisms and the human problems to which these facts and concepts must ultimately apply. Recent medical advances, by decreasing the preva- lence and severity of infectious diseases, have increased the relative importance of constitutional or inherited disease. It is to be hoped that knowledge of the etiology of human and analogous experimental mammalian inherited disease syndromes may be useful both for prophylaxis and for therapy. During the past quarter century much has been established about the nature of the self-duplicating molecules of deoxyribonucleic acid which transmit genetic in- formation from generation to generation and from cell to cell, along with a beginning insight into the nature of the relationship between these units of genetic transmission and their intracellular products. In experiments with microorganisms many gene- controlled biochemical pathways have been traced. It is very much to be hoped at this particularly stimulating stage in the development of genetics that widespread attention will be turned toward solution of problems of genie action in highly differen- tiated forms, particularly in mammals. Observed inherited characteristics in them are the raw material of many areas of biology : embryology, anatomy, physiology, bio- chemistry, endocrinology, immunology, and pathology, for example. The techniques of these disciplines may profitably be applied to analysis of mammalian genie action, 217 218 PHYSIOLOGIC GENETICS and the answers obtained contribute to understanding in these fields. At present the house mouse is the most widely studied mammalian species other than man, because of the large number and variety of named genes and the availability of many inbred strains with genetic homogeneity and well-established physiologic characteristics. The purpose of this paper is discussion of the special problems encountered in studies of the physiologic genetics of the mouse. f The fact of differentiation leads to complications never encountered in micro- organisms. Tracing the pathway between site of original action of the gene and observed character is frequently a major problem. When the site and time of original genie action have been established, it is often difficult to devise methods of analyzing metabolic processes within the affected cells. Examples of profitable approaches surmounting these obstacles have been selected from recent literature and current research projects. It is hoped that presentation of these pertinent examples, and discussion of their relationship to fundamental genetic questions, will lead to some basic generalizations regarding the methodology of mammalian physiologic genetics. GENES, PROTEINS, AND ENZYMATIC ACTIVITY It is probable that the primary activity of most, if not all, genes is determination of the specificity of an intracellular macromolecule. Very few of the inherited charac- teristics recognized in mammals represent these immediate genie products. In the few cases, however, where this may be true, that is, where the observed effect of a genie substitution is change in the structure of a protein molecule, the fact of cellular dif- ferentiation may simplify analysis of genie action. The production of this protein may be limited to particular types of cells and may represent a very large part of the total metabolic activity of these cells, greatly facilitating biochemical and physical chemical analysis. Determination of the globin structure of hemoglobin appears to fall in this class,102' 104, 1181 and experimental genetic analysis of hemoglobin pattern in the mouse, described in this volume by Dr. Popp, shows great promise. Little insight has yet been gained into the developmental processes channeling the metabolism of hematopoietic cells into this limited range of activity, a problem which presents a challenge for future investigations. The action of the well-known albino series of genes, affecting intensity of hair pigmentation in the mouse and other mammalian species, may very well be determina- tion of the structure of a tyrosinase molecule. Genie substitutions at this locus definitely alter tyrosinase activity,382 which could mean either alterations in concentration of identical enzymes or qualitative alteration of the enzyme molecule similar to that seen in certain tyrosinase mutants598 in Neurospora. Evidence that this series of genes t The research reported in this paper, which originates from the Physiological Genetics Group at the Roscoe B. Jackson Memorial Laboratory, has been supported by a contract between the U.S. Atomic Energy Commission and by grants to the laboratory from the U.S. Public Health Service and the American Cancer Society. GENIC ACTION IN THE MOUSE 219 may determine enzymatic structure comes from recent studies213 of tyrosinase activity in mice carrying a new allele of the albino series, Himalayan (ch), discovered recently in the Jackson Laboratory.487 This mutant almost certainly contains a qualitatively different tyrosinase from those found in mice with any of the other alleles of the albino series. If one gene in an allelic series is responsible for a qualitative enzymatic difference, it is probable that the entire series acts by determining enzymatic structure. Gene-induced difference in enzymatic activity is not always, of course, attributable to change in enzymatic structure. Recent studies of the mutant allele dilute (d) and dilute-lethal (dl) have demonstrated a deficiency of the enzyme, phenylalanine hydro- xylase, associated with genie substitutions at this locus.214 Further studies have indicated, however, that the genie action involved is an inhibition of activity of an enzyme formed quite independently of the genes at the dilute locus. FUNCTION OF GENES AND TISSUES A relatively large proportion of the analyzed effects of substitutions of single genes in the mouse has been traced to differences in the structural differentiation of certain specific tissues only. For these genotypes, the majority of tissues of an affected animal, except in terminal stages of a lethal condition, cannot be distinguished from those of an unaffected littermate. The choreic movements and deafness associated with six different independent mutant genotypes in the mouse have been traced to dedifferentiation of Corti's organ, degeneration of the spiral ganglion, and abnormali- ties in the stria vascularis.248, 249, 4" Since effects of these mutants compensate each other, in crosses, it is clear that there are at least six different ways in which tissues of the murine vestibular apparatus can fail. The locomotor difficulties, whole-body tremor, muscular spasms, and early mortality found in wobbler-lethal mice (wlwl) all appear to result from degeneration of the myelin sheath surrounding nerve processes.273 The retina of a mouse with retinal degeneration (rdrd) develops normally for the first ten days after birth, then begins to show degeneration of the rods, while those of normal littermates continue differentiation.1258, 1313 For each of the mutant types so far described in this section, histologic difference from normal has been described which involves an inability of affected tissue to com- plete normal differentiation or to maintain normal structure. It is probable that each defect is due either to a metabolic error specific to the abnormal tissue or one imposed upon this tissue by a correlative influence from some other part of the body, that is, either local or distant site of original genie action. For many tissues it is difficult to obtain critical evidence as to this cellular localization, although the limitation of visible difference to one tissue suggests greater probability of local genie action. A somewhat equivocal suggestion as to the site of genie action in retinal degeneration may be derived from an experiment involving organ culture in vitro of genotypically normal and rdrd eyes explanted at the stage (10 days) when degeneration is first 220 PHYSIOLOGIC GENETICS visible.808 The degeneration continued in rdrd eyes, while normal eyes showed fairly normal differentiation. This suggests local genie action. There was, however, some delay in the rate of rdrd degeneration in vitro, which may have resulted from retardation of autonomous development but which might also indicate that explantation removed the eye from an humoral toxic effect in vivo. Although this experiment provides a suggestion as to a site of original genie action, there is no clue as yet for any of these mutants as to the nature of their metabolic error. A somewhat different situation exists in the study of hereditary muscular dystrophy (dydy) in the mouse. Skeletal muscle is certainly the primarily affected tissue in this disease since the nervous system appears entirely normal.869 Detailed histopathology demonstrates great similarity to other hereditary muscular dystrophies, and gives evidence that there is a continuing attempt at muscular regeneration even in in- dividuals severely affected.1383 Studies of C14-glycine incorporation and turnover corroborate this finding.215 Progressive wastage results from an excess of destruction over synthesis. Parabiosis experiments, with conjoined normal-dystrophic pairs, indicate clearly that there is no humoral factor responsible for muscular breakdown.1013 The defect is indigenous to the muscle itself, thus almost certainly a result of local genie action. All of the differences observed are undoubtedly the result of difference in a single genie pair. The mutation was first recognized as a deviation within an inbred strain, and the distribution and frequency of dystrophy in pedigrees and in offspring of ovaries transplanted from dystrophic females clearly demonstrated in- heritance as a unit autosomal recessive.869, 1281 Evidence is now at hand, from out- crosses, repeated backcrosses, and linkage tests, that this substitution of a single genie pair creates essentially the same syndrome of disease in combination with a great variety of genetic backgrounds.805 The defect is already apparent histologically at two weeks postnatal, the first age at which the entity can be diagnosed from behavior. At earlier stages study of muscular disease is difficult because normal muscle has not yet assumed its adult form; this finding suggests that in muscular dystrophy, as in previously described syndromes, the tissue defect may involve a failure to complete normal differentiation. In contrast to the disease entities previously described, however, there is a bewildering array of evidence of deranged metabolism. K/Na balance is abnormal,47' 683 creatine/creatinine balance is abnormal,683, "6 muscle lipid is increased,1200 distribution of lipoproteins and glycoproteins deranged,968 myosin concentration decreased,969 contractibility decreased,1148 relaxation time lengthened,1148 and levels of activity of many enzymes altered.10, 216, 217, 541, 1034, 1035, 1074. i3i4. 1372. 1468 jt appears probable that many of these alterations occur when- ever muscle is degenerating and thus are results rather than causes of the basic defect. There is no guarantee that the character which is associated with a particular genie substitution is the final stage in a path of genie action ; it may be an intermediate step, and the recognized character may have extensive metabolic consequences. In a situation with multiple metabolic changes, it is difficult to determine which defect is primary. One helpful approach is retrograde analysis, attempting to find a metabolic GENIC ACTION IN THE MOUSE 221 deviation present at an especially early stage when other reactions are still normal. This concept underlies some of the especially promising current investigations on murine muscular dystrophy, but the answers are not yet forthcoming. GENES, HORMONES, AND PATTERNS OF RESPONSE Localizing the initial site of genie action presents special problems in cases of endocrine defect, because of reciprocal relations between different hormones. This has proved particularly true in recent studies of pituitary dwarfism in the mouse, the first gene whose action was analyzed by substitution therapy.1222, 1236 The defect is definitely in the anterior pituitary, but the particular type of cell primarily affected is still in doubt to some extent. Gonadotrops are present, although abnormal in ap- pearance, in the dwarf pituitary, and their functional capacity has been proven con- clusively in transplantation experiments.1223 All observers agree dwarfs show a great deficiency in typical murine acidophils, but recent investigations using sophisticated histologic techniques321, 100°- 1049, 1368 disagree as to whether there are undifferentiated potential acidophils. There is also great reduction in typical thyrotrop cells. Uptake of radioactive iodine suggests but does not prove that the thyroid of dwarfs is especially sensitive to thyrotropin, which favors the interpretation of initial deficiency of thyro- trops and thyrotropin.1367 Recent experiments in which rate of growth was studied following reciprocal transplants placing pituitaries of littermate normal and dwarf mice 14 to 18 days of age into the sella of hypophysectomized normal and dwarf hosts showed a comparable rate of growth of normals and dwarfs with transplanted normal pituitaries and a reduced rate in normals with dwarf pituitaries.155 This evidence indicates the defect is in the pituitary rather than the hypothalamus, since the organ functions autonomously following transplantation. It also favors the interpretation of primary deficiency of acidophils and growth hormone. It is to be hoped that in the near future a transplantation approach, combined with histologic study of changing cellular types in the implanted pituitary, may help to identify the types of pituitary cell initially affected in dwarf mice. Genetically controlled differences between inbred strains have also been reported for androgen level and responses,189, 191, 192, 194 estrogen level and response,191 and thyroxin level and response.190, 193 Since these differences have a polygenic basis, they do not provide good material for retrograde analysis, but the methods used merit description. Secreting glands were extirpated or inactivated, and sensitivity of target organ measured by addition of graded doses of exogenous hormone. Normal circu- lating level of each hormone was calculated from the dosage producing the end-organ condition normal for the strain. There is also much evidence of genetically controlled difference in reaction of the adrenal to gonadectomy. The adrenals of some strains fail completely to respond to this stimulus, others show hyperplasia, and still others show cortical carcinoma 193, 271. 1407. 1408 222 PHYSIOLOGIC GENETICS GENES IN EMBRYONIC DEVELOPMENT AND DIFFERENTIATION Many skeletal abnormalities and derangements of organs have been shown to result from genie action during embryonic development. Retrograde analysis, studying the mutant phenotype at progressively earlier stages, is clearly an effective tool for locating place and time of initial genie action but does not always help in determining its nature. Frequently there is evidence of inductive failure. An abnormality frequently associated with the Short-Danforth gene (Sd) is reduction or absence of the kidney, which has been demonstrated to result from reduced length of the ureteric outgrowth. In some cases there is no contact with competent tissue, and no induction of meta- nephros.440 In addition to the previously mentioned genes causing choreic move- ments through tissue dedifferentiation, a series of genes with very similar behavioral effects act in a very different pattern by preventing normal induction of the middle ear, each gene acting in a different and specific way.499, 818 The most extensively studied series of genes acting through induction is the Brachyury or T series. In addition to the normal wild-type allele, one dominant (T) and numerous recessive (tx) alleles are known; additional instances of mutants have frequently been found in populations of wild mice. Most of the homozygous types are lethal before birth, the time of death varying greatly among alleles, but a few are viable. Only T leads to tail reduction in combination with the wild-type allele, and all Ttx combinations lead to short or absent tails. The homozygous lethal alleles of the T'-series fall into five classes according to time and nature of original genie action.76 Primary action of T seems to be inductive failure at approximately the ninth or tenth day of development resulting from degeneration of the chorda-mesoderm.204- 502 The /12/12 homozygote shows the earliest lethal effect, failure of blastocyst formation as- sociated with defective trophectoderm.1221 In t°t° homozygotes the inner cell mass fails to differentiate into embryonic and extra-embryonic ectoderm.439 In a group of five relatively early-acting tw alleles (collected from the wild), the first microscopically visible defect is pyknosis in the basal plate of the neural tube around the seventh day of embryonic life. Homozygotes of four relatively late-acting tw alleles show their first abnormality on the ninth or tenth day of development, as pyknosis in the ventral portion of the hindbrain, the notochord and mesoderm being normal.76 These interesting findings have many embryologic implications for which the reader is referred to the original papers. Several points are, however, worth mentioning in connection with general principles of genie action. Bennett and Dunn77 point out that although the first microscopically visible defect in all t homozygotes is in ectoderm, it is still impossible to decide between local genie action deranging metabolism of the embryonic ectoderm and genie action in the endoderm leading to improper nutrition and consequent death of ectodermal cells. In each case, however, the ectodermal degeneration is associated with failure of induction of mesodermal tissues. The neural tube of both TT and twtw embryos is capable of inducing cartilage in vitro™ In GENIC ACTION IN THE MOUSE 223 attempting to bring together the differing effects of this multiple allelic series, Bennett, Badenhausen, and Dunn76 suggest there are "some grounds for suggesting that at least four different lethal /-alleles affect pathways which lead to progressively higher types of neural differentiation. The mutation T . . . appears to affect a different pathway in differentiation, that leading through the chorda-mesoderm. It is not easy to reconcile this diversity in physiological effects with 'unity of action' of a locus that appears on genetical grounds to be a unit not resolved by recombination." In the face of this confusion, it is comforting to remember that tracing the widely differing paths affected by T and t alleles has nevertheless been very helpful in explaining their complementation in the viability of 77 hybrids. Some genes with shape-determining effects in embryonic life act through mech- anisms other than induction. An interesting example is short ear, in which a primary effect appears to be reducing growth of cartilage. Since cartilaginous growth is most conspicuous in fetal life, many effects of short ear are apparent at this time. However, recent experiments with healing of broken ribs have shown that a metabolic deficiency in cartilage growth persists into adult life in short-eared mice.486 MURINE HEREDITARY ANEMIAS Analysis of genie action in the causation of six different types of inherited anemias in mice is a major concern in current physiologic genetic investigations at the Jackson Laboratory. Genes at three of the loci, Dominant spotting (W, W't Wv),503- 50i- 1091' 1102. 1106 gteei ^ s[d^ 83. 1155 an(j Hertwig's anemia (anan)7*1 cause varying degrees of macrocytic anemia. Homozygous flexed-anemia (ff) animals suffer from a transitory macrocytic siderocytic anemia limited to fetal and neonatal life.505' 506' 885 Animals homozygous for either jaundice {jaja) 1280 or hemolytic anemia (haha) 83 suffer from severe hemolytic disease with abnormal nucleated erythrocytes and extensive postnatal jaundice. Animals of five of the available genotypes die shortly after birth, and several other genotypes are semilethal. Each anemia seems to present a different defect in hematopoiesis, and each is present before birth. We are very hopeful that some of these anemia-producing genes may provide especially favorable material for identifying the metabolic error in a tissue primarily affected and possibly may even give clues as to factors limiting genie action to these tissues. The murine anemias named above which we are studying do not exhaust the list of available types. Another dominant- spotting allele has been reported (Wa).907 A very interesting independently in- herited anemia, diminutive (dm) , with associated skeletal defects, has also been described recently.1279 It is essential, of course, to have an accurate description of the erythron and the hematopoietic tissue in each mutant genotype. It is also important to measure ac- curately quantitative differences between the effects of different alleles within the same series and between genes of different series, avoiding difficulties from variations in the genetic backgrounds on which these genes are segregating. As with various entities 224 PHYSIOLOGIC GENETICS discussed in the general section on genes and tissue function, it is important to deter- mine the site of initial genie action in each case. It appears probable that most of the defects are indigenous to bloodforming tissue, but this can only be established by transplants between genotypes affected differently. For these reasons, the development of congenic histocompatible strains differing essentially only in the anemia-producing genes is an important part of our work.1105 Since animals of several important genotypes are available only in fetal and new- born stages, it is also very important to develop methods for working with extremely small quantities of tissues and for studying metabolism of fetal hematopoiesis. Forma- tion of hemoglobin is, of course, an essential part of all ery thropoiesis ; and, since many human hereditary anemias have been traced to specific hemoglobinopathies,619' 938 it seems essential to study the nature of hemoglobin associated with different anemic states. Studies of the reactions of normal and affected animals to particular physio- logic stimuli or stresses may be very helpful in identifying metabolic deviations. Several of the genes under study have extensive pleiotropic effects in other tissues. Analyses of these effects will undoubtedly increase knowledge of pathways of genie action, but may also yield clues as to the nature of original genie action in hematopoietic tissue. Attempts are being made to develop each of these approaches in the study of murine anemias, but, except for experiments with the W series, they are still in the stage of potentiality rather than performance. CHARACTERIZATION OF ff-SERIES ANEMIAS The hematologic phenotype of animals differing in M^-series genes ranges from an ostensibly normal picture in ww, Ww, and Wjw animals,1091, 1102, 1106 to slight macrocytic anemia in Wvw,1091 and severe macrocytic anemia in animals of all double- dominant genotypes. Order of increase in severity of afflication is ww (normal) = Ww = Wjw < Wvw < WVWV < WWV = W'WV < WW = WW1 = WjWj. (Animals of the last three genotypes are almost invariably lethal in early postnatal life.) The bone marrow of adult WVWV and WWV individuals has almost exactly the same cellularity as that of their normal littermates,970 although it produces only slightly more than one-half the normal number of erythrocytes, suggesting a delay in matura- tion of erythroid cells. Careful microscopic study of the marrow confirmed this suggestion, since the ratio of early to late stages in erythropoiesis was significantly higher in the anemic animals.1110 The first evidence suggesting a biochemical basis for this arrest came from C14-glycine-incorporation experiments using anemic and normal littermates.16, 1097 There was no difference between genotypes in time of appearance of erythrocytes with labelled globin, but in the anemic mice there was a great delay in appearance of cells with labelled protoporphyrin. Subsequent experi- ments showed a similar delay in appearance of labelled protoporphyrin in erythrocytes ofanemics following injection of radioactive (5-amino-levulinic acid,15 an intermediate on the path leading to protoporphyrin. This delay in heme synthesis, which is still GENIC ACTION IN THE MOUSE 225 under investigation, seems a critical feature in action of Miseries genes. Animals of severely affected genotypes are anemic because of a metabolic defect, which either specifically delays the synthesis of protoporphyrin or nonspecifically arrests erythroid cell maturation at a stage when synthesis of protoporphyrin is an important metabolic activity. TISSUE LOCALIZATION OF ACTION OF THE JF-GENE Genie action leading to the hematopoietic defect of Miseries anemic animals definitely occurs in the hematopoietic cells themselves, rather than being imposed from another part of the body. This has been demonstrated repeatedly by successful implantation of hematopoietic cells from normal ww fetal liver into adult WWV and juvenile WWV and lethally anemic WW mice.84' no9 The implanted cells function autonomously according to their own genotype, and the blood picture of the host changes gradually but permanently to that of a normal mouse. It is interesting that an initial very small inoculum of rapidly metabolizing cells eventually overwhelms a large body of indigenous defective marrow. These experiments are also an interesting demonstration that hematopoietic cells from the fetal liver at least can implant and function in an adult manner in the marrow spaces of an adult. RADIATION RESPONSE OF NORMAL AND ANEMIC MICE The responses to irradiation of normal ww and anemic WWV animals are extremely different. A dose of whole-body irradiation (200 r) which has very little effect upon the blood picture of normal ww mice causes severe and prolonged reduction of the hematocrit level, and, in some cases, death in littermate WWV anemic mice.85- 1109 The hematocrit level of surviving WWV mice returns to normal suddenly 3 to 4 weeks after radiation treatment. Quantitative microscopic study of the marrow of normal ww and anemic WWV mice at successive intervals after 200 r whole-body irradiation revealed no difference between genotypes in initial destruction of marrow cells.1098 The cellularity in both genotypes decreased sharply for the first two days after irradia- tion. The marrow of ww individuals then regenerated rapidly and had returned almost to pretreatment level by the fourth day after irradiation. That of WWV mice, however, regenerated very slowly, and showed no visible increase in cellularity by the eighth day after irradiation. Further evidence that radiation sensitivity depends in this case upon the genotype of bloodforming tissue is found in studies of the reaction of implanted WWV mice, with a normal blood picture, to increasing doses of X irradiation. Their 30-day ld50 dose corresponds closely to that of normal ww mice.83 There is even evidence that single doses of W or Wv (in Ww and Wvw animals) significantly affect radioresistance.83 226 PHYSIOLOGIC GENETICS RESPONSE OF NORMAL AND ANEMIC MICE TO ERYTHROPOIETIC STIMULI Studies of the responses of ww normal and WWV anemic mice to known erythro- poietic stimuli have yielded valuable information both on the pattern of M^-gene action and on the nature of hemostatic mechanisms. Twelve moderate-sized daily doses of a purified erythropoietin807 which has induced extra erythropoiesis in normal animals of six different species,696 were administered to normal ww and anemic WW0 animals. In all cases the normal ww mice responded to the treatment with greatly increased hematocrit level, marked reticulocytosis, and increased total blood volume.1104 The WWV mice, however, were completely unaffected by the injections. This genotypic difference in reaction to a known erythropoietic stimulus depends upon the genetic nature of the bloodforming tissue. The response to erythropoietin of WWV mice implanted with ww cells is exactly comparable to that of ww mice.697 There is at least one erythropoietic stimulus to which ww and WWV mice respond equally. If normal and If-anemic mice are subjected to reduced atmospheric pres- sure503 or to lowered oxygen tension at normal pressure,697 animals of both genotypes respond with reticulocytosis and increased hematocrit level. The difference in response of WW0 mice to different stimuli demonstrates clearly that there must be more than one fundamental erythropoietic stimulus and suggests that the basic genie action in Miseries anemias may be related to the phase of erythropoiesis affected by treatment with Borsook-type extracted erythropoietin. It is very tempting to speculate that the maturation arrest of WWV mice, their special radiation sensitivity through delayed marrow regeneration, and their inability to respond to erythropoietin may all be different aspects of the same basic phenomenon. ANALYSIS OF JF-SERIES AND STEEL PLEIOTROPISMS In addition to suffering from the macrocytic anemia already described, animals of all double-dominant TT-genotypes {WW, WWV, W'WV, etc.) lack pigment in the hair and are almost completely sterile. The sterility results from failure of the pri- mordial germ cells to multiply during their migration to the gonadal ridge, between the eighth and twelfth days of embryonic life.879- 880 In this analysis the primordial germ cells were visualized in sectioned whole embryos by alkaline-phosphatase staining. The association of reduced germ-cell number with the double-dominant genotypes was determined on a statistical basis rather than by genotypic identification of individual embryos. Only matings between two heterozygous parents produced embryos with defective germ-cell numbers at 9, 10, 11, and 12 days, and the proportion of defective embryos at each age was close to 25 per cent. The germ-cell defect seems to be com- pletely determined by the twelfth day of embryonic life, since 12-16 day fetal gonads transferred to a neutral site with a rich blood supply developed autonomously according to their genotype.1107 In this study using older fetuses as gonad donors, individual genotypic identification was possible. All double-dominant fetuses were pale at 12-16 days due to their hematopoietic defect. Luxate, a third-chromosome gene approxi- GENIC ACTION IN THE MOUSE 221 mately 18 crossover units from the WMocus, provided another check upon fetal genotype. Homozygous luxate (Ixlx) fetuses were identifiable as early as the twelfth day of develop- ment by the shape of their abnormal hind feet.168 This gene was placed in test matings in coupling with Wv, so that more than 96 per cent of all fetuses with abnormal hind feet {Ixlx) would also be expected to be severely anemic and potentially sterile (WVWV). Thus, a closely linked gene with early clear-cut expression proved to be a very useful tool in retrograde analysis. An interesting long-term consequence of the paucity of germ cells in WVWV mice is that all of the females eventually develop ovarian tumors.1101 The lack of pigmentation in WW and WVWV mice is essentially 100 per cent white spotting, involving absence of melanoblasts in the hair follicle.1207 Melanoblasts, which normally migrate from the neural crest to all parts of the body between the eighth and twelfth days of embryonic life, differentiate from explants of the flank area of normal vow 10-day embryos, but do not appear in explants from the same area of WW littermates.100 The pigment defect of homozygous defective Mi- series mice is thus completely determined very early in embryonic life. Homozygous SISl embryos and neonates74 and adult SldSld individuals83 show a triad of pleiotropic effects in bloodforming tissue, germ cells, and hair pigmentation almost identical with that found in IT-series homozygotes. At least 7 separate in- stances of mutation at the H^-locus are known to have occurred spontaneously, in addition to frequent Miseries mutations in radiation experiments. Four separate instances of SI- locus mutations are known. In each case, all three aspects of the triad were simultaneously altered. It is almost impossible to avoid the conclusion that each of the genes at both of these loci acts as a unit, controlling a single process. The process controlled by the W locus and that controlled by the SI locus may be closely related, possibly as different steps in the same synthetic pathway. In which of the three severely affected tissues is this series of processes initiated ? It is known to be important in fetal liver and adult marrow hematopoiesis, but no information is available as to possible yolk-sac hematopoietic defect before the twelfth day of embryonic life. Circulation begins at approximately 9.5 days.1247 The reduced multiplication of germ cells in WW and WVWV embryos is apparent at 9 days and very marked at 10 days, the melanoblast defect at 10 days. The very early appearance of these anomalies makes it relatively improbable that they are dependent for expression upon an anemia resulting from defective yolk-sac hematopoiesis. It seems very much more probable that the W-Sl series of processes is independently of great metabolic significance in primordial germ cells, in melanoblasts or their em- bryonic precursors, and in hematopoietic cells. Unity of genie action for these genetic loci may involve processes specifically important in the metabolism of three different tissues. MATERIALS AND METHODS IN MAMMALIAN PHYSIOLOGIC GENETICS Review of the examples of genie action in the mouse discussed in this paper may lead to certain generalizations on methodology. The first tool in search for time and 228 PHYSIOLOGIC GENETICS place of original genie action is a retrograde analysis. Transplantation and extirpation may be very useful in determining organ localization of the primary defect. Com- parison of growth of normal and affected tissues or organs in vitro may also prove very useful if culture conditions are sufficiently physiologic to allow deduction as to relation of observed differences to genotype. Linked genes may assist in genotypic identifica- tion, particularly in fetal material. Detailed microscopic analysis, sometimes com- bined with other approaches, helps to distinguish the cells primarily affected. Replacement therapy, isotope incorporation, and tests of functional capacity are useful in determining the nature of intracellular processes affected by particular genie substitutions. Comparison of the effects of different genes in a multiple allelic series and study of dominance relations may provide valuable clues, as may pleiotropic effects of single-gene substitutions. Studies of genie action are usually limited to unit genes with clear-cut effects, since their effects may most easily be traced. These unit genes tend to affect one or a very few types of cells, probably because the reactions they control are especially important in these cells. (It should be recognized that our methods of detection favor recognition of unit genes with tissue-limited effects; others may either be cell lethal or so diffuse in their effect as to escape detection. Many gene-controlled reactions must alter slightly the metabolism of many kinds of cells.) The characters observed as a result of genie action may be very close to original genie action, as in determination of hemoglobin pattern, or very far removed, as in choreic behavior as a result of defective induction of the middle ear or ovarian tumorigenesis as a result of deficiency of primordial germ cells. It is seldom possible to predict the number of processes between original genie action and observed characteristic; nor is it always possible to be sure when one has reached the end of the road. Although the examples in this dissertation have tended to be restricted to effects of single-gene substitutions, characteristics of inbred strains form an important part of available material. Comparison of mice from different inbred strains has demon- strated existence of many inherited characteristics depending upon the interaction of polygenic factors the individual effects of which cannot now and may never be identi- fied. Since it is probable that the interacting genes work in different ways, it is unlikely that the paths of genie action lying behind a multigenic character can be traced very far. Provided the limitations are understood, however, it is frequently possible to design excellent experiments identifying terminal stages of the pathway. Chai's work with endocrine level and response, Heston's studies with genetics of neoplasia, and Gowen's studies of disease resistance are excellent examples of such analyses. These cases must be studied if physiologic genetics is to be put to use in the service of man. Any study which traces a segment of a pathway between gene or genotype and observed character, however far this segment may be from original genie action, is a contribution to physiologic genetics. Although this point has not been stressed in our presentation, it is very clear that for successful analysis of the action of a unit gene it is very desirable to have it segregating GENIC ACTION IN THE MOUSE 229 against a genetically homogeneous background. Histocompatibility is essential for transplantation. Quantitative evaluation of effects of particular genie substitutions depends upon uniformity of the base line used for comparison. Uniformity of genetic background may come about in three different ways.483 With the increasingly wide- spread use of inbred strains, many of the stocks in which deviants are detected may be inbred, so that from its first appearance a new mutant may be segregating against an inbred background. The best way to maintain high congenicity is by repeated back- cross of the mutant heterozygote to the strain of origin. If a new mutation is found in a genetically heterogenous stock of animals, a new inbred strain may be produced by successive brother-sister matings with forced heterozygosis for the mutant allele, or the mutant allele may be placed on an existing inbred background by many repeated backcross generations. It may be worth mentioning that there are at present in the Jackson Laboratory alone 67 stocks designed to place and maintain specified mutant genes on inbred backgrounds.751 Twenty-five of these mutants are maintained con- genic with C57BL/6J, which makes for excellent uniformity between experiments and provides very favorable material for new genotypic combinations and comparisons, and for double-genic substitutions in transplantation experiments. There is evidence, however, that this one inbred strain is not the ideal background for all mutant genes. As a final plea in methodology of mammalian physiologic genetics, I would like to encourage very widespread use of controlled genetic material by investigators in other biomedical disciplines. The analyses of genie action cited in this paper include many excellent examples of such utilization. Physiologic geneticists are forced, by the diversity of the paths of genie action which they encounter, to be jacks of many trades and face the very real possibility of being masters of none. The studies of W- series anemias have involved active participation by pathologists, biochemists, embryo- logists, and physiologists. Multidisciplinary approach to analysis of the action of a single set of genes has been very useful in this case. Similar collaboration between geneticists and other types of investigators may prove profitable for many other studies. DISCUSSION Dr. Burdette: Thank you, Dr. Russell. Dr. Russell's paper will first be discussed by Dr. D. L. Coleman. Dr. Coleman : I would like first to commend Dr. Russell on her stimulating and comprehensive discussion of genie action in the house mouse. I would like to comment further on two points to which she has alluded in her talk. First, I would like to clear up a possible misunderstanding. I do not think that the tyrosinase picture is quite as clear-cut as she has indicated. However, the best evidence at this time does suggest the situation which she has described. The other point I would like to discuss relates to my particular studies on the dilute mouse214 and attempts to demonstrate how some of the methods which she has described 230 PHYSIOLOGIC GENETICS can be used. In the study of the dilute gene, we were very fortunate in that mutation to the intense color has occurred several years ago in the DBA stocks. Thus we had genetic homogeneity with an intense animal, DD, a heterozygote, Dd, and the normal dilute animal, dd, all available within the same strain. We were fortunate also here in having a multiple allelic series to work with since there is another allele of this locus, the dilute lethal. This allele, although not available on the DBA background, was in the process of being inbred on another stock. Also, we made use of pleiotropism. Both dilute lethal and dilute mice have diluted pigment, but the dilute lethal dies at about 3 weeks and is subject to spontaneous epilepti- form seizures. The DBA strains are also subject to seizures under audiogenic stimuli and it seemed that there should be some relationship between this type of seizure and the pigmentation defect if dilute were just a lesser dose of dilute lethal. The diluted pigmentation further suggested a possible abnormality in aromatic amino-acid meta- bolism. Thus, one of the first things tried was incorporation studies of radioactive tyrosine (the normal pigment precursor) into the pigment granules of both dilute and intense mice. It was found that the rate of pigment formation was the same in both genotypes when tyrosine is used as the pigment precursor. Next the enzymes involved in the formation of tyrosine were examined. The enzyme, phenylalanine hydroxylase, which forms tyrosine from phenylalanine was found to be deficient in dilute strains of mice, having an activity of about 50 per cent in dilute mice, dd, and about 14 per cent that of the normal in dilute lethal. This, at first glance, provided a ready explanation for the diluted pigmentation. However, it was pointed out by Dr. Russell that there is not less pigment in the dilute animal but rather that the granules are in a clumped formation. Also, calculations showed that a leaky enzyme which allowed a 50 per cent production of tyrosine would allow normal pigment formation under most of the conditions that we know, especially when one considers that some tyrosine is derived from the food. Thus, it appeared that we were working with a secondary effect of the original genie action. Further studies on the actual amount of the enzymes present indicated that there was no actual difference between the amount found in dilute and nondilute animals; one function of the dilute gene seemed to be the production of an inhibitor of the enzyme, phenylalanine hydroxylase. In any event, the situation is somewhat analogous to that found in phenylketonuria, diluted pigmentation and inhibition of this enzyme with a subsequent accumulation of phenylacetic acid and other phenylalanine metabolites. Phenylacetic acid is a com- pound toxic to the central nervous system which suggests an explanation for the seizure in dilute mice. There are several areas of research which we now are attempting. Could phenylalanine or a metabolite in abnormal concentrations cause an abnormal development of the brain or neural crest which then leads to these seizures? Could such abnormal concentrations cause an irreversible change in the mode of pigmentation, thus preventing the animal from ever forming normal pigment ? Or, on the other hand, could the decreased levels of tyrosine be critical at an early period of develop- ment, thus causing these changes ? The final answers to these problems will only be GENIC ACTION IN THE MOUSE 231 obtained by using some of the other methods of which Dr. Russell spoke, more specifi- cally, retrograde analysis and possibly transplantation. Dr. Burdette: Do you find very often that the same sort of pathway is present clinically in patients as in the murine condition ? Dr. Russell: In the mouse there is a similar triad of pleiotropic effects880, 1097' ii55, 1207 jn two distinct hereditary anemias. One is the W-series anemia, the other the ^/-series anemia. We know the two anemias are not identical since they are produced by completely nonallelic genes. However, it does seem to me quite probable that these two genes may very well affect different steps in the same synthetic process. It is possible that there are also human anemias of maturation arrest very similar to these conditions in the mouse. These humans might also fail to respond to erythro- poietin. One reason for thinking this is that there are many essential anemias in which affected individuals excrete erythropoietin; and, if they are excreting large amounts of this substance, it is probable that they cannot respond to it, even though they are producing it. If histocompatibility could be sufficiently controlled in man, there might be some purpose to therapy of human anemia by implantation of blood- forming tissue. However, one would have to know a great deal more about human histocompatibility than is now known before one could possibly determine if this method would be useful. The answer to Dr. Burdette's question will be more apparent when more information concerning human histocompatibility is forthcoming. Dr. Wright: The ordinary albino guinea pig has no pigment at birth but later develops considerable pigment on feet, ears, nose, and even back. This is definitely a temperature effect, as Dr. George Wolff and others1401, 1437 showed; there are two other alleles, cr and cd, both of which lead to increased intensity after birth. This darkening of the cd and the cT genotypes is definitely a temperature effect again as Dr. Wolff has demonstrated. Thus there are three alleles among the five in a guinea pig that presumably have thermolabile products. They are low in pigment-producing capacity up to birth. They immediately begin to produce much more afterwards which can be prevented by high temperature. Most of the other changes in color are independent of temperature. Sootiness in yellow guinea pigs, however, and also yellow in the rabbit, as demonstrated many years ago by Walter Shultz,1176 are apparently dependent on a temperature effect. The thermolability in these cases is entirely independent of that of c genotypes. Dr. Pilgrim: Have other heat-labile tyrosinases been found in other Himalayan albinos or in other species ? Dr. Russell : I do not know of any extracted tyrosinase experiment in another mammal. Of course there is thermostable and thermolabile tyrosinase in Neurospora which is the inspiration for this investigation in the mouse. Dr. Ginsburg: Nachtsheim found that the blue-eyed white rabbit is highly susceptible to seizures, both sound-induced and spontaneous.929 However, in a survey of the races, including various pigment types in Dr. Paul Sawin's very representa- tive rabbit colony, susceptibility to these types of seizures has been found in almost all 232 PHYSIOLOGIC GENETICS of his races of rabbits, including various pigment types. Genetic susceptibility to seizures in the house mouse has been found in a number of pigment types, including black animals.1343 Inhibition has not been studied very much. However, in the epidermal cells of the skin of the guinea pig there is an easily demonstrable sulf hydril inhibitor which does not correlate quantitatively with differences in pigmentation, but which does definitely inhibit the formation of pigment.433 This has also been verified by Dr. Rothman in human skin.1078 I do not know whether it has ever been found for the mouse. It may relate to Dr. Coleman's point on inhibition. I do not think this has ever been studied in relation to genes except in the guinea pig. Dr. Coleman: I did not mean to imply that susceptibility to audiogenic seizures and defects in pigmentation are always related. There are many other possibilities here. There is one mutant I have worked on at the Jackson Laboratory which does not have the dilute gene, that is, it has the normal intense pigmentation (DD) but has a lowered phenylalanine-hydroxylase activity which is about the same level as that found in the DBA. This animal also goes into seizures of an identical pattern to the DBA, suggesting again that the phenylalanine hydroxylase level is important in the induction or seizures in these animals. Although no pigment defect is observed, this also suggests that more than one gene is involved in the control of phenylalanine hydroxylase. Dr. Ginsburg : I did not disagree with the observations on phenylalanine hydroxy- lase activity, which are extremely interesting and need additional investigation. There did seem to be an impression that this activity was correlated with the pigmenta- tion, and I wish to point out that seizures occur in mice and rabbits of various pigment types and that inhibitory mechanisms of relevance to the problem of pigment formation are known but neglected. Dr. Herzenberg : Has anyone in this audience any information on the following question? It has been suggested that more genes of physiologic importance may be found in the mouse by applying sensitivity of various inbred strains to drugs or loading them with sugars and seeing what the excretion levels of these sugars might be. Has anyone any information about whether this has been done ? Dr. Russell: Water is a fairly good drug, and there certainly are fine examples of fairly simple, genetic differences in reactions to water which have been found recently. There are a number of kinds of polydipsic mice ; 267 the imbibition of alcohol varies between strains.820 The only thing I would like to say in closing is to express the hope that I have been able to give, through these examples, an idea of how one may go about studying the action in particular genes in mice, why it is a complicated process, and how I believe that differentiation is, in a sense, a tool as well as a problem. F. Clarke Fraser, Ph.D., M.D. METHODOLOGY of EXPERIMENTAL MAMMALIAN TERATOLOGYtt Experimental mammalian teratology deals with anatomical defects arising from errors of development caused by exposing pregnant mammals, or their embryos, to environmental noxious agents, or teratogens. (Mutant genes causing developmental errors may also be considered teratogens, but the analysis of their effects is usually categorized as developmental genetics and is beyond the scope of this paper.) Why should teratology be included in a volume on mammalian genetics? Be- cause an environmental teratogen must act on a developing organism, and the response that it produces will depend on, among other things, the genotype of the organism. Thus, in studying the effects of an environmental agent on development, it is important to take the genetic constitution into account, and much can be learned about develop- ment and its errors by studying the interaction of an environmental agent with a variety of genotypes. TYPES OF ANIMAL USED FOR TERATOLOGIC STUDIES A wide variety of mammalian species have been used for teratologic studies. These include rats, mice, rabbits, guinea pigs, hamsters, pigs, and dogs.681 There is no apparent reason why other species should not also respond to teratogens. Choice of a suitable species for experimentation depends on many factors, not the least of f Dedicated to Professor L. C. Dunn with admiration and affection. X Financial support received from the National Research Council of Canada and The National Foundation is gratefully acknowledged by the author. 233 234 PHYSIOLOGIC GENETICS which is the experimenter's purpose. If one intends to analyze the effects of the teratogen in relation to a variety of other factors such as developmental stage, dosage, and maternal physiology, relatively large numbers of embryos will be required for the appropriate statistical analyses. One of the small laboratory rodents may therefore be chosen, since they are relatively economical to house and maintain, and have relatively large litters. Of course, it is necessary to choose a species that responds in the desired way to the teratogen (one cannot study cortisone-induced cleft palate in the rat, for instance) . It may also be useful to study the interaction of genotype with teratogen, in which case mice have the advantages of being genetically well known and available in a variety of highly inbred lines. The use of inbred lines has many advantages. For embryologic studies it may be worth while to search for a strain in which the teratogen to be used produces a high frequency of the malformation to be studied. It also removes the variable of genetic heterogeneity. Some workers1118 have made use of F1 hybrids between different inbred strains, thus obtaining genetically uniform embryos with the additional ad- vantages of hybrid vigor. The study of differences between inbred strains and crosses between them in response to a teratogen is a useful way of analyzing the interaction of genotype and environment in producing malformations. The existence of strains in which a particular malformation occurs spontaneously in some animals provides an opportunity to study the interaction of genotype and intra-uterine variables that determine why some animals in a litter are affected and others not. Workers who are mainly interested in the pathogenesis of malformations may choose a species the embryology of which is well known, or in which the embryos are relatively large, permitting gross anatomic as well as microscopic study. Still others may wish to produce malformations in order to study their diagnosis or treatment, in which case a large species such as dog, sheep, or monkey would be advantageous. It might be useful, for instance, to produce puppies with congenital malformations of the heart and great vessels, to be used for improving the techniques of diagnosis and surgery of such conditions, but this sort of application of experimental teratology has so far not been exploited. Fowls, amphibia, insects, and other groups have their own special advantages for teratologic work, not the least of which is the fact that embryos can be treated directly, without the complication of a uterine barrier, but consideration of these lies beyond the scope of this article. CONTROLS Although it should not be necessary to discuss the necessity of proper controls for any experiment, some of the variables that can confuse the issue in teratologic studies are sometimes overlooked and will therefore be mentioned here. First, there is the fact that spontaneous malformations do occur in laboratory animals, and these must be distinguished from those resulting from the teratogen being EXPERIMENTAL MAMMALIAN TERATOLOGY 235 used. Second, there is the possibility that some factor in the experimental procedure other than the teratogen being studied may itself be teratogenic. For instance, some teratogenic procedures may be so stressful to the mother that she will stop eating during the treatment, and maternal fasting is itself teratogenic in some situations. Third, there are a number of variables such as maternal weight,677 diet,1190 and season of the year629 that have been related to the frequency of malformations, and these must be taken into account when comparing the results of different series. Finally, there are genetic differences between strains and substrains that demand caution in com- paring results from different laboratories, or even from the same laboratory at different times. AGENTS USED FOR TERATOLOGIC STUDIES There are now on record a great number of agents with teratogenic properties. Those that can be classed as metabolic (which includes nearly all of them except irradiation) have been discussed recently by Kalter and Warkany681 in an exhaustive and useful review. It is difficult to classify teratogens in any logical way, since the modes of action of most of them are not accurately known ; for instance, agents usually considered as physical may act through biochemical pathways and vice versa. Wil- son 1392 has grouped teratogens acting in mammals under the headings of physical agents (X rays, hypothermia, hypoxia, elevated C02, puncture of amniotic sac) ; maternal nutritional deficiencies (lack of vitamin A, riboflavin, folic acid, pantothenic acid, vitamin B12, thiamine, and vitamin D, and fasting); growth inhibitors and specific antagonists (nitrogen mustard, thiadiazole, triazines, other alkylating agents, urethan, azaserine, 6-aminonicotinamide, 8-azaguanine, thioguanine, 6-mercap- topurine, 2-6-diaminopurine, 6-chloropurine), infectious agents (influenza-A virus, attenuated hog-cholera virus, Newcastle virus) ; hormone excesses and deficiencies (androgens, estrogens, insulin, cortisone, vasopressin, adrenalin, alloxan diabetes) ; and miscellaneous drugs and chemicals (trypan blue, excess of vitamin A, antibiotics, chelating agents, phenylmercuric acetate, nicotine, salicylates). Choice of a teratogen depends on the purpose of the investigator. One may wish to know, for instance, whether a particular drug, or other environmental agent, may be teratogenic in humans. In this case the choice of teratogen is decided by the experimenter's question. If the investigator wishes to study the pathogenesis of a particular malformation, he will wish to choose a teratogen which, in the appropriate organism, will produce a high frequency of the malformation to be studied. In this way he can be relatively sure that the embryo examined during early development would have been malformed at birth. Alternatively, he may prefer to study the arrays of malformations produced by exposure to the teratogen at various embryonic stages, on the assumption that the types of malformations produced by teratogens with various metabolic effects may yield information about the nature of the develop- mental mechanisms involved. 236 PHYSIOLOGIC GENETICS In any case, it will be useful to know the precise stage at which the embryo is exposed to the teratogen. For this reason an agent such as irradiation, that reaches the embryo immediately, may be chosen in preference to one such as cortisone, for which an indeterminate delay occurs between the time the agent is applied to the mother and the time it, or its metabolic consequences, reaches the embryo in effective quantities. Treatment with specific metabolic inhibitors (such as 6-aminonicotinamide) followed shortly afterward by a protective substance (in this case, nicotinamide) may also be used to achieve a short teratogenic episode, precisely timed.1008 The use of analogs as teratogens has the further advantage that their metabolic effects can be inferred from their chemical nature, and study of the embryonic effects of specific inhibitions, at particular stages of development, may provide information about the biochemical properties of the embryo. Further factors influencing the choice of teratogens will become apparent later in this discussion. DEVELOPMENTAL STAGE AT WHICH THE TERATOGEN IS USED In studying the effects of an agent on development, it is obviously useful to know the gestational stage at which the teratogen is applied. This is usually done by applying the teratogen at a given time after mating, but sometimes by counting back from parturition, assuming gestation length is known from previous observations. The latter is an unreliable method. The time of mating can be established, within limits, by placing the female with a male for a known period of time and observing her at the end of this period for signs of insemination, such as a vaginal plug in mice or rats, or the presence of sperm in the vagina. The frequency of successful matings can be increased by exposing the female to the male when she is in the appropriate stage of estrus, as established by vaginal smear or other signs, depending on the species. It may also be useful (in mice, at least) to maintain the females in a room with a regular light-dark cycle, so that their estrus cycles become synchronized, and they can be exposed to the males at the appropriate time in the cycle. No matter how precisely the time of mating is known, there is no assurance that all embryos are exposed at the same developmental stage, since within one litter there may be variations in time of ovulation, fertilization, implantation, and post- implantation development, so that no matter how accurately the time from insemina- tion is measured, there is often quite marked variability in developmental stage from one littermate to another. Variation in developmental rate between litters and between strains (or even sublines within strains) must also be taken into account, by using adequate numbers of animals and appropriate controls. Some confusion exists in the literature as to the terminology of timing gestational stages. Some authors refer to the day on which signs of mating are observed as the first day of pregnancy, or day 1 . Others refer to it as day 0. Care should be taken to specify which interpretation is meant in any particular case. The latter system is preferable. EXPERIMENTAL MAMMALIAN TERATOLOGY 237 It may be well to point out here the invalidity of the popular notion that a given morphogenetic process is most sensitive to alteration by a teratogen at the time when the process occurs. This is not necessarily true. Each teratogen usually has a period of maximum efficiency in producing a particular malformation (the critical period) , but this period may differ from one teratogen to another and often precedes the stage when the morphogenetic process involved takes place. X irradiation, for instance, produces cleft palate in mice with maximum frequency when given about 1 1 days after insemination,1118 whereas 6-aminonicotinamide is most effective on day 13, about a day before the palate actually closes.448 Presumably, normal palatal closure depends on the normal sequence and interaction of a number of previous develop- mental events, and interference with any one of them will interfere with closure of the palate. Since X irradiation and 6-aminonicotinamide have different critical periods, they presumably interfere at different points in the web of interacting processes leading to closure of the palate. DOSE In general, the teratogenic dose of an agent is somewhat below that which causes resorption or abortion of the litter. However, there are quite wide variations in the range between the lethal and the teratogenic923 doses for different teratogens. TERATOGENS AND THE ANALYSIS OF MALDEVELOPMENT Admittedly, some teratological experiments are begun simply because an agent is at hand and an experimenter is curious about what it might do. Others, of course, are conceived to answer specific questions about the nature of malformations and, by inference, about normal development. A number of such approaches will be discussed in the following pages. I. Is a given agent capable of producing malformations in humans? — Since it is usually extremely difficult to demonstrate the existence of an environmental teratogen in human beings, a reasonable first approach to the problem is to see whether the suspected agent is teratogenic in experimental mammals. The work that gave the first great impetus to the field of experimental mammalian teratology — the demonstration by Warkany1357 and his colleagues that specific maternal nutritional deficiencies caused malformations in embryonic rats — was inspired by the desire to know whether maternal nutritional deficiencies might cause human malformations. This approach will not, of course, give a definitive answer to the question that (as Warkany has repeatedly emphasized) must rest ultimately upon observations on human beings. If an agent is found to be teratogenic in experimental mammals, this raises the possibility that it may be teratogenic in human beings, but so far very few experimental teratogens have been shown to cause human malformations. On the other hand, if an agent is found not to be teratogenic in experimental animals, even 238 PHYSIOLOGIC GENETICS after having been tested in a wide variety of species and strains, the existence of marked species differences in response to teratogens prohibits the conclusion that it is non- teratogenic in human beings. 2. What are the pathogenetic mechanisms underlying malformations? — It is often impossible to infer correctly, by observing a malformation at birth, how it got that way. How- ever, if a malformation can be produced with a high frequency by a teratogenic agent, treated embryos at various stages can be compared with untreated controls, and the sequence of events from the first deviation from normality to the full-blown malforma- tion observed at birth can be observed. This approach has been used to elucidate the pathogenesis of a number of malformations. For instance, Monie et al.889 have demonstrated how a variety of urinary tract anomalies produced in rats by a maternal deficiency in pteroylglutamic acid could be explained by retarded development of the urinary tract and vertebral column. Giroud and Martinet436 have traced the patho- genesis of anencephaly produced in rats, by maternal treatment with large doses of vitamin A, from failure of the encephalic tube to close, through formation of a brain that is in effect turned inside out, and its subsequent degeneration. Warkany et al.1358 have analyzed, by this approach, the origin of myelomeningocoele produced by maternal treatment with trypan blue in rats. Failure of the neural tube to close is followed by overgrowth and eversion of nervous tissue and later by degeneration of the neural plate and formation of a fluid-filled space between the pia of the neural plate and the dura covering the vertebrae. Thus, the myelomeningocoele is essentially a cyst in the subarachnoid space in this case. Many other types of malformations that can be produced at will by teratogenic procedures deserve to have their origins worked out by this approach. It must not be concluded, however, that the pathogenesis demonstrated for a particular type of malformation produced by one teratogen at one stage of develop- ment is the same for all malformations of this type. It has been demonstrated, for instance, that cortisone-induced cleft palate in mice results from a delay in movement of the palatine shelves from their original position on either side of the tongue to their final position above the tongue, and that this delay seems to be due to interference with the mechanism within the shelves that provides the force necessary for this move- ment.1354 Cleft palate following amniotic puncture, on the other hand, is probably due to increased resistance of the intervening tongue.1330 Diminished shelf width, or increased head width, are other possible causes for the failure of the shelves to meet in the midline at the proper time.405 It is often impossible to tell, from the appearance at birth, which of these mechanisms caused the cleft in a particular case. Here again, extrapolation of experimental findings to human beings should be supported by observations on human embryos, but the experimental observations are useful in illustrating possible pathogenetic mechanisms to be looked for in human malformations. 3. What are the biochemical properties of an embryo at various stages of development? — The teratologic approach to this question was first formulated by Warkany and his group, who put female rats on a diet deficient in vitamin A. They showed that the offspring EXPERIMENTAL MAMMALIAN TERATOLOGY 239 of these rats had characteristic patterns of malformations, and that the array of mal- formations could be modified greatly by adding vitamin A to the diet at certain stages of pregnancy. For instance, when vitamin A was added to the diet before the thirteenth day of gestation, virtually no ocular malformations occurred; but when it was added after the fifteenth day, there was no reduction in the number of ocular defects. Mal- formations of the aortic arch, on the other hand, were prevented by addition of vitamin A before the twelfth day, but cardiac defects were not prevented when the vitamin was added as early as the tenth day.1394 It could be inferred, therefore, that certain processes concerned with organogenesis required more vitamin A than others (since some organs develop normally in embryos from unsupplemented animals) and that the requirements vary from one developmental stage to another. The synthesis of analogs to a number of biochemical compounds made it possible to refine this approach, by using an analog of a particular nutritional element to produce a temporary inhibition of the activity of that element. There is some question as to whether substitution of an inactive analog (which might conceivably have toxic effects in its own right) is strictly analogous to a sudden deficiency of the compound concerned, but the technique does provide a useful analytical tool. Nelson and her group, for instance, have used a maternal diet deficient in pteroylglutamic acid (PGA) supplemented with a PGA analog and succinyl-sulfathiazole (to inhibit PGA synthesis by the intestinal flora) to produce malformations in rats.940 A 36-hour period on the diet followed by high levels of vitamin supplementation to terminate the deficiency was teratogenic, but a 24-hour period was not. The type and frequency of malforma- tions produced by this transitory deficiency varied with the time of instituting the deficiency and with its duration and severity, providing an opportunity to study the requirements for folic acid at different stages of embryogenesis. In mice, folic acid analogs have produced malformations in the offspring of treated, pregnant females without the necessity of using a deficient diet.1327, 1332 A further refinement to this approach to the analysis of the biochemistry of morphogenesis made use of the nicotinamide antagonist 6-aminonicotinamide (here- after called 6-AN) which is teratogenic in mice even when a corrective dose of nicotina- mide is given as little as two hours after the analog is injected.1008 The compound forms a diphosphopyridine nucleotide (DPN) analog that is inactive in some DPN- dependent enzymatic reactions,227 and it is reasonable to suppose that this is the basis of its teratogenicity. By varying the dose of 6-AN and the dose of the nicotinamide supplement, it should be possible to obtain useful information about the relative requirements of various organogenetic processes for nicotinamide. For instance, when a standard dose of 6-AN is given 9.5 days after insemination and a standard dose of nicotinamide is given simultaneously, no malformations occur in the offspring. When the same dose of nicotinamide is given two hours after the analog many of the offspring have cleft lip. When twice the amount of nicotinamide is given, again two hours after the analog, the frequency of cleft lip is reduced. This suggests that a single dose of nicotinamide is not sufficient to correct the metabolic block produced by the analog; 240 PHYSIOLOGIC GENETICS if it were, doubling the dose would not reduce the frequency of defect. At other stages, and for other types of malformation, a double dose of nicotinamide two hours after the standard dose of 6-AN does not produce any fewer malformations than the single dose, showing that in such a case a single dose of nicotinamide is enough to correct the inactivation produced by the analog.1008 Thus the nicotinamide, or DPN requirements of the maternal-fetal system, appear to vary from stage to stage of embryo- genesis, and this approach provides an opportunity to study these variations in a roughly quantitative way. Further information might be obtained by comparing the arrays of malformations produced by several DPN inhibitors. It is known that 6-AN forms an analog of DPN which is inactive in a variety of DPN-dependent enzymatic reactions but not in all of them.277 Presumably malformations caused by treatment with 6-AN result from inhibition of one or more of the DPN-dependent reactions, but it is not possible to say which reaction, when inhibited, leads to which malformation. If other DPN analogs that blocked other DPN-dependent reactions were used, presumably a different array of malformations would result. If a given reaction were blocked by both analogs, the same malformation should result from treatment with either one. Thus by using a battery of analogs, and seeing which malformations were produced in common by which analogs, it might be possible to infer which enzymatic system was blocked in order to produce the defect. As the number of analogs increases, and their biochemical effects are better understood, they should provide excellent tools for studying the biochemistry of normal and abnormal morphogenesis. The immunologic aspects of development, now being energetically studied by experimental embryologists, 316 provide another promising approach to the biochemistry of morphogenesis that has so far been little exploited by mammalian teratologists. Gluecksohn-Waelsch441 showed that female mice immunized with extracts of brain produced offspring with an increased frequency of central-nervous-system malforma- tions, whereas extracts of heart were ineffective. Wood1402 confirmed the report of Guyer and Smith513 that lenticular antiserum injected into pregnant rabbits produced defects in the eyes of the embryos (though the claim of Guyer and Smith that these changes was heritable has not been confirmed). Further studies of this type are needed, particularly in view of the preliminary report by Blizzard et a/.110 that con- genital absence of the thyroid in human beings may result from maternal antithyroid antibodies. It would also be interesting to investigate further the suggestion that excessive amounts of specific proteins administered to the embryo would cause specific inhibitions or stimulations of development in mammals as they do in some other organisms. 4. What are the biochemical effects of teratogens on development? — As previously suggested, it may be possible to infer, from the biochemical nature of some teratogens, the probable metabolic pathways on which they act to produce their developmental effects, although even with specific analogs the picture may not be entirely clear. With other teratogens, such as cortisone, for instance, the biochemical effects may be so varied and widespread EXPERIMENTAL MAMMALIAN TERATOLOGY 241 that it is impossible to foretell which effect is related to the production of a malformation. A useful approach to this question involves the use of combinations of teratogens. The assumption is made that two teratogens which in combination give the same frequency of a malformation as that produced by the one with the higher effect given singly (a nonadditive effect) act on the same metabolic pathway to produce the mal- formation, whereas when the combined teratogens produce a frequency of defects which is the sum of the frequencies produced by each one singly, different pathways are involved. This is well demonstrated by the work of Runner and co-workers on the embryonic effects of maternal fasting in mice. Malformations of the vertebrae and ribs were prevented by giving small quantities of glucose or casein during the fasting period, and (to a lesser extent) certain amino acids and acetoacetate. This suggested that protection was provided by supplying substrate for the citric acid cycle. Maternal treatment with insulin, iodoacetate, or a PGA antagonist produced a similar array of malformations, and could reasonably be postulated to interfere with the citric-acid cycle.1088 Further evidence came from the results of fasting combined with one of a variety of other teratogens.1090 When pregnant females were fasted and exposed to hypoxia, the frequency of defects in the offspring was approximately the sum of the frequencies produced by each teratogen separately. This is reasonable on the assumption that the two teratogens decrease two substrates, involving separate metabolic pathways. In Runner's hypo- thetical scheme, hypoxia affects both the ectodermal and mesodermal components of the inductive system leading to differentiation of the axial skeletal system. Fasting in addition reduces the glucose substrate necessary for the ectodermal component, so the combined treatment should (and does) give a higher frequency of defective offspring than either one alone (fasting 24 per cent, hypoxia 47 per cent, combined 75 per cent) . On the other hand, iodoacetate combined with fasting gives almost the same frequency of defects (66 per cent) as iodoacetate alone (62 per cent), as would be expected on the basis that if glycolysis is blocked by the iodoacetate, reducing the glucose substrate by fasting will make no difference, since it involves the same (blocked) pathway. Synergistic effects (for fasting and cortisone on cleft palate frequency)678, 878 are more difficult to interpret, especially if the malformation concerned falls into the class of quasicontinuous variations,501 as postulated for cleft palate.405 Here, two agents which altered the threshold in an additive manner could produce an apparently synergistic effect as measured by the frequency of induced malformations, by moving the threshold in from the flat tail of the curve to the more steeply sloping portion. 5. What are the relations of mother and fetus with respect to teratogens? — The fact that a teratogen must pass through the mother to act on the fetus in most cases is a problem that is not always given sufficient attention. In fact, for many teratogens, it is not clear whether the effects on embryonic development result from direct action of the teratogen on the embryo or from secondary metabolic effects of the teratogen on the mother. Giroud et a/.435 have reported that maternal riboflavin deficiency in rats is accompanied by a decreased concentration of riboflavin on the maternal liver and an even greater 242 PHYSIOLOGIC GENETICS decrease in the embryonic liver, and that a maternal excess of vitamin A results in some increase of vitamin A in the embryonic liver.434 The teratogenic effects of radiation are not due to secondary effects of maternal whole-body irradiation.1391 Trypan blue has never been observed in the tissues of the embryo, but has been demonstrated in the yolk-sac fluid of rabbit embryos on the seventh, eighth, and ninth days of pregnancy.357 Wilson et al.1393 have presented evidence to suggest that the yolk-sac epithelium in the rat protects the embryo after the eighth day of gestation by absorbing and immobilizing the dye. Variation in the protective efficiency of the uterine barrier from stage to stage of gestation is an interesting aspect of teratology that needs further investigation. The physiology of the mother undoubtedly influences the effect of the teratogen in some cases. For instance, the frequency of cortisone-induced cleft palate in mice decreases with increasing maternal weight.677 This not only means that this variable must be taken into account when designing experimental controls, but raises the question of its biochemical significance. What aspect of the mother's metabolism is involved, and does it influence the mother's reaction to the teratogen, or have some effect on the embryonic developmental pattern that influences the embryo's response ? Or both? Reciprocal cross differences in response to a teratogen suggest the importance of the maternal environment in determining the embryo's reaction to the teratogen. For instance, the frequency of cortisone-induced cleft palate404 was much higher in F1 hybrids from a cross between A/Jax female mice and C57BL males, than from the reciprocal cross (the A/Jax inbred strain being more susceptible than the C57BL). Backcrosses ruled out a permanent cytoplasmically inherited factor as the source of the difference.679 Differences in maternal metabolism or placental transmission of the drug were possible explanations, although the latter seems unlikely if the placenta is constituted from fetal tissues, since the two types of hybrids are genetically similar. Another explanation was suggested by studies on the developmental pattern of the embryos. In crosses that produce embryos with a high frequency of cleft palate following maternal treatment with cortisone, the palate, in untreated animals, tends to close later than it does in crosses that are more resistant to the cortisone treatment.1329 The palate closes later in A/Jax than in C57BL/6 embryos, and later in A/Jax $ x C57BL (J than in C57BL/6 $ x A/Jax $ embryos. Thus there is no need to invoke a difference in the way the mother handles the cortisone to account for the reciprocal cross difference in cleft palate frequency, although this has not been entirely ruled out. The difference in response to the teratogen can be accounted for simply as the result of a difference in developmental pattern resulting from the interaction of maternal and fetal genotype. The techniques for transfer of ovum and embryo would be an elegant way to analyze further the interactions of embryonic genotype, maternal, and cytoplasmic factors in determining the embryo's response to environmental teratogens;485, 826 but such an approach so far does not seem to have been used for this purpose. Extrinsic variables influencing the probability that a given embryo will have a EXPERIMENTAL MAMMALIAN TERATOLOGY 243 malformation may be classified as individual factors, affecting littermates differently (local variations in blood supply, implantation, and so forth) and background factors, affecting all embryos within a litter (maternal physiology, variations in size of uterine artery, and so forth). Within inbred strains, it may be possible to detect such back- ground factors by comparing the frequencies with which defective offspring occur within litters with the distribution expected if the probability of being malformed is constant from embryo to embryo. If there is a tendency for clustering, that is, for an excess of litters with many defective offspring and of litters with none, a maternal variable is suggested. A tendency for some mothers to have more defective offspring than others (in the absence of genetic segregation) suggests that the maternal factor is a relatively permanent constitutional factor, whereas fluctuations in the maternal environment may be implicated if the variance is greater between litters than between mothers. 6. What are the intra-uterine variables related to the production of malformations? — One of the most difficult problems to attack experimentally in teratology is the fact that, when a teratogen is applied to a genetically homogeneous litter in the same uterus, some embryos may have no malformations and among the malformed ones there may be considerable variation in nature and severity of the defect from one littermate to another. In some cases this can be accounted for on the basis of quasicontinuous variation — a continuous distribution separated by some developmental threshold into discontinuous parts.501 In the case of cleft palate, for instance, the time of closure of the embryonic palate can be considered a continuously distributed variable. If move- ment of the shelves from their vertical position on either side of the tongue to the horizontal position above the tongue is sufficiently delayed, the width of the head will have increased so much that the shelves cannot meet in the midline when they do reach their final position. Thus the point at which they can no longer meet separates the continuous distribution of times of palatal closure into a discontinuous one- — cleft, or not cleft.405 However, this interpretation only pushes the problem back one step. What are the factors that determine where on the continuous distribution an individual embryo will lie ? Why does shelf movement occur so late in one embryo that the teratogenic procedure pushes it over the threshold, while in its genetically similar littermate shelf movement occurs early enough to keep it on the side of normality? Is some other variable perhaps involved, such as variation in shelf width? So far, no relevant factors have been identified. In addition, there may be variations within a litter in the amount of teratogen that actually reaches the embryo, and this aspect of the question remains almost entirely unexplored. The problem of intra-uterine variability is perhaps even more important in the case of the spontaneous malformation that appears with a low but characteristic frequency in certain inbred strains, for instance, the cleft lips that occur in about 10 per cent of newborns in the A/Jax strain, or the microphthalmia that occurs in some sublines of the C57BL strain. Here the situation may be more analogous to the situation in humans ; a particular grouping of genetic factors increases the probability that an 24-4 PHYSIOLOGIC GENETICS embryo will have a particular malformation, but whether or not a given embryo actually has the defect depends on intra-uterine variables that so far remain an almost complete mystery. It has been suggested that one of the variables might be segregation of genetic modifiers that may persist in spite of the intense inbreeding that occurs in the development and maintenance of inbred lines. However, the microphthalmia that occurs in the G57BL/6/Fr subline of mice is just as frequent in the offspring of un- affected mothers as it is in the offspring of their microphthalmic sisters, which argues strongly against genetic segregation in this case (Fraser, unpublished data) . Trasler1328 has reported that the spontaneous cleft lip that occurs in the A/Jax strain is more likely to appear in the embryo adjacent to the ovary than in embryos at other uterine sites. This directs attention to the nature of the relevant factors that make the juxta-ovarian site different from other sites, but so far no such factors have been identified. It would seem that the genetic constitution of an A/Jax embryo (and mother, or both) makes its lip-closing mechanism susceptible to disturbance by rather subtle variations in the uterine environment. What are the variations and what is there about an A/Jax labial primordium that makes it so sensitive to environ- mental disturbance? Answers to these questions will eventually require coming to grips with the problem of intralitter variability in biochemical terms. Perhaps some light could be thrown on the subject by observing the effects of agents with known pharmacologic properties, particularly antimetabolites, on the frequencies of defects occurring spontaneously in inbred lines. This approach has been well demonstrated by Landauer748 in chickens. 7. Interaction of genes and teratogens. — It should be obvious from the foregoing discussion that the genetic constitutions of the embryo and its mother are influential in determining the response of the embryo to a teratogen. This fact, which will be no surprise to geneticists, is significant for a number of reasons. For one thing, it may account for the differences in nature and frequency of defects reported by different workers using the same teratogen but different strains or substrains of animals. It also emphasizes the fact that even when an environmental teratogen is clearly implicated as a cause of congenital malformations, the genetic constitution may determine whether an embryo exposed to the teratogen will be malformed. This, for instance, is a possible explanation for the fact that some offspring of mothers having rubella in the early weeks of pregnancy do not have the characteristic malformations shown by others. As previously mentioned, the study of inbred strains that differ widely in the frequency of a malformation produced by a teratogen, and of crosses between them, can be useful in clarifying the intricate interaction of factors that determine whether or not a given embryo is malformed. Our studies of cortisone-induced cleft palate in the A/Jax and C57BL strains, Fx hybrids and backcrosses to the A/Jax strain demonstrated the importance of both fetal and maternal genotype in determining the embryonic response to the teratogen. Studies of the embryology of cortisone-induced cleft palate in strains with both a high and a low frequency of induced clefts demonstrated, far better than a study of either strain separately would have done, that delay in movement EXPERIMENTAL MAMMALIAN TERATOLOGY 245 of the palatine shelves from the vertical to the horizontal plane was the cause of the clefts observed at birth.1354 Furthermore, comparison of the normal time of palatal closure in these strains and crosses showed a correspondence between the normal time of palatal closure and the frequency of induced cleft palate, thus demonstrating how a genetically determined difference in normal developmental pattern was related to the observed differences between strains in response to the teratogen. Differences between inbred strains in response to teratogens can also be useful in elucidating the metabolic pathways involved. For instance, the frequency of cleft palate produced by a transitory inactivation of nicotinamide is higher in the A/Jax strain than in the C57BL/6 strain, and in the A/Jax x G57BL/6 than in the C57BL/6 x A/Jax hybrids,448 just as it is when cortisone is used as a teratogen. On the other hand, galactoflavin produces more cleft palates in the C57BL/6 strain than in the A/Jax strain.680 This shows that the susceptibility of the A/Jax palate to cortisone and 6-AN is not just a nonspecific instability to any environmental insult. It also suggests that galactoflavin interferes with closure of the palate at a different metabolic point than cortisone and 6-AN, and that perhaps cortisone and 6-AN act on the same pathway. Since 6-AN is known to interfere with DPN synthesis, this implies that cortisone's teratogenicity may reside in its effect on DPN synthesis, or on some related process. Of course we would not want to draw conclusions from these few comparisons, but the approach appears useful. By observing the effects of a variety of teratogens on a panel of strains and seeing which ones produce the same patterns of strain differences in frequency of malformation, it should be possible to deduce which ones are affecting the same metabolic pathways, and get some idea of which pathways are involved. SUMMARY This review of methodology in experimental mammalian teratology has con- sidered the following aspects: choice of experimental animal, choice of teratogen, gestational stage of treatment, dose of teratogen, and contributions of teratologic studies to a better understanding of the causes of malformations. Possible ways in which teratological methods can make such contributions include : 1. Experimental screening for teratogenicity of agents suspected of causing malformations in human beings. 2. Production of specific malformations with known and controllable frequencies, permitting embryologic studies of the pathogenetic processes leading to the defects observed at birth, and testing of diagnostic and therapeutic procedures on malformed animals. . 3. The use of antimetabolites and immunologic preparations to study the bio- chemistry of morphogenetic processes and the biochemical pathways through which teratogens act. 246 PHYSIOLOGIC GENETICS 4. The uses of reciprocal crosses and other methods to investigate the nature of maternal-fetal interactions influencing developmental patterns and their response to teratogens. 5. The use of inbred strains to study the intra-uterine variables that influence the probability that a given embryo, predisposed by its genotype or a teratogen to be mal- formed, will actually have the malformation. 6. Study of the genetic differences in response to teratogens to clarify the inter- actions of genes and extrinsic environmental agents in determining normal and abnormal developmental patterns. Walter E. Heston, Ph.D. GENETICS 1359 genetic nutritional changes in cells can be identified. By using cells from one of the highly inbred strains so that the point at which they become malignant can be determined by successive transplantation of the cultured cells back into the strain of origin, it may be possible to associate some of these nutritional changes with the neoplastic change. DISCUSSION Dr. Andervont: Those who have worked in cancer research during recent years recognize the importance of inbred animals that have proved so invaluable. They are used to ascertain the response of different inbred strains to a standard dose of carcinogen or the response of a single strain to graded amounts of a carcinogen. In essence, geneticists have made possible a quantitative approach to the problems of the relation- ship between host and cancer-inciting factors. This contribution alone is sufficient to justify the use of inbred animals, but, as Dr. Heston has pointed out, cancer is a complex disease. He used mammary cancer of mice to exemplify this complexity because it is an excellent example of the reactions of the host to a combination of genetic, viral, and environmental influences. Geneticists have given most of their attention to the host, with the result that the most clearly defined factor in the occurrence of the disease is the genetic constitution of the host. Their efforts have yielded important contribu- tions, such as the discovery of the mammary-tumor agent, but they have also un- covered many new problems. The inbred strains display a remarkable difference in susceptibility to the virus as well as to the production of breast cancer by hormonal stimulation in the absence of the virus. It is of some interest that, according to evidence now available, those strains susceptible to hormone-induced breast cancer are also susceptible to the virus. This does not imply that all strains susceptible to the virus are also susceptible to hormone-induced tumors, but only that those which respond readily to the administration of estrogenic hormones are also susceptible to the virus. GENETICS OF NEOPLASIA 265 In contrast, there is no well-defined correlation between susceptibility to the virus and susceptibility to chemically induced tumors. For example, mice of strain DBA/2 are susceptible to both virus and chemically induced breast tumors, whereas those of strain IF are resistant to the virus but susceptible to a chemical carcinogen. As Dr. Heston stated, strain C3H mice are susceptible to both virus and hormone-induced tumors but resistant to carcinogen-induced tumors. These striking variations in susceptibility to induced breast tumors raise the problem of whether we are dealing with a single disease or different diseases. If mammary cancer in mice is classified according to its etiologic agent, then we are dealing with different diseases. Hepatoma arising in mice, too, is the result of the interplay of several factors. A virus has not been implicated but, as Dr. Heston described, heredity is very important. Hormonal influences are involved because the spontaneous tumors occur more fre- quently in males of all inbred strains susceptible to their development. Diet exerts a pronounced influence on their development. Experiments performed with strain C3H mice as test animals reveal the com- plexity of these tumors. Males develop more spontaneous tumors than do females, but when the tumors are induced by the administration of an azo dye the females are far more susceptible than males. However, castrated males are as susceptible as females and can be made resistant again by injection of androgens. When tumors are induced by the administration of carbon tetrachloride, both sexes appear to be equally susceptible. However, a single strain of inbred mice exhibits remarkable variation in susceptibility to this tumor. Such variations suggest to the oncologist that he is dealing with a group of diseases which eventuate in the same final manifestation. At least the tumor can be studied within a single inbred strain when the genetic factor remains constant. Any discussion of Dr. Heston's paper would be incomplete without some mention of the contribution the methodology of genetics has made to recent advances in the field of tumor viruses. During the past seven years at least six viruses have been implicated in the origin of leukemia, or leukemia-like lesions, in laboratory mice. To those attending this symposium the interesting feature of these viruses is their range of specificity for inbred strains of mice. Much work remains to be done along these lines, but, to date, strain DBA is susceptible and strain C3H is resistant to one virus, whereas all strains tested thus far are susceptible to another. These two viruses represent the extremes of the six. You will recall that the murine mammary-tumor agent varies in concentration in different strains but that a susceptible strain reponds to the virus from other strains. If the specificity of the leukemia virus for certain strains is established firmly by future studies, then the establishment of inbred strains will have enabled the virologist to detect tumor viruses specific for certain genetic constitutions within a species. Carrying this thought one step further, it is possible that, within a genetically heterogenous species, some tumors are induced by viruses which are so specific that they produce tumors only in certain individuals. On the other hand, the ability of murine leukemia 266 PHYSIOLOGIC GENETICS viruses to elicit a variety of leukemias in their hosts suggests the possibility of a single virus producing a variety of tumors within a species. The logical conclusion to this discussion is that the methodology of genetics has contributed, and will continue to contribute, much to the solution of the cancer problem. Dr. Nalbandov: A year ago in Russia I learned of a method which impressed me and which may interest you. It involves applying uniform standard stresses (such as electric shock) to inbred strains of mice (C57 and A) and classifying the individuals according to their responses: nervous (unstable nervous system) to nonnervous (stable system). According to V. K. Fedorov (Pavlov Physiol. Inst. Koltushi, Leningrad) mice with an unstable nervous system died very rapidly after inoculation with cancers, whereas mice with a stable nervous system died after a more prolonged period of time and showed many metastases. Mice made neurotic by repeated electrical shocking were more susceptible to cancer (both spontaneous and inoculated) than were their non-neurotic counterparts. In dairy cows responses to light, sound, and electrical shock stimuli were measured and cows classified into four groups from very stable to unstable. The milk yields in 300 days were 6,193 kg. for the most stable group I; 5,516 kg. for group II; 4,918 kg. for group III; and 4,708 kg. for the least stable group IV.730 Dr. Pilgrim: I do not know what methods they were using, but most people who work with mice probably consider the C3H one of the calmest and easiest strains to work with and one with the highest incidence of cancer. Dr. Nalbandov: That is not the point. They subject them to a uniform stress in order to determine the degree of stability of the nervous system. Dr. Heston: Were the growth curves of the mice followed? Cancer is a disease of the healthy, and almost anything one does to make the mouse less healthy, if such a term is permissible, and which will reduce growth rate will decrease the occurrence of tumors.570 For example, a deficient diet will decrease the occurrence of tumors and a growth-promoting diet will increase their occurrence.24, 572, 759, 1311, 1312 Various genes affect susceptibility to pulmonary tumors. The gene which increases growth rate of skeleton and muscle is associated with an increase in these tumors, whereas those that decrease growth rate are associated with a decrease in pulmonary tumors.556, 566 Dr. Klein: Dr. Heston, you said that in serial backcrossing the agent was elimi- nated due to the reactivity of the females by antibodies. What is the evidence for this ? Dr. Heston: No one, to my knowledge, has shown that the mouse can produce antibodies against the mammary-tumor agent. When Dr. Andervont26 gave the agent to low tumor strain C57BL females these females did not get tumors, but they did not eliminate the agent they received, for they were able to transmit it to foster nursed, susceptible young that later developed tumors. Their own C57BL young, however, could not transmit any agent. In our own segregation studies, 571 among the females resulting from backcrossing Fx females to C57BL males one would have expected that some would not have genes for susceptibility to the agent; yet, although many of these backcross females did not get tumors, none of them eliminated the agent com- pletely, for all of them transmitted it to susceptible test females that later developed GENETICS OF NEOPLASIA 261 tumors. In testing their own second-backcross offspring, however, it was found that certain ones of these first-backcross females did not have any offspring that could transmit the agent. It thus appears the C57BL females and certain backcrosssegregants lack the genes for propagation of the agent and although they do not eliminate what agent they receive, it does not multiply and is lost completely (presumably by dilution) in the subsequent generation. Dr. Klein : I read the paper and was quite convinced the agent was lost, but I was not sure whether the agent was lost because it lacked something in the host or because it was eliminated by a host reaction. Can you distinguish between these two possibilities ? Dr. Heston: I think it is lost from some process like being diluted out and not by being destroyed by the animal. Dr. Yerganian: With respect to Dr. Heston's remarks concerning the role of underlying variables, an interesting sequence of events has taken place since 1954, when we provided the Chester Beatty Institute with a subline of partly inbred Chinese hamsters having then undergone four generations of inbreeding. Shortly thereafter, this new colony provided a subline to the Radiobiological Research Unit at Harwell. I have been informed by Dr. C. E. Ford that the subcolony at Harwell is now in the thirteenth generation of brother-sister matings and has developed an extensive number of pituitary and pancreatic carcinomas among both the control and X-irradiated populations. In our hands, the parental line (BUY) is now in the seventeenth genera- tion and has a relatively high incidence (7 per cent) of diabetes mellitus by 200 days of age. I was wondering if genetic drift would have any underlying contributions towards this presumably unrelated dichotomy and, if so, whether or not cancer may then be regarded as a form of metabolic dysfunction which, in the Harwell colony, is expressed in a malignant pattern, whereas a slight alteration in the genetic complex in question within the Boston colony is expressed metabolically as diabetes mellitus. In both instances, the target cells and organs are identical. Dr. Chai : I am in agreement with Dr. Heston's concept that tumors are threshold genetic characters. To this I should like to add (as a speculation) that development of tumors depends not only on genes with major effects but also on the genetic background and interaction between the genes. There are many published data which can be so interpreted. For instance, there were differences in incidence of mammary tumors between the F \ and F2 hybrids in crosses involving cancerous strains of mice.793 These differences can be explained by the threshold concept as the level of threshold fell on certain points in the frequency distributions of the genotypes. They can also be explained by the differences in genetic background and possibly by different inter- actions between the genes. There are many threshold characters, including some hereditary diseases and deformations, for which the incidences are different on different backgrounds, and the genetics is complex and not all clear. Most of the tumor data in experimental animals come from highly inbred and not from noninbred animals. 268 PHYSIOLOGIC GENETICS This raises the question as to whether the level of homozygosity in an individual affects tumor incidence. Dr. Burdette : Dr. Lynch, do you have a comment ? Dr. Lynch: I enjoyed hearing Dr. Heston's survey of this subject to which he him- self has contributed a very great deal. The production of strains of mice has entered the modern age. For example, I have been trying to produce a new strain of leukemic mice, one quite different from any used so far. After a good many generations of in- breeding during which with but one exception either one or both parents had the disease, the leukemia was lost rather suddenly. Whether it was due to the loss of a virus or to a genetic change affecting a virus or the host, I do not know. We are trying to intro- duce a virus into the strain to see whether that will be effective in bringing back the high incidence of leukemia. With respect to the production of some of the tumor strains, we are in a viral age. Dr. Burdette: Historically, the two most controversial, opposing ideas of carcino- genesis have been the somatic-mutation and viral hypotheses. In recent years, differences between them have not seemed quite so great as formerly with the advent of more information regarding cellular and viral DNA and RNA. Would you elabor- ate on how what is known about the similarities between viruses and genes may be related to carcinogenesis at the cellular level ? Dr. Heston : There are new and exciting things in this area that may have some bearing on the problem of cancer. It has been interesting to consider both lysogeny and transduction, although I think that some of the investigators of cancer may have used these terms rather loosely. Lysogeny may be involved in the induction of tumors if it can be shown that the cancer virus does enter the cell and become an integral part of the genetic mechanism of the cell and in so doing causes the cell to become malignant. This would be similar to lysogeny as described for bacteriophage. Such a process would not be outside the limits of the somatic-mutation hypothesis of cancer if we include in this hypothesis any change in the hereditary mechanism. of the cell. A situation similar to transduction might be involved in the spread of cancer if, after the virus has entered the cell and caused it to become malignant, some of this information is then carried over to neighboring cells, causing them in turn to become malignant. Such things are interesting to think about, and they are stimulating (and I hope they will continue to stimulate) good research. A. V. Nalbandov, Ph.D. GENETICS of REPRODUCTIVE PHYSIOLOGY The scientific literature is replete with examples of heritable differences in reproductive performance between strains or breeds of animals or races of man. Much of this knowledge about hereditary differences in prolificacy had been accumulated before the advent of experimental endocrinology which occurred about 1927. In the infancy of endocrinology the notions concerning the hormonal mechanisms govern- ing reproductive events were vague or incomplete, and it is only relatively recently that a beginning has been made in sorting out the heritable manifestation of hormonal effects and correlate the cause-and-effect relationships between genes and hormones. BASIC CONCEPTS OF ENDOCRINOLOGY First, agreement is desirable on some of the basic concepts of endocrinology which define the manner in which hormones affect their target tissues. Some of these inter- relations are simple, one-step effects in which a single hormone is known to act directly on its target organ where it produces certain metabolic modifications. Other inter- relations are chain reactions which may involve single hormones as the metabolic modifiers between steps, or, in the more complex relationships, may require a plethora of hormones which may be necessary to produce an exceedingly complex metabolic end result. Both the single-step responses and the complex chain reactions are known to be, or can be presumed to be, under genie control. The importance of genie control may vary depending on the step which is being affected and depending, of course, on the importance of this step in the ultimate metabolic response. 269 270 PHYSIOLOGIC GENETICS It is generally agreed that hormones do not initiate biochemical reactions per- formed by cells, but that they can govern the rates at which these reactions take place. If, for instance, a hormonal deficiency is induced by surgical or pharmaceutical methods, cells retain a modicum of their inherent ability to perform their highly specialized tasks; but now these tasks are performed slowly, inadequately, and in- efficiently. If the level of the missing hormone is raised by exogenous administration of the hormone, the rate of reactions can be raised in proportion to the amount of hormone injected. It is assumed that the genetically determined rates of hormone secretion similarly set the rates at which the inherent cellular reaction take place. All cells within a body come in contact with all endogenous hormones which are carried by the common blood stream bathing the cells. However, in spite of this general distribution via the blood stream, hormones react only with their specific, genetically conditioned end organs. The biochemical reasons for this ability of the different cells to discriminate between hormones and to respond only to their own specific trophic substances remains largely unknown, although it is presumed to be a matter of activation of mechanisms which are permissive of optimal intracellular interaction between enzymatic systems and substrates. Examples of such specialized activation of end organs are plentiful. Gonadotrophic hormone, for instance, is a specialized growth hormone which increases the metabolic activity of the gonads but has no such effect on somatic cells or on the cells of other glands. Conversely, somatotrophic hormone is a growth hormone which stimulates growth of somatic cells but has no effect on the metabolic activity of gonads. This apparent autonomy of hormonal systems should not lead to the conclusion that the efficiency of any one hormone-end-organ reaction is independent of other endocrine interactions in the body. While there is a specific trophic hormone for al- most every reproductive event and almost every end organ, optimal responses are possible only in euhormonal internal environments. For example, only gonadotrophic hormone can stimulate growth of gonads, but optimal growth of gonads can be achieved only in an endocrine environment in which all other glands (thyroids, adrenals, and so forth) are functioning at a satisfactory level. For this reason it is necessary to distinguish between primary, secondary and, occasionally, tertiary deficiencies. For instance, lowered fertility or even sterility of animals may be erroneously attributed to a deficiency of gonadotrophic hormone, while in reality it may be due to the inability of a normal level of gonadotrophic hormone to produce optimal gonadal responses because of a deficiency of hormones other than gonadotrophins. Since hormones govern rates of reactions it can be presumed, and in some cases demonstrated, that genes or genie complexes determine the rates at which hormones are secreted. Rates of hormonal secretion are frequently the only phenotypic ex- pressions which can be measured and used as a guide to the genotypic differences known to exist. Estimates of rate of hormonal secretion can be obtained in a variety of ways. In some instances the relationship between genotype and endocrine phenotype is simple, as in cases in which only one hormone is known to be involved and in which GENETICS OF REPRODUCTIVE PHYSIOLOGY 271 this hormone is known to act directly on the end organ. Size of comb of chickens is an example of such a simple relationship. More often the pathway is indirect and in- volves several hormones. Examples of the latter type include the whole reproductive process, growth, lactation, and, in fact, most of the phenomena governed by hormones. To complicate the picture still further, it is now known that in addition to the endocrine system the nervous system is equally important in the control of phenomena which earlier were thought to be directed by the endocrine system alone. In spite of these difficulties, valuable information can be obtained by using the various techniques which provide a guide to rates of hormonal secretion. ESTIMATES OF RATES OF HORMONAL SECRETION Bioassays of glands yield information on the concentration of hormones in them. This method has the disadvantage that the gland-giving animals must be sacrificed or mutilated. Furthermore, endocrinologists are not certain whether such bioassays estimate the rate at which hormones are secreted, the rate at which they are stored, or whether they simply indicate the amount of hormone remaining in the gland after its physiologically effective quantity has been released into the blood stream. Obviously the value of such bioassays hinges on which of these three alternatives is being estimated. While this question has not been resolved to the satisfaction of all endocrinologists, there is much evidence which shows that such assays do indeed estimate the rates at which hormones are being secreted.930 It would be most desirable to obtain information on the amounts of the various hormones circulating in the blood, but no good and easy methods are available to make such estimates either by chemical or biological means. This is because hormones, once they enter the blood, are destroyed or inactivated within minutes or, at most, within a very few hours. Because of this difficulty, and because the concentration of hormones in the peripheral circulation is extremely low, it becomes necessary to use special techniques which sample the blood as it leaves the more accessible glands or end organs. These techniques are cumbersome and difficult and have only limited application in the study of populations of the size required for adequate genetic analysis. In addition to these direct methods of obtaining information on the rates at which certain hormones are secreted, various methods permit indirect estimation of rates of hormone production. To obtain such estimates, a hormone is allowed to act on its target organ and the degree of stimulation of the target organ is used as an index of the rate of secretion of the hormone. Differences in rates of ovulation in females belonging to different genetic strains provide good examples of the variable rates of hormonal secretion by pituitary glands. Rates of ovulation, expressed either in numbers of eggs recovered from the female ductal system, or in numbers of corpora lutea found in the ovaries, have been used to demonstrate pronounced genetic differences between strains of domestic and laboratory animals. Litter size is an indirect measure of rate 272 PHYSIOLOGIC GENETICS of ovulation, but, in defining genetic differences, it must be used cautiously, since litter size is not always determined by rate of ovulation alone. This will be illustrated in a number of examples later. The genetic controls of such characteristics as rate of ovulation are probably as complex in mammals as they are known to be in chickens. Thus, the ability to lay many eggs depends on the combined action of five or more component characteristics each of which is known to be controlled by genes and influenced by hormones. Chickens which lay the highest number of eggs must show early sexual maturity, the ability to lay many eggs in succession without rest periods (long clutches), and they must be non- broody.539, 540 It is highly probable that the original analysis of the numbers of genes involved in the control of the fecundity of fowl is greatly oversimplified and inadequate and that the number of genes governing the expression of each of these characters is larger than originally thought. This possibility does not alter the fact that each of these five characteristics is known to be affected either by single hormones or interacting hormone complexes which are involved in determining the general level of fecundity in fowl. No comparable analysis has been made with regard to the genetic control of a similar array of component parts of the reproductive cycle of mammals. Other indirect measures of rates of hormonal flow can be obtained from observa- tions on the live animals in which rate of growth, rate of lactation, onset of puberty or menopause, and the like reflect endocrine states and are indicative of differences in genotypes. Still another method of establishing gene-controlled differences between strains of animals involves the measurements of their sensitivity to the injection of exogenous hormones. In some instances these methods require that the animal be sacrificed following treatment in order to obtain the target organ for analysis or weighing, while in others, the necessary measurements can be obtained from the living animal as in the case of the sensitivity of combs of male chickens to androgens, the sensitivity of the vaginae of rats or mice to estrogen, or the ability of the thyroid to take up radioactive iodine. Using one or the other of the techniques mentioned, it is possible to obtain reason- ably reliable direct or indirect estimates of the rate at which the different hormones are secreted. Much information has been accumulated in the literature showing conclu- sively that heritable differences in the rate of hormonal secretion exist between strains of animals. It is not the purpose of this paper to make a complete survey of the litera- ture illustrating these points and only a few of the more unusual or interesting examples will be presented. These examples have been selected because they illustrate the problems and principles which confront the scientist interested in this general area of physiologic genetics. Neither is it possible to present an exhaustive review of the gamut of chemical or methods of biological assay which can be used to obtain estimates of the various hormones contained in glands, tissues, or fluids of animals. The interested reader is referred to the current endocrine literature as well as the standard texts on endocrinology. Hormone Assay,323 which is somewhat out of date, and The GENETICS OF REPRODUCTIVE PHYSIOLOGY 273 Hormones1®01 contain methods for biological assay as well as chemical determination of hormones in tissues and fluids. CORRELATED CHANGES IN RATES OF HORMONAL SECRETION It appears to be generally true that genetic selection for the increased or decreased rates of secretion of one hormone results in increased or decreased rates of secretion of other hormones produced by the same gland. There are many examples of this principle in the literature. Body size is generally determined by the cooperative actions of somatotrophic, thyrotrophic, and perhaps adrenotrophic hormones, the rate of somatotrophin secretion probably being the primary determining factor responsible for the differences in the body size between two breeds of rabbits. Similarly, rate of ovulation is a reflection of the rate of secretion of gonadotrophic hormone complex. In one breed (table 51) there is a close association between small body size and small Table 51 Comparison of various hormone-controlled characteristics of large and small breeds of rabbits Breed Body weight (kg.) Rate of ovulation Litter size Gestation length (days) Polish Large race 1.66 4.30 5.52 10.73 4.48 8.13 31.1 31.5 Data from Venge. 1341 litter size, the former presumably due to a low rate of secretion of somatotrophic hor- mone, the latter presumably caused by a deficiency of gonadotrophic hormone. In the large race, the opposite association of the presumed rates of hormone secretion is noted. If one compares the rates of twinning, lactation, and growth in cattle, one finds that the highest rates of these characteristics (all of which are due to the highest rates of hormonal secretion) appear to be closely associated. Thus, the Holsteins are highest in all of these characteristics, while the Jerseys are lowest (table 52). The fact that this association does not seem to hold for body weight and lactation in beef cattle in comparison to Holsteins can be explained on the basis that large body weight in Hol- steins was achieved by selection for a high rate of secretion of somatotrophic hormone in euthyroid, and perhaps even hyperthyroid animals, whereas selection in beef cattle was in favor of hypothyroidism, that is, a low basal metabolic rate and a phlegmatic, easily fattened animal. One of the most interesting examples of this type is provided by MacArthur's831 experiment. Using mass selection and avoiding inbreeding, he selected mice for only 274- PHYSIOLOGIC GENETICS Table 52 Rank order of various breeds of cattle with regard to three hormone- controlled PHYSIOLOGIC CHARACTERISTICS Rank order in Breed Per cent twins Lactation Body weight Holstein Ayrshire Guernsey Jersey Beef cattle 1 3 2 4 5 1 2 3 4 5 1 2 3 4 1 one criterion — body size. His foundation stock weighed about 23 grams, and, after selecting for 21 generations, his small race weighed about 12 grams while the body weight of the large race was 40 grams. Keeping in mind that the only criterion for selection in this experiment was body weight, it is interesting to note that at about the twelfth generation, the small race had a rate of ovulation of 7.2 eggs and a litter size of 5.3 young, while the large race had a rate of ovulation of 14.1 eggs and a litter size of 10.5 young. Although these data suggest that selection for one hormone- controlled characteristic usually leads to an increase in the rate of secretion of other hor- mones secreted by the same gland, data proving this by actual assay of glands or the determination of hormonal levels are not readily available. In another study46' 1066 a comparison was made of the hormonal contents of the anterior pituitary glands of two strains of swine one of which was selected for a slow rate of gain, the other for a rapid rate of gain. A comparison of the concentration of Table 53 Comparison of the hormonal content of the adenohypophyses of two genetically different strains of swine Line selected for No. of corpora lutea tis Testi No. of weight of embryos chicks (g.)f Body weight of pigs 1 56 days old (kg.) Width of epiphyses of rats (micra) + Rapid growth Slow growth 14.5 10.5 11.62 8.53 15.7 25.2 48.7 24.9 243.4 ± 198.6 + 15.6 8.1 | Indicates gonadotrophic hormone content and X content of growth hormone of adenohypophyses. gonadotrophic hormone and growth hormone in a standard quantity of pituitary tissue was made at comparable ages in both strains (table 53). There was a significant and clear-cut difference with regard to the concentration of growth hormone. Because GENETICS OF REPRODUCTIVE PHYSIOLOGY 275 it was found that the anterior pituitary glands of the rapidly gaining strain contained significantly more somatotrophin per unit pituitary tissue than did the glands from the slowly gaining strain, it was possible to account for the differences in the rates of gain between the two strains. However, even though there was also a clear-cut difference between the fertility (that is, litter size) of the two strains (table 53), the assay of the hypophyses for concentration of gonadotrophic hormone showed an inverse relationship to rate of ovulation and to size of litter. Since the assay method used measured the total gonadotrophic hormone content rather than the two component parts of the gonadotrophic complex (consisting of the follicle-stimulating hormone FSH, and the luteinizing hormone LH), it is conceivable that the poorer reproductive performance of the slow strain was due to an abnormal ratio between FSH and LH which was perhaps different from the ratio usually found in normal animals and which was incompatible with optimum reproductive performance. From the genetic point of view it is clear that the two hormones under discussion were present in the pituitary glands of the slow and fast strains in significantly different concentrations; but the endocrine interpretation of the inverse relation between gonadotropin and fecundity of these two strains is not obvious. The slow strain showed a significantly higher incidence of cystic ovaries and generally impaired fertility in comparison to the fast strain. In this case, then, the higher gonadotrophic hormone potency of hypophyses of the slow strain is indicative of abnormal reproductive performance rather than of greater fecundity. This example illustrates the fact that estimates of hormonal concentrations are not always reliable indicators of the efficiency with which the animal utilizes its hormones. Such estimates are useful only when considered in conjunction with the overall performance of the animal. It is interesting to speculate on the possibility of producing strains of animals in which a high rate of secretion of one hypophyseal hormone is associated with a low rate of secretion of another trophic hormone. This would create genetic strains in which, for example, large body size is combined with small litter size, or small body size is associated with a litter size significantly larger than that observed in the larger strain. As a general rule, breeds of chickens with large body size are poorer egg producers than are breeds of chickens with a smaller body size. Although this suggests an inverse relationship between the hormones concerned with the control of these physiologic differences, it is possible that the principle cited earlier to explain differences between beef and dairy breeds may apply. There are examples which show that beneficial effects of genie complexes control- ling hormonal action at one step of the reproductive process may be partly or completely offset by adverse genie effects acting at another step. The work of Fekete350 shows that between two strains of mice ovulation rate is inversely correlated with reproductive efficiency and litter size. Despite a significantly higher rate of ovulation, the litter size of mice of the DBA strain is significantly lower than the litter size of mice of the C57BL strain (table 54). Analysis of this situation has shown that DBA ova are as viable as the ova of the other strain, but that the higher loss of ova in DBA mice is 276 PHYSIOLOGIC GENETICS Table 54 Comparison of rate of ovulation and reproductive efficiency of two inbred strains of mice Average no. Average no. Percentage of eggs of eggs per of young per developing Strain ovulation litter into young DBA 8.2 4.8 58.3 C57BL 6.7 5.6 83.9 due to an inhospitable uterine environment in which DBA eggs find themselves. A similar situation which results in lowered fertility in cows, and which could be ascribed to an unfavorable uterine environment, has been described by Hawk et a/.535 The comparative rates of the efficiency of reproduction of the two strains of mice present a good example of the necessity of analyzing each step of the reproductive process separately before deciding which stage is adversely affected by genetic mechanisms. Obviously, the genetically determined low size of litters of DBA mice must not be ascribed to a genetically low rate of secretion of gonadotrophs hormones. Several other examples of genie action at secondary or tertiary steps are known in reproduction, lactation, and growth. The nature of the adverse uterine environment on implanting ova is not known, but it is known that definite differences exist among strains of animals in the intensity with which uteri respond to their trophic hormones and in the types of responses produced. Drasher288 demonstrated significant differences in sensitivity of uteri of mice of five different inbred strains to a standard dose of estrogen (table 55). She also made comparisons between the immediate effect of estrogen and the residual Table 55 Effect of 3 gamma of estrogen injected into 5 strains of inbred, castrated mice killed 13 days or 73 days after castration Days killed after castration Strain 129 C57BL/6 C3H/He C3HeJH DBA/1 13 Uterine Wt. (mg-) 127.5 63.0 58.5 74.7 80.6 Total RNA 592 88 35 281 264 Total DNA 893 378 682 522 668 Total N 1903 888 835 1012 1060 73 Uterine Wt. (nig.) + 13.9 + 0.8 -26.8 -40.7 -64.9 Total RNA -81 + 273 -5 -95 -108 Total DNA -381 -80 -357 -325 -536 Total N + 594 + 242 -307 -477 -855 GENETICS OF REPRODUCTIVE PHYSIOLOGY 277 effect which could still be detected about 60 days after initial treatment. Drasher found that uteri of DBA mice were much more sensitive to estrogen than were the uteri of mice of the C57BL strain. However, when the effects of estrogen on the uteri of these two strains were compared some 70 days after the initial treatment, it was noted that uteri of females of the G57BL strain still showed a carryover effect which manifested itself in a smaller loss in uterine weight, as well as in a smaller loss in the number and size of cells than those noted in the DBA strain. Whether the significantly different rate at which the injected estrogen was metabolized by the two strains is related to the significantly different hospitality of the uteri of these strains noted by Fekete is unknown. It seems redundant to cite extensive evidence to show that highly significant differences have been found with regard to the sensitivity of different strains of animals to exogenous hormones. Apparently, degree of inbreeding has little to do with either the sensitivity or with the variability of response.189 Some strains may be less sensitive to one hormone but more sensitive to another. Munro et al.920 compared the sensitivity of various end organs of five different breeds of chicks to the various hormones present in a crude anterior pituitary extract (table 56). This preparation contained gonado- Table 56 Order of breed sensitivity to the injection of exogenous hormones (organ weights expressed as percentage of body weight) Comb Thyroid Gonad Breed 6 6* ? ..-;;>:;>" • WH 20- / / ''.**'' fc> ,R 5 II 15 AGE IN WEEKS From top to bottom, the breeds are the cocker spaniel, beagle, Shetland sheepdog, wire-haired fox terrier, and "barkless" basenji. Controlling environmental contributions to variation. — In all genetic experiments it is essential to separate those differences between individuals produced by biological inheritance from those produced by environmental differences. In the case of behavior, the range of environmental effects is much greater than for such characters as coat color or blood type. Some characteristics of an organism such as body size are, of course, determined in large measure by environmental factors. However, the in- vestigator of physical characters need not usually pay as much attention to the previous history and to the conditions of measurement as the behavioral geneticist. Maternal effects upon behavior, for example, extend beyond the uterine period through lactation and, in some species, far beyond weaning. Maternal effects may be transmitted by means as diverse as chemicals diffusing through the placental barrier, composition and amount of milk, amount of sheltering of the young, and age of weaning. Thus, in a recent study of inheritance of behavior in the dog, Fx males were backcrossed to the parental stock females in order to compare backcross and Fx generations reared by the same mothers. Such a design helps to separate environmental and genetic sources 288 PHYSIOLOGIC GENETICS of variance. The F2 generation in our experiment was reared by Fx mothers whose hybrid vigor created a quite different maternal environment. It would have been desirable in this case to have fostered F2 puppies upon mothers of the parental strain. With the dog this is not always easy to accomplish, since cross fostering can be success- fully carried out only between females in approximately the same stage of lactation. Another means of obtaining estimates of the effects of random and environmental factors is comparison of successive litters from the same mating. Such litters will, on the average, have similar heredity, although random variation will occur if the parents are heterozygous. Another solution of the problem of equalizing or eliminating maternal effects is hand rearing. Under such circumstances, of course, it is possible that the behavioral patterns usual in the species will not appear, since the eliciting stimuli may be omitted in the hand-rearing situation. Thus, the greater control of environmental variation may detrimentally affect the appearance of the behavior of interest. Isolation of subjects to insure equality of environmental conditions suffers from the same drawback. It is important in studying the development of behavior to place the subject in an expressive environment.410 Such an environment is one designed to favor the elicita- tion of species-specific behavioral patterns. It is conceivable that the technique of semi-isolation408 may prove to have some value in behavioral genetics. In this method, animals are isolated at weaning and held in cages which permit minimum stimulation. They are removed, however, at regular intervals and placed in a highly stimulating environment. This method insures control of development while providing essential conditions for psychologic development. The measurement of behavior. — As stated earlier, behavior may be defined as the acti- vity of the intact organism. Behavior is complex and obviously can vary in more than one dimension. Behavioral analysis of a character such as fighting in the mouse begins with a set of descriptions of the various behavioral patterns involved. When two mice are placed together, the measurements consist of the latency, frequency, and intensity of the fighting pattern. Latency and frequency can be measured with a stopwatch and counter. An enormous amount of behavioral work can be done with no more elaborate instruments than an arena, timer, and counter. The intensity of fighting behavior is more difficult to measure, as one cannot usually apply direct measures of force. This is a technical and not a theoretical difficulty. Presumably each blow and each bite involves the expenditure of a definite amount of energy, but the application of a measuring instrument would interfere with the behavior. Rating scales for intensity have been used, but these create problems of reliability and of equality of intervals on the rating scale. Latency, frequency, and intensity of behavioral patterns are often not independent measures. A pair of mice that fight often are apt to fight quickly and vigorously; but since the correlations between these measures are less than unity, one might expect to arrive at somewhat different genetic conclusions depending on the particular measure employed. BEHAVIORAL DIFFERENCES 289 Another difficulty is encountered when several behavioral patterns are used to identify a particular trait. For example, sniffing of the female genitals is part of the male sexual behavior of guinea pigs.510 This behavioral pattern, however, actually appears more frequently in low-sex-drive males than high-drive males. The latter proceed directly to more active courtship and copulation. As indicated above, the measurement of behavior tends to result in a quantitative analysis of the effects of genetics. However, behavior may also be analyzed in terms of the presence or absence of specific patterns, and the possibility of unitary characters is not excluded. Choice of conditions for testing. — -Conditions of stimulation are critical for the extension of group differences. This is expressed diagrammatically in figure 39, which shows Fig. 39. Hypothetical relationship between stimulus and intensity of response in two populations, A and B. I 2 3 4 5 6 7 8 9 10 II 12 STIMULUS INTENSITY an hypothetical relationship between stimulus and response in two populations, A and B. Note that below a stimulus threshold, 3 for B and 4 for A, no response is elicited in either population. On this measurement, therefore, B would be considered the more responsive. As intensity of stimulus increases up to 6, the differences between populations also increase. Above 6, they converge until they are equal at a stimulus intensity of 8. At still higher levels of stimulation, responses are on a plateau and a physiologic limit has been reached. Obviously, measurements of strain differences which follow this model are depen- dent upon the particular level of stimulus in the situation for testing. Measurements of threshold stimuli have not been widely used in behavioral genetics, although any test which involves the presence or absence of a behavioral pattern probably involves such a threshold. Measures taken at a physiologic limit have the advantage that 290 PHYSIOLOGIC GENETICS stimulus intensity can vary over a rather wide range without changing the difference between the strains. It is clear, however, that such a measure tells only part of the story and that a more dynamic concept of behavior is obtained when we consider the quantitative stimulus-response relationship over a wide range of intensities. That is, A and B may be compared with respect to the slope of a curve of learning or extinction or in terms of a psychophysical function. Such measures may have complex genetic elements, but they are apt to be more meaningful in relation to the adaptive function of behavior. Genotypic versus phenotypic orientation. — The preceding remarks point up a major difference in the methodology of behavioral genetics, genotypic versus phenotypic orientation. In the former, an investigator starts with a known genotypic difference and studies its effects upon behavior. In such a situation a gene, a chromosome, or a whole genotype is analogous to a treatment applied to an organism. The genotypically oriented investigator does not usually stop with demonstrating a correlation between genotype and behavior. He is also interested in tracing the path between gene and character through intervening physiologic mechanisms. In using mammals he has available two general methods. The technique of strain comparison makes use of populations with differences at a large number of loci, some of which are known but the majority are unknown. The experimenter using this method has an excellent opportunity to find behavioral differences, but genetic analysis is apt to prove difficult except in terms of a coefficient of heritability. Some success has been obtained in analyzing results of crosses between strains by standard biometric techniques using transformed scores,140, 410 but these methods sometimes yield ambiguous results. More precise genetic analysis is possible when a single genie difference can be estab- lished between the experimental populations. One of us1182 used the method of repeated backcrosses to eliminate genes linked with brown and white eye color in Drosophila. The gene bw appeared to have no important effect upon phototaxis. The white-eye gene did affect phototaxis. Earlier work on the effect of genes on the behavior of Drosophila often failed to take into account the numerous loci in addition to the one with conspicuous morphologic effect. The preparation of stocks of this type in mammals is laborious, but the behavioral geneticist can find a number of inbred strains of mice maintained in forced heterozygosis at a particular locus. The difficulty of such methods is that the genes in question may have no behavioral effects, or, at least, no effects detectable on a background of en- vironmentally induced variability. One can, of course, force the issue by using genes which produce major defects in sensory and motor organs, but the behavioral effects in this situation are not of great psychologic interest. Here it may be pointed out that behavioral techniques potentially provide a sensitive method for the detection of cryptic mutations — those without readily de- tectable morphologic defects. When heritable differences in behavior appear between recently separated sublines of established inbred strains, it is conceivable that a single BEHAVIORAL DIFFERENCES 291 locus may be involved. A possible instance of this sort has been reported by Denen- berg246 who found that different sublines of C57BL/10 mice differed in rate of avoidance learning even when reared under identical conditions. Genetic analysis of this pheno- menon would be most interesting. We have no means of knowing how frequently such cryptic mutations may occur, and how successful a systematic search with a number of reliable behavioral measures may be. The problems of behavioral differences may also be attacked from a phenotypic orientation. The behavioral patterns which can be chosen for investigation are of course almost infinite, and the choice will depend upon personal interests. Behavioral patterns which are important in adaptation have especial interest. Many biologists and psychologists are much more interested in the heritability of important adaptive patterns than in determining the effects of a rare mutant gene upon a standard genetic background. One can be quite sure that findings on the inheritance of choice of mate, dominance and submission, or problem-solving ability will not be trivial insofar as the welfare of the species is concerned ; but the very fact that these behaviors are so important makes it unlikely that they will have a simple genetic base. Thus the investigator in this area must sacrifice genetic clarity in order to work with characters of major importance for survival. The distinction between the phenotypic and genotypic orientation is, of course, not absolute, but a matter of degree. Both approaches have advantages and dis- advantages and both will contribute to the development of behavioral genetics. Statistical analysis. — One of the special problems of the analysis of the inheritance of behavioral patterns is the assigment of variance to genetic and environmental com- ponents. It is relatively easy to determine the heritability of a trait in any particular environmental situation, but such a figure has little general significance because of the responsiveness of behavior to environmental change. The method may, however, be highly important in determining whether or not there is a major genetic component in a behavioral trait, and Broadhurst136 has demonstrated how it may be applied to a simplified cross-breeding experiment. On the other hand, the detailed analysis of Mendelian mechanisms through analy- sis of variance frequently fails because of complicated interaction between genetic and environmental factors. Scott, Fuller, and King 1188 have demonstrated how a relatively simple genetic mechanism may interact with environmental factors to produce a highly complex manifestation of a phenotype. The annual seasonal breeding cycle of the African basenji dog appears to be determined by a single recessive gene, but the expres- sion of the character is also controlled by decreasing diurnal length. Fx hybrids with cocker spaniels show a tendency to run six-month nonseasonal cycles like the cocker, but also to respond in part to the seasonal changes in light, so that their breeding cycles are much more variable than those of either parental strain. With more complex genetic mechanisms, the interactions become so complex as to defy analysis in most cases. Bruell140 has suggested that it is possible through the use of means and medians to determine whether or not a behavioral trait is consistent 292 PHYSIOLOGIC GENETICS with Mendelian inheritance, without attempting to measure the effect of segregation on variance. Scott1183 has suggested a method of analysis of segregation independent of variance, based on the point of maximum separation between the two parental strains and a comparison of ratios between the two backcrosses. The method assumes the existence of a threshold, plus the additive effect of genetic and environmental factors which lead to crossing the threshold, and it can be used where such assumptions are justified. It will give an estimate of the number of genetic factors involved and can be used to predict an expected ratio in which correspondence with actual data can be tested by the usual statistical methods. FUTURE DEVELOPMENTS IN BEHAVIORAL GENETICS A major problem in behavioral genetics is the investment in manpower needed to determine a behavioral phenotype. A long period of testing under carefully control- led conditions may be required to obtain a score which can become part of a genetic analysis. Mechanization of tests may in the future make possible more elaborate experiments. Hirsch and Boudreau584 devised a geotactic maze for Drosophila which sorted out the population automatically. Bruell140 has automated various pieces of apparatus for the study of murine behavior. It might be hoped that the operant con- ditioning procedure (which is almost the limit of automation) would prove useful for behavior genetics, but in actuality the long period of time required for shaping behavior in the operant apparatus seems to preclude such applications, and procedures so far used have been designed to minimize genetic variations. It does, in fact, seem doubtful that the more complex functions of learning can be automated to the extent that large numbers of subjects can be run through tests with little involvement of humans. Many behavioral patterns are more accurately and reliably recorded by the human eye than by elaborate apparatus. Nevertheless, ingenuity and the desire for new tests may well help the investigator with his problems of manpower. The demonstration of differences in behavior between strains should continue, but it is to be hoped that such demonstrations will merely be the prelude to more ela- borate genetic analyses. The modern tools of biometric genetics have actually been applied only sporadically to behavioral characters. Experiments by Bruell140 and by Broadhurst136 indicate a trend toward more sophisticated designs. Possibly the most significant advances will be made in the relation of genes to behavior through biochemical pathways. Ginsburg431 has outlined the general scheme for such studies. The relationship between investigations of this sort and the search for a biochemical factor in mental disorder in man704' 705 is obvious. Behavioral phenotypes are more difficult to measure than ordinary phenotypes, but a large number of investigators have demonstrated that the problems are not insuperable. In fact, the difficulties in studying the genetics of behavior are fairly similar to those encountered in studying the inheritance of complex physiologic characters. The methods, too, BEHAVIORAL DIFFERENCES 293 are similar in that emphasis may be placed alternatively upon the effects of a well- defined genetic entity or upon the genetic contribution to the variance of a character continuously distributed. The concept of thresholds which appear so important to behavioral genetics is familiar to students of growth and was formally described by Wright1451 in his discussion of Polydactyly in guinea pigs. Thus the unique nature of behavioral genetics lies predominantly in the need for more extensive and detailed control of the life history of the subjects and of the use of special methods developed by psychologists and animal behaviorists for the objective measurement of behavioral patterns. DISCUSSION Dr. Burdette: Dr. Benson Ginsburg of the University of Chicago will open the discussion of the paper of Dr. Scott and Dr. Fuller. Dr. Ginsburg: One section of the paper by Drs. Scott and Fuller should be empha- sized by repeating it. "The preceding remarks point up a major difference in the methodology of behavioral genetics, genotypic versus phenotypic orientation. In the former, an investigator starts with a known genotypic difference and studies its effects upon behavior. In such a situation a gene, a chromosome, or a whole genotype is analogous to a treatment applied to an organism. The genotypically oriented in- vestigator does not usually stop with demonstrating a correlation between genotype and behavior. He is also interested in tracing the path between gene and character through intervening physiologic mechanisms." I wholeheartedly agree with this point of view and cite it as the reason that the Scott-Fuller paper really does belong in a section devoted to physiologic genetics. My own view toward so-called behavioral genetics follows from this position: behavior is a biologic aspect of organisms under genetic control and with an evolutionary history. I do not think that I am any more or less of a geneticist since turning from pigment studies to behavior. It is simply that one is studying another phenotype belonging to a different and more fundamental aspect of organisms. I believe that genetics occupies a central synthesizing position in the biological sciences and that it contains the organizing principles for thinking about evolution; for thinking about ontogeny; and for thinking about methods of investigating the ways in which organisms come to be the way they are, both phylogenetically and ontogenetically, including having the capacities to interact with each other. Physiologic genetics is thus involved in the study of the capacities of the nervous system, the endocrines, and the way in which all the capacities of the organism behave, including those making possible the com- plexities of group organization, development, and interaction. At the level of the gene, genetics occupies the same central position in a broader context. The new laws of physics and chemistry will probably come from a study of these biomolecules, the genes, and the ways in which they are able to duplicate them- selves and control cellular activities. In this manner, genetics occupies a very central 294 PHYSIOLOGIC GENETICS and important position on the threshold of the future of all sciences, from the physical to the psychologic. Turning to the psychologic, I would like to make a few points in relation to the position that to a psychologist, a rat is a rat and a mouse is a mouse. This should not be so. One can, biologically speaking, parse the creature, mouse, into genera, species, races, sexes, phenotypes, strains, and genotypes. If one takes almost any aspect of the psychologic literature, for example, the effect of early experience, one finds that general principles are developed that purportedly apply to all rats or all mice, if not to all infrahuman mammals, so that such effects as those of early manipula- tion on adult emotionality can be described and predicted. 1162 If, however, one goes to the inbred mouse and samples different strains, what one finds is something quite different. A majority vote of a population of mixed strains or of random-bred mice may follow the general rules if the minority votes are discarded. Even with genetically controlled materials, one can find situations (genotypes) in which the rules are followed. One can also find genotypes in which, with exactly the same experimental techniques, the behavior, measured in the same way, is exactly the opposite to that predicted by the general rules. In still other genotypes, the same experimental manipulations will make not a particle of difference to the later behavior.430 Environment or nurture has been nicely classified, controlled, and manipulated by psychologists and sociologists; but nature remains to them merely a rat, a mouse, or a mammal. Another example approaches more closely the kind of thing that Dr. Russell mentions and which, as Dr. Heston pointed out, is still not as close to the gene as we would like to get, but, nevertheless, much closer. If one considers various genotypes within the house mouse, either on the background of an inbred strain as in Dr. Cole- man's work or by comparisons between strains, one finds differences in the ability of the nervous system to handle common substrates. One of the things that we dis- covered in our group through collaboration with investigators at the Roscoe B. Jackson Laboratory years ago was that the controversy over the role of glutamic acid in behavior was a situation of precisely the kind that I have been describing. The ability of this simple substance to affect the nervous system and thereby behavior depended on the genotype within a behavioral phenotype. Within that phenotype some strains existed in which glutamic acid would give all of the positive effects claimed in the literature, but for other strains this was not true. The ability of this substrate to affect the nervous system and through this, the behavior, was a function of the genotype.432 Moreover, the way in which it affected the nervous system was also a function of the genotype. In mice of the DBA/1 strain, the threshold of audiogenic seizures can be depressed by administered glutamic acid. In other high-seizure strains, one cannot necessarily do this. Along with alleviation of the seizures, one can improve learning performance on mazes which are located on the thresholds of its ability and, therefore, represent a mild stress. If this effect is compared to the effect of the same agent on another high- seizure strain, DBA/2, it is found that, whereas the effect increases with dose and then remains elevated in DBA/1 mice, other ways of handling the glutamic acid metabolically BEHAVIORAL DIFFERENCES 295 are present in 'DBA/2 mice and the strain very quickly returns to its own behavioral equilibrium. In this case the return is maladaptive because glutamic acid helps the organism. The initial doses produce the effect, but an escape occurs with subsequent doses. The difference between the effect of glutamic acid in the two strains is clearly due to a difference in genetic capacity. Another important point concerns the definition of a natural unit of behavior or phenotype. Is gross bodily activity, which is the sum of various behaviors, any more a natural unit than fighting under confined conditions? The ethologists have attacked the problem of the natural unit by making the behavior misfire. Imprinting to an inappropriate object identifies an entire behavioral complex and provides one method for extracting a natural biological unit, and this, in a sense, is a kind of natural phenotype. Another way of defining a natural unit is to grasp whatever genetic difference exists, whether it be a strain difference or a genie difference, whether it involves climbing up on a pole, turning an activity wheel, agressiveness under confinement, or whatever, and consider that this is that part of the iceberg that is above water and that only by analyzing what genetic complex is behind it and then working back to the causal mechanism as well as forward again to produce the phenocopy, can the part of the ice- berg that is below water be found. A behavioral phenotype is the result of research and not something that is given at the beginning. This, to me, is behavioral genetics. It involves a physiologic-genetic approach to behavioral phenotypes. It is in no way a new science. If one reads the earlier genetic literature, one finds that when Mendelian traits were described, many of them were, broadly speaking, behavioral. They ranged all the way from mutations that affected the sense organs, such as rodless retina,692 to position preference in coach dogs.695 That behavior is under some genetic control has been known for a long time, and this so-called phenotypic approach has not advanced our knowledge of it very far. The physiologic-genetic approach is needed to bring about such an advance. Darwin had a prototype of a strain difference in behavior that he refers to in The Expression of the Emotions of Man and Animals. He describes a little hybrid girl who was half French and half English. When she was still very young, she shrugged her shoulders in a completely un-English way, although reared in an English environment. On the phenotypic side, we have not progressed much beyond this in so-called behavioral genetics to date. We shall not progress unless a physiologic-genetic approach is followed as outlined in these comments. Dr. Fuller: The two previous speakers have been discussing two different viewpoints. Dr. Scott emphasized the phenotypic orientation towards behavioral genetics, in which one starts with behavioral patterns which are defined as a phenotype and works backward to find out how they are inherited. The advantage of this approach is that it deals with behavior important to the organism and significant in evolution. However, one may have to sacrifice genetic clarity for the sake of behavioral significance. In the genotypic approach one uses genes as a form of treatment. The effects of the 296 PHYSIOLOGIC GENETICS treatments may be assayed by measuring behavior which is comparatively trivial, although it serves well for analysis of the genetic effects. The two methods are not really contradictory but are, rather, supplementary. Dr. Scott illustrates examples of genetic populations which diverged and converged in behavior as they developed. Such convergence or divergence of means does not necessarily mean that genetic effects are becoming less or greater as the animals grow older. In some instances we have been able to show that the contribution of heredity to differences as measured by interclass correlation stays relatively constant as animals age or as they practice a skill. Looking only at means may be deceiving. Finally, I would like to close by stating that behavioral phenotypes are more difficult to measure than ordinary phenotypes, but a large number of investigators have shown that the problems of behavioral genetics are not insuperable. In fact, the diffi- culties of studying the genetics of behavior are very similar to those encountered with complex physiologic characters. Great progress may be expected from studies of behavioral effects of well-defined genetic entities. Another important area is the determination of the heritability of continuously distributed traits. The concepts of a developmental threshold is familiar to students of growth and also appears important in behavioral genetics. The unique factor of behavioral genetics is its need of extensive and detailed control of the life history of its subjects and its use of special methods for the reliable objective measurements of transitory phenomena. Dr. Scott: Dr. Ginsburg argues that behavioral genetics is no different from physiologic genetics, and I agree that one of the basic scientific questions in this field is how heredity affects behavior. However, I think that behavorial genetics goes beyond physiologic genetics in that a higher level of organization is being investigated and consequently phenomena are encountered which are not apparent on the lower level. Dr. Wright has already illustrated the complicated types of interaction involved in the inheritance and production of pigment. Adding another level of organization makes Dr. Wright's system appear relatively simple. BIOCHEMICAL GENETICS Raymond A. Popp, Ph.D. MAMMALIAN HEMOGLOBINS t Variability among mammalian hemoglobins was recognized many years ago,33' 51- 285' 732' 1047 but recent advances in methods for their identification, separa- tion, and more complete characterization have enabled biologists to utilize differences between hemoglobin to probe more deeply into the challenging and tantalizing prob- lem of the mechanisms of genie function. Physical methods for distinguishing mam- malian hemoglobins have been the subject of many papers since 1940. Technologic advances in protein chemistry are now being applied to analyze the chemistry of hemoglobins as well as other molecules of biological importance. Chemical analyses of the content and sequence of amino acids in some peptide chains have been reported for a variety of mammalian hemoglobins within recent years. The chief objective of this review is to cite methods that have been applied in studies of mammalian hemoglobins and to compare and evaluate the usefulness of each method for survey, preparative, and/or analytical procedures. GENERAL COMMENTS Attempts to elucidate the nature of genie action have been based on the premise that the specific physical and chemical properties of macromolecules are reflections of the function and specificity of genes. The molecules chosen for such studies should, as far as can be determined, be primary products of genie action, have similar biological functions and similar physical and chemical properties, yet occur as intra- or interspecies f Some of the analyses in this paper were made possible through the cooperation of Dr. Norman G. Anderson and his group. 299 300 BIOCHEMICAL GENETICS variants that can be easily isolated from other biological contaminants without de- naturation in quantities sufficiently large for comparative physical and chemical analy- ses. Among other molecules, mammalian hemoglobins seem to satisfy these criteria. Hemoglobin is a conjugated protein composed of heme prosthetic groups, which are united to the polypeptide chains of a globin. The physical structure, four heme units associated with four globin coils, appears to be similar for all mammalian hemoglo- bins.998 Heme is compsed of four pyrole groups with one molecule of iron and is similar in all mammals. Each of the four polypeptide chains of the globin molecule is composed of approximately 150 amino acids. In contrast to heme, the globin moiety differs among species and frequently occurs in more than one form within a species. Establishment of homogeneity or heterogeneity of hemoglobin may require the use of many physical and chemical methods, since small interspecies variations fre- quently cannot be revealed by use of a single method. Intraspecies heterogeneity is often more difficult to demonstrate; however, the genetic implications that might be derived only through complete characterization of intraspecies differences become the reward for the added effort required to elucidate such variations. Ultimately, only a complete analysis of the amino-acid sequence will establish unequivocally the homogeneity or heterogeneity of some intraspecies hemoglobin variants. Use of pooled samples should be avoided if at all possible, since properties of individual samples may be obscured. However, pooling of samples may be permitted if strains of laboratory animals are used in which homogeneity of individuals has been established. The objectives of the investigator should be decided at the outset, since the informa- tion being sought usually dictates the analytical procedures. Simplicity is desirable, but simple methods frequently yield equivocal results. Every method has its limitations and sources of error, and the investigator should be acquainted with them. Moreover, hemoglobin is a relatively labile protein and may therefore be altered during some preparative or analytical procedures. Hence, it may be necessary on occasion to establish that minor hemoglobin variations observed with use of a particular technique reflect actual hemoglobin differences per se and are not modified forms or complexes of hemoglobin with other molecules, such as haptoglobins. The validity of the results is strengthened, however, if similar results are obtained by more than one method. For the purposes of presentation, the methods are classified according to whether they are used to study a physical or a chemical property of the molecule; some methods could be placed under either category. An evaluation and discussion of each method is presented, and several references are cited for the application of each method to the analysis of mammalian hemoglobins. PHYSICAL METHODS Electrophoretic analysis Electrophoresis is commonly used to determine the isoelectric point and electro- phoretic mobility of biological materials. Knowledge of the isoelectric point is of MAMMALIAN HEMOGLOBINS 301 value not only in characterization of proteins, but also aids in choosing physical con- ditions necessary for the isolation and purification of proteins. The properties of proteins that enable them to be separated in an electrical field have been discussed.4 When electrophoretic analyses indicate a single component in the system, the fraction may nevertheless not be pure by other criteria. Moving-boundary electrophoresis. — The moving-boundary apparatus developed by Tiselius1318 has been modified many times to increase its resolving power and thereby its usefulness as a tool for research. An important modification has been the develop- ment of an apparatus with four electrophoretic cells that can be used simultaneously.1010 The essential components of the instrument, the U-shaped electrophoretic cells contain- ing two electrode compartments for application of the electric field804 and an im- proved optical system for hemoglobin 648 to permit localization and visualization of the shape of the moving solutes, have been described in the articles cited. The methodology for moving-boundary electrophoresis is thoroughly described by Longsworth.803 Electrophoretic mobility, as experimentally defined, represents the distance traversed by the boundary or particle under observation per unit time per unit volt. The electrophoretic mobility of mammalian hemoglobins is in the order of 10-5cm2/ sec/volt at pH 7.0. The migration of molecules is affected by the hydrogen-ion concentration, ionic strength of the buffer, chemical composition of the buffer with regard to valence of the ions and viscosity of the buffer, and the surface charge density of the molecule under investigation. Thus, the physical conditions should always be stated precisely. Ideally, as many different moving boundaries are formed as there are different types of molecules in the solution. Observations on many proteins reveal that interactions between different molecular species that affect their electrophoretic mobilities are relatively rare. However, components in the buffer may interact with proteins, such as hemoglobins and enzymes, producing a shift in their isoelectric points.994, 1195 Buffers used for studies on hemoglobin by the moving-boundary technique are sodium phosphate with dithionite, 0.1 ionic strength, pH 5.7-8.0,994 and cacodylate- NaCl, 0.1 ionic strength, pH 6.5.994, 1211, 1381 More rapid separations can be achieved by using buffers of lower ionic strength; Beaven et al.ei used sodium phosphate, ionic strength 0.05, pH 8.0, and Itano and Robinson649 used potassium phosphate, ionic strength 0.01, pH. 6.85. The lower ionic strength is satisfactory for qualitative analyses, but quantitative analyses may not be highly accurate under such conditions. Differences in the moving-boundary electrophoretic patterns of murine carbonmonoxyhemo- globins are illustrated in figure 40. Moving-boundary electrophoresis is not a practical method with which to survey for differences among hemoglobins. The required instrument is expensive, only a few samples can be analyzed in one day, large volumes of hemoglobin solutions are needed, and considerable time is required to prepare the samples. Nevertheless, moving-boundary electrophoresis, along with techniques of X-ray diffraction997, "8 and chemical analysis 1053, 1054 has been a valuable analytical tool for studying problems 302 BIOCHEMICAL GENETICS of theoretical interest to hemoglobin chemists. The structural arrangement of the four globin units524' 650' 651, 652' 1065' 1210 has been approached by subjecting mixtures of electrophoretically distinguishable hemoglobins to conditions of low or high pH to dissociate hemoglobin into half-molecules and subsequently to allow the half-mole- cules to reassociate at neutral pH. The mixtures of reassociated molecules are then analyzed by the moving-boundary technique or other methods, that is, paper, starch-gel, and agar-gel electrophoresis or column chromatography can be used, for presence of fractions other than the two original components. Presumably, half-molecules of structurally similar hemoglobins can hybridize upon molecular reassociation at neutral pH. Hybridization of human and canine hemoglobin is reported in the latter two Fig. 40. Comparison of murine carbonmonoxyhemoglobins analyzed by moving- boundary ELECTROPHORESIS. ■ i>! Ml . 1 1 i ... *rr t*t Patterns were photographed after eight hours of electrophoresis; phosphate buffer, ionic strength 0.1, pH 7.5, 158 volts, and 20 milliamps. Left (A). Single hemoglobin of strain C57BL mice. Right (B). Diffuse hemoglobin of strain 101 mice. studies cited above, suggesting that canine hemoglobin contains configurational analogs of the alpha and beta chains of human hemoglobins. If the ionic change on the poly- peptide chains of the hemoglobin differ, it may be possible to establish the plane of cleavage of the dissociated half-molecules. The results obtained with human hemo- globin indicate that the hemoglobin molecule dissociates into non-identical half- molecules at low pH,1210 the two alpha chains separating as a unit from the two beta chains; however, at high pH the hemoglobin molecule dissociates into identical half- molecules.524 Each of the half-molecules of hemoglobin will dissociate further into two subunits, but the reaction is irreversible since such units will not reassociate at neutral pH as do the half-molecules.524 • 616 Zone electrophoresis. — A more compact and less expensive instrument than that required for moving-boundary electrophoresis was necessary before electrophoretic MAMMALIAN HEMOGLOBINS 303 analysis could become a routine laboratory procedure. Zone electrophoresis may be carried out using many types of supporting media. In reviewing zone electrophoresis, the methods are classified by the kind of supporting medium that is employed. Theo- retical aspects of electrophoresis discussed above are also applicable to zone electro- phoresis. Comparative studies on the electrophoretic mobility of mammalian hemo- globins are commonly carried out with zone electrophoresis, but exact determinations of electrophoretic mobilities are best determined by the moving-boundary techniques. Frequently the latter method also- offers higher resolution. Because of its simplicity, zone electrophoresis is generally preferred as a system when surveying populations of animals for hemoglobin differences. 1. Paper electrophoresis: Methods using paper as a supporting medium, first described by Wieland and Fischer1387 and Haugaard and Kroner,525 developed more rapidly than other methods of zone electrophoresis. Designs of several types of equipment have been illustrated elsewhere111 and the methodology has been outlined very well.203, 648' 76° For optimal results and reproducibility, the factors that influence the migration of hemoglobin during electrophoresis should be constant throughout the paper, that is, the paper porosity must be uniform and vapor pressure, quantity of hemoglobin applied, electrical current, temperature, and electroosmosis should be constant.1337 The filter papers commonly used as supporting media are Whatman no. 1 , 3 cm. wide, 3MM and 531 or Schleicher and Schuell 2043 A for hanging strip methods, and Whatman 3MM, 10 to 20 cm. wide for the sandwich technique. More hemoglobin can be applied to thicker papers and the presence of minor components can therefore be detected more readily.50 Moreover, thicker paper gives better reproducibility among runs, but thinner paper is more often used when quantitative determinations with recording instruments are to follow. Vapor-pressure equilibrium should be reached before electrophoresis is begun, the case used for hanging strips should be air tight to maintain the vapor pressure, and the glass plates used for the sandwich technique should be sealed with silicone. Simpler instruments without cooling devices may overheat at high currents, and the resulting evaporation of buffers from the paper may produce distortions of electrophoretic patterns and inconsistent results. The addition of inert substances, such as glycerol,822 inhibits evaporation of buffer from the paper. Pretreatment of the paper with the buffer may possibly reduce the adsorption of proteins and reduce electroosmosis by neutralizing the negatively charged carboxyl ions of the cellulose paper. The buffers commonly used for paper electro- phoresis of hemoglobins are barbital, pH 8.6 and ionic strength of 0.025-0.06, and phosphate, pH 6.5 and ionic strength of 0.1. 446 After electrophoresis, the proteins are fixed to the paper by heat denaturation at 1 10-120° C. for 15-30 minutes. For quantitative analysis, the temperature and period of time for heat denaturation should be controlled, since the capacity of proteins to bind dyes is affected by different conditions of heat denaturation.111 The hemoglobin on the paper strips can be stained by one of the following methods: bromophenol blue,311 304 BIOCHEMICAL GENETICS Amido black 10B,360 or light green.48 If desired, either the stained or the heat- denatured, unstained paper strips can be placed in a recording device to determine the mobility and relative amount of hemoglobin present. Several sources of error are inherent in any type of device chosen; these factors have been reviewed elsewhere.712 Hemoglobin solutions studied are not pure; for example, they generally contain car- bonic anhydrase and methemoglobin reductase. Staining procedures are not specific for hemoglobin but rather stain proteins. It is necessary to prove by other means that a stained peak, especially a minor one, is hemoglobin. A few additional reports, not mentioned above, concerning the use of paper electrophoresis for the investigation of hemoglobin variants in many mammals, are easily available.153, 331, 392, 443, 656, 1039, i34o. 1384 Population studies are reported in these papers; the inheritance of some of the variant forms has been established. Conventional paper-strip electrophoresis is not an efficient technique for obtaining large quantities of a pure fraction of hemoglobin from blood that contains more than one type of hemoglobin. Continuous flow, paper-curtain electrophoresis111 has been successfully used to separate serum fractions, but reports were not found of its use for isolation of hemoglobins. In our laboratory we found that hemoglobins are tightly adsorbed to the curtain and that good resolution of the fractions is difficult unless they are electrophoretically quite different. 2. Starch-block electrophoresis: Starch-block electrophoresis is often used for preparative procedures when larger quantities of an electrophoretically pure fraction are needed than can be obtained by paper-strip electrophoresis. The technique was first described by Kunkel and Slater.739 It is perhaps less suitable for routine analysis than is paper electrophoresis, although comparable electrophoretic patterns are obtained with either technique. It has the advantage that large quantities, up to 1,000 mg., of hemoglobin can be applied; moreover, adsorption of proteins on starch is less pro- nounced than on paper, which facilitates removal of individual components by elution. It should be mentioned that electrophoresis on sponge rubber, from which materials can easily be removed by squeezing, has also been investigated,882 but its usefulness for isolation of hemoglobins has not been reported. The procedures for starch-block electrophoresis have been set forth previously.737 The starch block is usually prepared by pouring a barely liquid paste of washed potato starch and buffer, usually barbital buffer, 0.05-0.10 ionic strength, pH 8.6, into a mold (38 x 10 x 1.5 cm.). Blotting paper is placed at each end of the tray to remove excess liquid and is retained until no more buffer remains on the surface of the starch. A slot is made in the starch block and the sample, as either a solution or a starch paste, is added with a pipette. Minor components (A2 and A3) of normal human hemoglobin were first isolated by use of starch-block electrophoresis.736' 740, 856 The technique has been used clini- cally in the diagnosis of the Thalassemia trait, an inherited condition in which the quantity of A2 hemoglobin is elevated from normal values of 1.2-3.5 per cent to ab- normal values of 4.2-6.8 per cent of the total hemoglobin.429, 738 An increase in the amount of A2 hemoglobin above 3.5 per cent is usually diagnostic for Thalassemia, MAMMALIAN HEMOGLOBINS 305 although the quantity of A2 hemoglobin may increase under the influence of other types of anemia.671 Cepellini,187 through the use of starch-block electrophoresis, has recently identified a new human-hemoglobin fraction (B2) which he suggests may be the product of an allele of the locus that controls the synthesis of A2 hemoglobin in humans. 3. Starch-gel electrophoresis: Resolution on starch gel is greater than that on paper. The adsorption of proteins on starch gels is low, similar to that for starch-block electrophoresis; the gels are easy to prepare and can be preserved for permanent records. Another advantage of the starch gels over paper is that the rate of migration is less dependent on concentration of protein. Furthermore, samples being compared can be run adjacent to one another on the same starch-gel block without appreciable interference by lateral diffusion of the proteins during electrophoresis. The design of the apparatus, preparation of gels, application of material, staining, and other details of the method for starch-gel electrophoresis have been described by Smithies.1228, 1229 The technique is not so simple as paper electrophoresis, but the increased resolution achieved more than compensates for its increased complexity. Prehydrolyzed starch has recently become commercially available,1227 thus removing a major source of variability. Twelve to 16 g. of starch per 100 ml. of buffer is heated until the opaque mixture becomes transparent and thick. The solution should not be removed at this point but is heated longer and shaken vigorously until it begins to become less viscous. The air bubbles are moved by placing the heated solution under reduced pressure for few seconds using a flask with heavy walls before the liquid starch is poured into plastic trays. A cover is placed on each tray removing excess starch and producing a uniform starch-gel block. Optimal gelling is achieved by placing the preparations in a refrigerator at 4-6° C. overnight. Boric acid-NaOH buffers, 0.015-0.04 ionic strength, pH 8.0-8.6, are most common- ly used, but other buffers, such as formate, 0.02 ionic strength pH 1 .9,918 phosphate,1228 and acetic acid-sodium acetate (unpublished) have been used with hemoglobin. The gelling property of the starch is influenced by the type of buffer and the amount of starch used ; thus, the percentage of starch may need to be changed when a new buffer is tried. Several methods have been used to apply samples to the starch-gel preparation. Samples to be inserted may be mixed with starch to make a thin paste that is pipetted into a slot made in the gel, or samples may be applied to filter paper that is placed into a transverse cut made in the gel. The slot method of application is recommended when larger samples are required; however, resolution under such conditions may be somewhat reduced. The quality of paper, when the technique of paper insertion is used, is important; if the paper adsorbs hemoglobin tightly, the bands are spread more. A thin plastic sheet is placed over the gel to reduce evaporation during electrophoresis. The electrophoretic patterns are better near the center of the gel ; therefore, the starch-gel slabs are sliced horizontally and the hemoglobin patterns at the inner surfaces are compared. It may not be necessary to stain hemoglobin owing to its red color, 30G BIOCHEMICAL GENETICS but the minor components of multiple hemoglobins become more readily visible following staining (figure 41) with a saturated solution of Amido black 1 OB in methanol, H20, and acetic acid (5:5:1). It is more difficult to estimate the quantity of different fractions of hemoglobin in starch-gels than on paper strips. Photoelectric devices have been used with preparations of starch gels made temporarily transparent by boiling them for 30 seconds in a 10 per cent solution of acetic acid.1342 Permanent preparations Fig. 41. Comparison of murine oxyhemoglobins analyzed by starch-gel ELECTROPHORESIS. B Red-cell lysates were electrophoresed for four hours; borate buffer; ionic strength 0.025, pH 8.5, 6 volts/cm., 6 milliamps. Hemoglobin was stained with Amido black 10B. Above (A). Single hemoglobin of strain SeC mice. Below (B). Diffuse hemoglobin of strain BALB/c mice. for photometric analyses can be made361 by mounting stained, transparent starch-gel blocks in agar. Quantitative analysis of eluted hemoglobin can also be made after freezing the gel, which makes the gel spongelike, and removing the pigment by applying pressure. The electrophoretic mobility of proteins is not identical in starch gel and on paper,1020 presumably due to greater adsorption of proteins on paper. Although two- dimensional electrophoresis — paper in one direction and starch gel in another at right angles to it — can be used for some protein separations, this system is perhaps not useful for hemoglobin. However, because of the greater resolution two-dimensional electro- MAMMALIAN HEMOGLOBINS 307 phoresis affords, it might be useful for distinguishing hemoglobin from hemoglobin- haptoglobin complexes through the use of the differential peroxidase activities that these substances exhibit.979, 1228 Application of starch-gel electrophoresis to the study of hemoglobin differences in mammals appears in several reports.49, 1016,1017,1072.1103 4. Agar-gel electrophoresis: The use of agar gel in electrophoresis, like starch gel, developed more slowly than paper, but agar is being used in place of starch by some investigators because it makes excellent preparations for quantitative analyses. The electrophoretic patterns in agar are very similar to those obtained by moving-boundary electrophoresis, as 1 per cent agar gel is essentially an aqueous medium, yet agar gel has sufficient structure to reduce free diffusion of macromolecules during and after electrophoresis. Most agars commercially available should be purified151 before use for best reproducibility of results. The systems used for agar-gel and starch-gel electrophoresis are very similar with one exception — liquid agar is usually layered about 3-6 mm. deep on photographic plates rather than poured into plastic molds. Slits for application of filter papers containing the samples are cut in the sheet of agar after it gels. Several slits can be made in an agar plate and as many samples of hemoglobin can be analyzed simul- taneously. The pH range in which agar gel can be used satisfactorily lies between 6 and 9, which is more restricted than that for starch-gel electrophoresis. Citric acid- sodium citrate buffer, ionic strength 0.025, pH 6.5,1064 and barbital buffer, ionic strength 0.025, pH 8.2,471 are commonly used for hemoglobins. For quantitative determinations, hemoglobin can be eluted from agar gel after freezing in a manner similar to that described for starch gels. The agar can also be dried following electrophoresis and hemoglobin stained with a solution of Amido black 10B dissolved in a 0.1m sodium acetate-1.2M acetic acid buffer containing 15 per cent glycerol, pH 3.5.360 The stained, washed, and dried agar film can be separated from the plate; the film is transparent and ideal for quantitative analyses in densitometric devices. Techniques of immune electrophoresis470 in agar gel have not been reported for the study of hemoglobin, but such methods could be applied in the study of hemo- globins. Column chromatography The theory of ion-exchange chromatography has been discussed by Boardman and Partridge.112 Pure ion-exchange adsorption is a function of the conditions of equili- brium between (1) the protein and the buffer, (2) the protein and the resin, and (3) the resin and the buffer. (1) Protein-NH2 + H© ^ Protein-©NH3 (2) Protein-©NH3 + Resin-COONa ^ Resin-COONH3-Protein + Na© (3) Resin-COONa + H© ^ Resin-COOH + Na© 308 BIOCHEMICAL GENETICS Exclusive of differential filtration, which may also influence separation, it can be deduced from the above formulae that optimal conditions for ion-exchange chromato- graphy should occur in a resin that is in equilibrium between its acid and salt forms near the isoelectric point of the protein. Column chromatography has been used for identification of hemoglobin differences and also for isolation of hemoglobin fractions. Boardman and Partridge112 first demonstrated the separation of sheep and bovine hemoglobins on ion-exchange resins, and Huisman and Prins612 developed the method for distinguishing several abnormal human hemoglobins. The reviews of Hirs583 and Moore and Stein891 should be con- sulted for a complete description of how to assemble a column for chromatographic separations. In brief, the components of the system are a reservoir for buffer and a column containing the ion-exchange resin through which the buffer and dissolved protein pass under gravitational force. Use of an automatic fraction collector permits continuous recovery of isolated fractions over periods during which the column need not be attended. The quantity of hemoglobin in the effluent fractions is determined by photometric assay, adsorption at 415, 540, or 575 mu. being commonly used. If the hemoglobins collected occur in different forms, that is, HbOa, HbCO, HbCN, or Hb®, a wave length should be chosen that gives equivalent extinctions for equal amounts of each form of hemoglobin in the mixture. Selection of a system of ion-exchange resin, buffer, />H, ionic strength, and elution rate is in part obtained by trial and error. Previous knowledge of the differences between the electrophoretic mobilities or isoelectric points of the hemoglobins to be separated is usually helpful, since the binding or adsorption of the molecule on a resin depends on the existence on the protein of a sufficient charge of sign opposite to that on the adsorbent. In general, there is a relationship between electrophoretic mobility and chromatographic behavior, but exceptions are not uncommon.610 Amberlite IRC-50 (XE-64 or 97), a carboxylic cation-exchange resin, is most commonly used for separating mammalian hemoglobins. More recently, Huisman et a/.611 have in- vestigated the use of carboxymethyl-cellulose, which has a higher capacity for the ad- sorption of protein, for separating some human and animal hemoglobins. Many ion-exhange resins have a tendency to bind proteins so tightly that denaturation or alterations of the molecule may occur during chromatography; an appreciable amount of methemoglobin is formed from carbonmonoxyhemoglobin unless chromatography is carried out in the cold at temperatures below 5° C. If proper ventilation is available, KCN can be added to the buffer to convert the methemoglobin formed to cyanmethemo- globin, which has chromatographic properties similar to those of carbonmonoxyhemo- globin. The buffer for ion-exchange chromatography should have the same ionic charge as the adsorbent, to avoid formation of^H variables at the point of interaction of buffer and adsorbent. The pH of the buffer should be chosen so that the protein is actually adsorbed or only a filtration process is accomplished. In general, adsorption of hemoglobin varies inversely with the pW of the buffer. As experimentally de- MAMMALIAN HEMOGLOBINS 309 termined,112 adsorption of hemoglobin below pH 7 is not due solely to electrostatic forces but is influenced by hydrogen bonding between the undissociated carboxyl groups of the resin and polar groups of the protein; that is, the positive charge of hemoglobin realized above pH 7 becomes less important below pH 7 for specific adsorption on the resin. The influence of secondary forces, such as hydrogen bonding, at low pH may be helpful in aiding separation of components chromatographically similar at neutral pH. As indicated by the formulae, adsorption can be reduced by increasing the sodium ion concentration of the buffer. The salt concentration is gener- ally increased stepwise as the fractions separate, such that elution is accomplished more rapidly and with smaller volumes of effluent. The elution rate used by most investigators is 4-6 ml./hr. for columns about 1 cm. in diameter. Specific conditions of buffers, pH, ionic strength, and flow rate for isolation of different components of human hemoglobins can be found in various articles.8- 207 Column chromatography has also been used as a preparative method to purify hemoglobin fractions used for further chemical analyses8' 616' 1395 and to detect and isolate hybrid hemoglobin molecules formed during hybridization.1345 Column chromatography through molecular sieves, such as Sephadox, is being adapted for the separation and analysis of peptides of enzymatic digests.578 C Utracentrifugation The ultracentrifuge is used to enhance the migration of molecules through a solution by increasing the intensity of the field of force. The sedimentation of particles in the ultracentrifuge depends upon volume, shape, and density of the particle; viscosity and density of the medium; and the intensity of the gravitational field. The sedimentation rate, described in terms of the sedimentation constant, s, can be used to calculate molecular weight by application of the formulae derived by Svedberg.1304 The molecular weights of hemoglobins (approximately 68,000) determined by this method agree with those obtained by diffusion methods.512 A description of the apparatus and methodology has been presented by Pickels,1005 and a monograph on ultra- centrifugation has recently been published by Schachman.1159 In general, the ultracentrifuge is not useful for distinguishing between mammalian hemoglobin variants since their molecular weights are usually near 68,000. A determi- nation of the molecular weight of a molecule is necessary for interpretation of some chemical analyses, such as amino-acid composition, peptide analysis, sulphydryl analyses, or end group analyses. Popp and St. Amand1017 and Gluecksohn-Waelsch442 have reported that the diffuse-type hemoglobin of the mouse contains a component of heavy molecular weight (figure 42), but additional investigation is required to ascer- tain whether the heavy hemoglobin is synthesized as such or is formed in vivo by dimeri- zation, including disulfide bonding. In support of the possibility that the component of heavy molecular weight of certain mouse hemoglobins may be formed secondarily by chemical bonding after synthesis, Gluecksohn-Waelsch442 found that increased quanti- ties of a heavy molecular component were formed during storage at 4° C, although this 310 BIOCHEMICAL GENETICS heavy component might not be the same hemoglobin as that present in fresh preparations. The physical structure of the hemoglobin molecule under various chemical conditions has also been studied by ultracentrifugation. Field and O'Brien358 showed that human hemoglobin undergoes dissociation into half-molecules at pH 3.5-5.5, and recently Hasserodt and Vinograd524 described a similar phenomenon at pH 1 1 . Fig. 42. Comparison of murine oxyhemoglobins analyzed by ULTRACENTRIFUGATION. A B Patterns were photographed after 80 minutes of ultracentrifugation; phosphate buffer, ionic strength 0.1, pH 7.5, 5° C. Left (A). Single hemoglobin of strain C57BL mice, S20 value, ~4.7. Right (B). Diffuse hemoglobin of strain 101 mice, S2o values. ~4.6 and 7.1. The dissociation is more complete at pH 1 1 , and less denaturation occurs. A dis- advantage of dissociation at high pH, however, is that the heme-labelling methods described by Singer and Itano1210 cannot be used to establish transfer of hemoglobin subunits during hybridization experiments, since methemoglobin of most species is rapidly denatured by alkaline hydrolysis. However, Vinograd and Hutchinson1345 have used C14-labeled hemoglobin to demonstrate molecular hybridization of carbonmonoxyhemoglobins dissociated at pH 1 1 . MAMMALIAN HEMOGLOBINS 311 Alkali denaturation Alkali denatures hemoglobin, producing a brown, insoluble, alkaline-globin hemochromogen. Korber732 demonstrated by this method that fetal hemoglobin is more resistant than adult hemoglobin to alkaline decomposition. Although fetal hemoglobins of other mammals also differ in alkali resistance from their adult counter- parts, greater resistance to decomposition does not apply to all fetal hemoglobins; that is, the fetal hemoglobins of sheep, goat, and cow are less resistant to alkaline de- naturation.134, 669 The number and percentages of different types of hemoglobin in a sample can often be determined from the rate of denaturation, since the decomposition of each hemoglobin in a mixture proceeds according to first-order kinetics.526 This approach has been used in studying the hemoglobin of monkey,1191 mouse,1379 rat,843 and sheep,1338 as well as human hemoglobins.202, 1385 Crystallography and solubility The classical monograph of Reichart and Brown1047 should be consulted for illustrations of the various types of hemoglobin crystals that occur in different taxonomic groups of mammals. Drabkin's method286' 287 of crystallization of hemoglobin has been used to obtain pure crystalline fractions of hemoglobins for analytical procedures, on the assumption that crystalline hemoglobin was homogeneous. However, Allen et al.8 and Clegg and Schroeder207 have shown by column chromatography that crystal- line human hemoglobin is, indeed, heterogeneous. Crystallography has also been used to differentiate between fetal and adult hemoglobins670' 703 and more recently has been developed as an auxiliary method for the identification of different types of murine hemoglobins (figure 43) in conjunction with electrophoretic and solubility properties.1015 These techniques have also been used in studies on the mode of inheritance of some hemoglobins of the mouse, as is mentioned later. Crystallographic and solubility studies are usually carried out in parallel; because of the sensitivity of the hemoglobin molecule to physical variations, the methods used separately may not be highly reliable. Determination of hemoglobin solubility is based upon the determination of the amount of protein remaining in solution at a series of salt concentrations. The results are influenced by the pH, ionic species, and temperature of the solution and by the concentration and form of the protein.212 The comparative solubilities of different hemoglobins are determined by establishing salting-out curves at constant pH, temperature, and hemoglobin concentration.261, 262 A number of hemoglobins have different solubilities under identical conditions and may be distinguished on this basis. In choosing a system for salting-out, the following factors should be considered: salts with a high buffering capacity are desirable, since hemoglobin solubility is greatly affected by change of pH; buffers containing multivalent anions, that is, phosphate, sulfate, and citrate, are more efficient for salting-out than those with univalent anions; 312 BIOCHEMICAL GENETICS the solubility of the salt must be sufficient to allow a precipitating concentration to be reached; and the pH of the buffer should be near the isoelectric point of the protein, at which point solubility is generally minimal. The most commonly used buffer for hemoglobin studies is potassium phosphate. The hemoglobin is usually converted to carbonmonoxyhemoglobin, since the latter is more stable than oxyhemoglobin. The amount of carbonmonoxyhemoglobin left in solution is determined photometrically. To compensate for daily variations in the physical conditions, standards are usually run along with each group of assays. Popp1014 has modified this technique by establish- ment of nomographs to enable quantitative determinations of mixtures of several murine hemoglobins within + 5 per cent. Fig. 43. Comparison of murine carbonmonoxyhemoglobins analyzed by CRYSTALLOGRAPHIC METHODS. A B Precipitates were formed within 21 hours; 3.08 M phosphate buffer, pH 6.5, 24° C. Left (A). Crystals of single hemoglobin of strain C57BL mice. Right (B). Amorphous precipitate of diffuse hemoglobin of strain 101 mice. Literature on the solubility of hemoglobin is not extensive65- 66> 478' 670' 687' 1068 in view of the fact that a solubility study may be performed with little effort and ex- pense in conjunction with other types of assay. A relationship between electrophoretic and solubility characteristics may exist, as was found for the sheep1338 and mouse.1015 Solubility may also be helpful in distinguishing hemoglobin types that are not electro- phoretically different. Itano645 showed that although human hemoglobins D and S are electrophoretically similar, the reduced form of hemoglobin D is more soluble in phosphate buffer than the reduced form of hemoglobin S. More recent studies have indicated other differences between these hemoglobins which will be discussed in the next section. In studies on the inheritance of single and diffuse hemoglobins among inbred strains of mice, Popp (unpublished data) has observed that several MAMMALIAN HEMOGLOBINS 313 strains possess single hemoglobins that are electrophoretically similar in starch gels, but three of these single hemoglobins have different solubilities in phosphate buffer (figure 44) . The solubility of a 50/50 mixture of SeC and C57BL hemoglobins is inter- mediate between that of SeC and C57BL. The quantity and form of crystals that Fig. 44. Comparison of murine carbonmonoxyhemoglobins analyzed by salting-out METHODS. 0.72-1 0.64- f-0.56- 0.48- UJ 5 0.40 n- 0.32 o o * 024 Ll. O 0.16- 0.08- 231 2.45 2.59 2.73 2.87 MOLARITY OF K2HP04-KH2P04 AT pH 6.65 3.01 Optical density of carbonmonoxyhemoglobin filtrates obtained in phosphate buffers of variable molarities were read at 575 m\x; physical conditions were 0.3 per cent solution of hemoglobin, 21 hours of incubation, pH 6.65, 30° C. develop in 21 hours at 30° C. in a 2.73 M phosphate buffer, pH 6.7, can also be used to distinguish the C57BL, (G57BL x SeC)Fl5 and SeC hemoglobins. Data are presented in table 58 on the inheritance of solubility characteristics of the single hemoglobins of strain C57BL and SeC mice. Previous studies1017 indicated that the hemoglobin locus that governs the molecular characteristics which determine the electrophoretic behavior of single and diffuse hemoglobins is closely linked to the chinchilla locus of linkage group I. Data in table 58 show that the chinchilla locus and the locus that controls the molecular characteristics which determine the solubility properties of C57BL and SeC hemoglobins segregate independently. Thus, at least two loci appear to be involved in directing the physical and chemical characteristics of hemoglobins in the mouse. Data on solubility, crystallography, and electrophoretic characteristics 314 BIOCHEMICAL GENETICS Table 58 Progeny resulting from Fx intercrosses and Ft backcrosses of C57BL and SeC classified for chinchilla and hemoglobin solubility Phenotype of Progeny! cch HbSeC cch HbSeC ccnHbc57BL CHbSeC C ' HbC57BL -HbSeC C HbC57BL Matings cch HbSeC cch HbC57BL cchHbCS7BL -HbSeC _HbCS7BL Number C HbC57BL C HbC57BL cch Hbsec " cch HbSeC C HbC57BL cch HbSeC 4 6 36 28 6 18 34 25 38 13 cch HbSeC " cch HbSeC reciprocal crosses f Symbols C and cch indicate full color and chinchilla, respectively; symbols HbC57BL and HbSeC indicate hemoglobin loci in strains C57BL and SeC, respectively. of hemoglobins obtained from test progeny also indicate that the diffuse-type hemo- globins differ among some strains of mice (Popp, unpublished data). CHEMICAL ANALYSES The physical methods described in the foregoing section are used to identify different hemoglobins in mammals and to study their mode of inheritance. Such methods in themselves, however, do not reveal the details of molecular structure which distinguish one hemoglobin from another. Analyses of the amino-acid composition and sequence are required to elucidate these differences. Differences in the protein chemistry of hemoglobin molecules, resulting from genie action, are of concern to bio- chemical geneticists. Some of the equipment and methods described above are also used for analysis of the composition of the hemoglobin molecule, with the principal difference that the submolecular components are being analyzed and compared rather than the native hemoglobin molecule. Amino-acid analysis Column chromatography, used for recognition of different hemoglobins, as described earlier, is also used to separate the amino acids of hemoglobin hydrolysates. The procedures of amino-acid determination on ion-exchange resins, developed by Moore and Stein,890, 892 has been applied by several investigators to assay the amino-acid composition of hemoglobins.647 Automatic recording apparatus1209, 1259 provides complete quantitative analyses within 24-72 hours. In a comparative analysis of human hemoglobins A, C, E, and F, Stein et al.1272 observed that the amino-acid composition of these hemoglobins is similar except that fetal hemoglobin contains an additional amino acid, isoleucine. Allen et al.8 noted a similar isoleucine difference between adult and fetal hemoglobin. Since different MAMMALIAN HEMOGLOBINS 31 o adult hemoglobins may have similar amino-acid compositions, total amino-acid analyses fail to disclose the nature of the differences between closely related hemoglobins. However, the analyses are necessary for estimating the number of peptides that can be expected from enzymatic digestion for peptide analyses. Sulphydryl analysis Attempts have also been made to detect differences in the number of available sulphydryl groups in hemoglobin by amperometric titration.630, 637 As described by Ingram, the number of available -SH groups is determined by measuring the num- ber of silver atoms per molecule bound as Ag-S-protein. A weak ammoniacal silver nitrate solution is added to the protein until Ag ions are no longer bound. The assump- tions are made that the available -SH groups in hemoglobin will react with metal ions such as Ag or Hg in a 1 : 1 ratio and that the metal ions will not react with non-thiol groups in the protein. Comprehensive studies have revealed minor differences in the number of thiol groups among hemoglobins of man, horse, ox, dog, cow, and sheep,637, 921 and comparative studies indicate a similar number of -SH groups for human hemoglobins A, S, and C. Although earlier reports using silver ions suggest that eight -SH groups are present,597 recent studies using mercury ions report only six -SH groups in human hemoglobins.13 Further application of Hg titration in the study of mammalian hemoglobins can be found in a recent report by Riggs.1056 Thus far, configurational differences in hemoglobins that could be produced through disulfide linkage have not been observed. Nevertheless, formation of inter- molecular disulfide linkages could produce hemoglobins of high molecular weight, such as are encountered in some strains of mice. Comparative results of sulf hydryl content of the native versus the denatured, heavy hemoglobin might be used to support or reject the possibility of intermolecular disulfide bonding. Results obtained, how- ever, should be interpreted with caution; Murayama921 has reported that not all the -SH groups in native hemoglobin may be titratable and that the end point determined by amperometric methods is temperature dependent. End-group analysis Efforts to establish the amino acids that occupy the terminal position of the poly- peptide chains of the globin moiety of hemoglobin have been more fruitful than sulfhydryl studies. A free alpha-ammo (N-terminal) group remains at one end of a polypeptide and a free carboxyl (C-terminal) group at the other end. These end groups are free to undergo a number of organic reactions. N-terminal analysis. — Several methods for N-terminal analysis of peptides and proteins have appeared in the literature. The method developed by Sanger1153 is based on the fact that 2-4-dinitrofluorobenzene will react with the free amino group forming a bond which is more stable than the peptide linkage of the amino acids in the protein. After addition of the dinitrophenyl group (DNP) the protein can be hydro- lyzed and a fraction of the DNP remains attached to the N-terminal amino acid. 316 BIOCHEMICAL GENETICS DNP confers a yellow color upon the DNP-amino acid complex, which is useful in following the fractionation and subsequently in identifying the N-terminal amino acid by methods of paper or column chromatography.111- 777 In addition to the terminal a-amino group, the <5- and e-amino groups of arginine and lysine, which are not involved in the peptide linkage, will also react to form DNP complexes. This must be considered in determining which labeled amino acid is actually in the terminal position. Keil698 has described a modification of Sanger's procedure for analysis of microgram quantities. The dinitrophenylation, extraction, and hydrolysis can be carried out in a single vessel, eliminating the possibility of loss of the peptide during extraction of the DNP complex from reagents and degradation products formed during the procedure. The free a-amino acid will also react with thioisocyanates (for example, phenyl- thioisocyanate) to form a phenylcarbamyl-peptide, a principle applied by Edman318 for the N-terminal analysis of proteins. In this complex, the terminal peptide bond adjacent to that upon which the phenylcarbamyl substitution takes place is more susceptible to acid cleavage than the other peptide bonds of the protein. Controlled acid hydrolysis splits off the N-terminal amino acid in a phenylthiohydantoin form soluble in 5 per cent NaHCOg, while the other peptide bonds of the insoluble peptide are left untouched. The amino acid is freed from the phenylthiohydantoin complex by alkaline hydrolysis and can be identified by paper or column chromatography. An important adaptation of N-terminal analysis has been its use in analyzing the sequence of amino acids in polypeptide chains. The method of Edman is suited for analysis of amino acid sequence through stepwise degradation of the polypeptide. Ingram638 has adapted the DNP-labelling method for stepwise degradation through catalytic reduction of the DNP peptide. The sequence of the amino acids in the pep- tides is established through identification of the amino acid released after each stepwise degradation procedure. Hunt and Ingram618 and Hill and Schwartz579 have analyzed the use of the two methods for studying the sequence of amino acids in peptide number 4 of human hemoglobins. With the use of the methods described above, the N-terminal amino acids and, in some cases, the sequence of adjacent amino acids have been established for hemo- globins of man,581' 616, 667, 1053, 1054, 1168' 1196 horse, dog, cow, pig, goat, sheep, rabbit, and guinea pig.785' 987' 988- 1156- 1157 Enzymatic methods for determining N-terminal residues with the use of leucine aminopeptidase are under development.580 Because of the rapid rate of the reaction, it is difficult to obtain information on the sequence of the amino acids released. Further- more, leucine aminopeptidase may not react on all native or even denatured proteins ; for example, performic acid oxidation was required before the N-terminal residue of albumin could be hydrolyzed by the enzyme. C-terminal analysis. — Procedures for the chemical analysis of C-terminal amino acids have developed more slowly than those for N-terminal analyses. The method developed by Akabori3 and Ohno960, 961 is performed by hydrazinolysis of the protein MAMMALIAN HEMOGLOBINS 317 which produces amino-acid hydrazides and amino acids; the latter are derived only from amino-acid residues possessing the free carboxyl group. Upon dinitropheny- lation the hydrazide forms di-DNP-amino acid hydrazide and the amino acid forms DNP-amino acid. The C-terminal, DNP-amino acids are then separated by fractional extraction and can be characterized chromatographically and estimated colorimetri- cally. In the analysis the dicarboxylic amino acids, aspartic and glutamic, may be contaminants in the extract of the C-terminal amino acids. The application of the method has been widely demonstrated, and a review of satisfactory results of analyses of C-terminal residues of a variety of proteins is presented by Haruna and Akabori.523 The method as described is not useful for sequence analysis since all the peptide bonds of the molecule are destroyed during the hydrazinolysis. Ohno962 and Niu and Fraenkel-Conrat945' 946 have modified the hydrazinolysis method for sequential analysis of the amino acids from the C-terminal end. The principle consists of partial, rather than complete, hydrazinolysis of the protein followed by dinitrophenylation of the peptides and extraction of the C-terminal, DNP-peptides from the other di-DNP- peptide hydrazides. The C-terminal peptides are then completely hydrazinized and the sequence inferred from the relative amounts of the hydrazinized amino acids present in the mixture. The method is not highly accurate since some amino acids are partially destroyed and the DNP-derivatives of dicarboxylic amino acid, that is, aspartic and glutamic, are difficult to distinguish from the true C-terminal amino acid. Enzymatic methods for obtaining C-terminal analysis appear to be as satisfactory as chemical methods. Use of carboxypeptidase-A and -B,437 protaminase,1370 and a Streptomyces griseus proteinase1157 have been reported. Another method (unpublished) can be used to identify the C-terminal peptide. Prior to removing the sample of hemoglobin, lysine can be tagged through injection of C14-labeled lysine. Autoradiography and specific chemical tests can be used to identify the peptides that contain lysine and arginine, respectively. Peptides that possess neither arginine nor lysine are presumably in the C-terminal position. Peptide analysis Knowledge of the chemistry of protein molecules expanded rapidly following development of quantitative methods for analyzing the amino-acid content of proteins. Amino-acid analysis fails, however, to disclose the exact nature of the chemical differ- ences that exist between closely related hemoglobins, such as A and S, and a search was made for another approach to enable investigators to elucidate any structural variations hemoglobins may possess. Scheinberg et a/.1164, 1165 reported that the differences observed for the electrophoretic mobilities of A and S hemoglobins at neutral pH "4 disappeared when these hemoglobins were subjected to electrophoresis in buffers at low pH. Prom their studies, they deduced that the electrophoretic difference be- tween A and S hemoglobins could be ascribed to a difference in the number of ionized carboxyl groups each possesses. Ingram633 reasoned that, if the difference involved even a single charged group, such a specific chemical difference might be 318 BIOCHEMICAL GENETICS more easily identified in a mixture of a smaller number of polypeptides released through enzymatic digestion than in a mixture of a large number of amino acids produced by acid hydrolysis. Enzymatic digestion with trypsin and chymotrypsin, followed by two-dimensional paper chromatography, was used by Sanger and Thompson1154 to establish the amino-acid sequence of the glycyl chain of insulin. This system was adapted to the study of hemoglobin peptides633 with the incorporation of electrophoresis into the procedure. The peptides of the trypsin digest were separated by paper electrophoresis in one direction and further characterized by chromatography at right angles to the direction of the electrophoresis. This procedure is called "finger- printing." Detailed methodology, along with an improved method for trypsin diges- tion, is described by Ingram.632 The improved method reduces the time required for trypsin hydrolysis from 40 to approximately 2 hours. The trypsin-resistant core that remains after digestion of hemoglobin can be hydrolyzed by chymotrypsin.617 If necessary, a drop of caprylic alcohol can be added to the solution without ill effects to reduce foaming created by nitrogen stirring. A thick chromatography paper, usually Whatman no. 3 or 3MM, is used as a supporting medium for electrophoretic and chromatographic separations of the soluble peptides of enzymatic digests. Care should be taken that the chromatography and electrophoresis are always run in the same manner relative to the machine direction of the chromatography paper. Follow- ing the method described by Ingram,632 electrophoresis is performed first, using pyridine : glacial acetic acid -.water (10:0.4:90) at pH 6.4, and ascending chromato- graphy second, using n-butanol : glacial acetic acid: water (3:1:1). However, equally good results are obtained by reversing the procedures;688 the peptides are separated by descending chromatography first, using n-butanol : acetic acid: water (4:1:5) and electrophoresis second, using pyridine -.acetic acid: water (1 : 10:289) atj&H3.7 (figure45). The various peptides of human hemoglobins have arbitrarily been assigned numbers for ease of reference.632 An illustration of the chemical differences in no. 4 peptide of A, S, and C hemoglobins has been presented elsewhere.634- 636 (It should be noted, however, that the amino-acid sequences are incorrect in the references cited above; the corrected sequences have recently been reported by Hunt and Ingram618 and Hill and Schwartz.579) Comparative studies on peptides of hemoglobins can also be done with instruments used for paper-strip electrophoresis. Resolution of the peptides is not so good as with two-dimensional separation; however, the application of specific biochemical tests on the separated peptides increases the sensitivity of the system. Such a system has been used by Benzer et al. 79 to establish that there are 3 varieties of human hemo- globin D, although the native hemoglobin of the 3 varieties are electrophoretically and chromatographically similar. That chemical alterations occur in peptides no. 23 and no. 26 for hemoglobins Da and D0, respectively, illustrates that chemical alterations in the hemoglobin molecule occur in places other than peptide no. 4, which contains the chemical alterations of hemoglobins S and C. It has been found that hemoglobin /also has a chemical alteration in tryptic peptide no. 23. 922 MAMMALIAN HEMOGLOBINS 319 Fig. 45. Comparison of murine hemoglobins analyzed by "fingerprinting." L CHROMATOGRAPHY (a) &(b © © ® © <© ® ® ® © © L CHROMATOGRAPHY (b) Tryptic digests were chromatographed with n-butanol : acetic acid: water (4:1:5) for 16 hours and electrophoresed in pyridine : acetic acid:water (1:10:289) buffer, pH 3.7 developed with ninhydrin. Left. Single hemoglobin of strain C57BL mice. Right. Diffuse hemoglobin of strain BALB/c mice. In 1957 Rhinesmith et a/.1054 established that the hemoglobin molecule is composed of four polypeptide chains of two different kinds, referred to as alpha and beta chains. Through the use of reversible dissociation and reassociation of mixtures of A and S hemoglobins Vinograd et a/.1346 showed that the alpha chains of the half-molecules of A and S hemoglobins were similar, whereas the beta chains were not. Thus, they argued that the chemical difference between A and S hemoglobins occurred in the beta chain. In view of the basis of the template theory of protein synthesis, it became of interest to know whether the other peptides in which alterations had been observed (that is, no. 23 and no. 26) were in the same chain as peptide no. 4. The alpha and beta chains can be separated electrophoretically in a detergent (sodium dodecylsulphate in veronal buffer), ionic strength 0.12, /?H 8.6, or by urea chromatography on ion-exchange resins (Amberlite IRC-50/XE64 or "CG50") using 2-8 M urea, pH adjusted to 1.9 with HC1.635, 1395 "Fingerprints" of peptide hydrolysates of each half-molecule showed that approximately one-half of the peptides of the hemoglobin molecule were present in the alpha chain, and the remaining peptides were in the beta chain. Peptides no. 4 and no. 26 were found in the beta chain, and the alpha chain contained peptide no. 23. 320 BIOCHEMICAL GENETICS (It should be noted that some confusion occurs in the earlier literature regarding which peptides are in the alpha and beta chains of human hemoglobins ; see Hunt and Ingram619 for schematic representation of correct assigment for alpha and beta chains of human hemoglobins.) The observations that chemical alterations occur in both the alpha and beta chains support earlier suggestions by Schwartz et al.1180 and Smith and Torbert1220 that per- haps there are two hemoglobin loci in man, one controlling the sequence of amino acids in the alpha chain and the other controlling the sequence of amino acids in the beta chain. A mutation may affect one or the other of these loci. Hunt 616 and Jones et al.667 established that the alpha chain of F and A hemoglobins are similar but F hemoglobin has a modified beta chain, called gamma, suggesting that separate loci exist for the synthesis of alpha, beta, and gamma chains of adult and fetal hemoglobins. A2 hemoglobin apparently differs from A in several peptides.1291 More recently, it has been found by Jones et al.668 that hemoglobin H lacks alpha chains. A fetal counterpart of hemoglobin H, Bart's hemoglobin, that also lacks alpha chains has been reported,620 and further investigations have shown that it is composed of four gamma chains.699 Thus, there is suggestive evidence, at least, for the presence of many different loci which control hemoglobin synthesis in man SUMMARY Methods commonly used in studies of mammalian hemoglobins are surveyed briefly. They include moving-boundary and zone electrophoresis, ultracentrifugation, alkali denaturation, crystallography and solubility determinations, and amino-acid, sulphydryl, end-group, and peptide analyses. Immunologic and spectrophotometric techniques, and oxygen- and carbon monoxide-combining capacity, which generally have more special applications, are not discussed in this review. The methods are described in such a way as to give the reader an idea of the facilities required for each analysis, and detailed descriptions are cited. It is hoped that the material presented will help the reader to choose the methods most suited to his own investigation and facilities. DISCUSSION Dr. Burdette : The discussion of Dr. Popp's paper will be opened by Dr. Elizabeth Russell who also is engaged in some interesting studies on murine hemoglobins. Dr. Russell: First let me congratulate Dr. Popp for making clear a great deal of complexity. I hasten to add that I do not pose as one who can tell a great deal about murine hemoglobins, because I am still very much a student in this area. I agree completely with Dr. Popp that multiple approaches from many different angles will be essential to understand how genes are acting in producing the varying hemoglobins found in mice. MAMMALIAN HEMOGLOBINS 321 It is extremely important to study murine hemoglobins, because there are so many potentialities in mice for experimental analysis. I hope studies in mice will help us to understand genetic differences in human hemoglobins for which experimental analysis is not possible. The kind of breeding tests which can be performed with mice are not possible in human populations at any rate. In connection with that I especially congratulate you on the finding of a difference of the segregation pattern of differences between the hemoglobin of SEC mice, which we have known formerly as an electro- phoretically single hemoglobin, and that of C57BL/6, which is another single hemoglobin. These two segregate independently of the albino locus, as I understand it. Differences between single and diffuse hemoglobins previously observed appear to be very closely linked with albinism. Now we are beginning to progress in using the potentialities of mouse genetics to help in the understanding of how these hemoglobin differences in mice actually arise through genie action. Recently I have been learning about column chromatography as a method for studying differences in murine hemoglobins. There are two reasons for using this method. One is that Dr. Richard Schweet of the University of Kentucky Medical School has used column chromatography very effectively in the study of hemoglobin synthesis.103 Since he has become interested in murine hemoglobins and wishes to do this kind of work with them, chromatography is an obvious method of choice. After observing his work with column chromatography, I would comment that the patience required for this method is considerable. As for the degree of resolution, it appears to be excellent in the separation of particular components of hemoglobin from different strains. A great advantage in column chromatography is that one can prepare mixtures of different hemoglobins and tell whether the same hemoglobin component is present in both strains. Perhaps "fingerprinting" could do the same, but I am reasonably sure that we will find out more by using both methods than by utilizing either exclusively. So far we have found that there are two, different, diffuse types of hemoglobin in the two inbred strains which we have studied extensively; that is, samples from the inbred flexed mouse and the AKR mouse give different hemoglobin patterns on column chromato- graphy. The two single ones with which we were dealing are alike. One is the C57BL/6 hemoglobin which is like that of SWR/J. This is fortunate in that one strain is albino, the other full color. This difference in a gene linked to a locus affecting hemoglobin will help us to use the same tool in different linkage experiments. The AKR/J hemoglobins have two major components and a minor. C57BL/6J and SWR/J have a single, major component and a minor. Extensive experiments involving mixtures and Fx hybrids must now be carried out if inheritance of murine hemoglobins is to be more clearly understood. I wish that there were a quick way of doing the hard job, but I really do not think there is any means of avoiding the extensive, difficult work required. Dr. Popp, have you studied N-teminal amino acids ? Something on this is known from Dr. Schweet's work also. In the C57BL/6 major component, there is an alpha 322 BIOCHEMICAL GENETICS chain and a beta chain. In both of the chains the terminal amino acid is valine as in human hemoglobin; the second amino acid in one chain is leucine, but in the other chain is not leucine. We know there are two chains, but we do not know any more than that about them. Commenting on the similarity of heme in all hemoglobins, let us call this a simi- larity, or perhaps identity, of the chemical structure of hemes. We are talking in theoretical terms about the synthesis of a protein molecule. We already know from humans of the possibility of independent alpha-chain synthesis and beta-chain synthesis. Let us remember further that in mice there are at least two independent genes which affect the rate of synthesis of heme. Thus, before the total, complex, hemoglobin mole- cule is formed, many kinds of genie action must occur. Dr. Bernstein : Dr. Popp, a number of methods that you elaborated on today require the assumption that hemoglobins are quite stable. Inasmuch as changes in oxygen concentration (oxygenation or partial oxygenation) of the molecule result in changes in charge and solubility characteristics, I wonder if you would comment on the relative stability of oxyhemoglobin, carboxyhemoglobin, and so forth, with particular reference to species differences. Of course I am concerned with the mouse. Dr. Popp: Cyanmethemoglobin seems to be the most stable form of murine hemo- globin. Although methemoglobin is quite stable, for many studies it is a less desirable state in that the electrophoretic mobility and chromatographic behavior of methemo- globin is slower than that of cyanmethemoglobin or carbonmonoxyhemoglobin. Murine carbonmonoxyhemoglobin is about twice as stable as oxyhemoglobin; however, studies of Douglas et al.285 and Anson et al.33 suggest that the conversion of murine hemoglobin to carbonmonoxyhemoglobin may be incomplete. Their data show that 30 to 40 per cent of the hemoglobin remained in the oxyhemoglobin state. This in itself is interesting, since hemoglobins of most mammals are readily converted to carbonmonoxyhemoglobin by simply bubbling carbon monoxide into the solution of hemoglobin. Dr. Russell: My impression, based on starch gels rather than on resin papers, is that one sees dense spots but resolution is not good enough to test for identity of hemoglobins in a mixture. Resolution may be better on resins; I would be glad to try this method, but its suitability may depend upon the question being asked. Dr. Burdette: Is the wave length used 415 mu.? Dr. Russell: Yes. Dr. Popp: We have run an absorption spectrum from 220 through 900 mu. using a Beckman automatic recording instrument. Areas of the spectra that looked slightly different were examined more carefully, using a Beckman spectrophotometer. The absorption spectra were indistinguishable even for hemoglobins of different electro- phoretic character, for example, single and diffuse hemoglobins of strains C57BL and 101, respectively. Willys K. Silvers, Ph.D. TACTICS in PIGMENT-CELL RESEARCHt The phenomena of genie action and interaction are so clearly presented by the permanent record which results from the elaboration of melanin granules in the hair matrices and their incorporation in the hair shaft, that the elucidation of the genetic aspects of melanogenesis greatly preceded a true understanding of the anatomical basis and biochemistry of pigment formation. Indeed, it was not until about 1940 that the extraepidermal origin of the pigment cell was unequivocally demonstrated in the classic experiments of Rawles.1040, 1041 We now are aware that pigment formation in mammalian skin and hair is restricted to specialized branched or dendritic cells — melanocytes — of neural crest origin, usually located in the basal layer of the superficial epidermis, in the case of skin pigmentation, and in the hair bulbs for hair pigmentation. Once it had been established that the many coat-color patterns found in mammals resulted from genie mutations and genie interactions, geneticists became interested in studying particular coat-color phenotypes, since it was evident that such phenotypes reflected or expressed, rather directly, the end results of specific gene-controlled pro- cesses. Interest in coat color was intensified when it was realized that only a single genie change was necessary to alter the phenotype drastically. Thus any study of mammalian coat color could not help but be concerned with the final results of a dynamic interplay of genie actions and interactions originating within the boundaries of a single type of cell, the melanocyte, and its milieu. Moreover, the fact that the entire genetically controlled sequence of chemical reactions in the synthesis of melanin from precursor tyrosine was restricted to the cytoplasm of the melanocyte, and could f Some of the work referred to in this paper was supported by a grant (C-3577) from the National Institutes of Health, U.S. Public Health Service. 323 324 BIOCHEMICAL GENETICS apparently only be greatly influenced through the local environment in which this cell occurred, also commended these cells as attractive subjects for experimental studies in a wide variety of biological fields. Although there is a great array of pigmentation patterns among different species of mammals, all of these patterns owe their origin to factors fundamentally similar. They are based on regional or local variations in the distributions of two basic pigment types, eumelanin (black, brown) and phaeomelanin (yellow, red). They include phenotypes in which pigment is completely or partially absent. Familiar examples are white spotting involving localized regions of the body and silvering or mottling due to variations in the pigmentation of individual follicles. Furthermore, it is probably true to say that almost all the diverse patterns of pigmentation found in mammals are phenotypically represented within certain domesticated species, particularly in dogs. Despite its presentation of a wide assortment of coat-color patterns which have, to a certain extent, been analyzed genetically,797 the dog is an unfavorable species for critical studies of genie action because of the difficulty evident in attempts to breed it and inbreed it on a large scale. Attention was, therefore, devoted to the small labora- tory rodents as convenient subjects with which to investigate these problems. Since the effect(s) of a particular genetic locus can only be recognized on the basis of variations (alleles) from wild type produced by mutations, the development of suitable laboratory animals has depended on the recognition and description of coat-color mutants and their preservation. DEVELOPMENT OF INBRED COLOR STOCKS A thorough, systematic investigation of the genetics of mammalian pigmentation, however, requires more than maintaining coat-color mutants. It requires the produc- tion of different stocks of animals each of which can be defined with respect to its coat- color genotype. Furthermore, since it has been shown that the expression of many genes concerned with melanin formation, or suppression of its production, may be drastically modified by other genes (modifiers) , it is obligatory to produce inbred color stocks in order to control genetically the phenotypic variations that are almost un- avoidable in noninbred material. For example, in the mouse the genetics of white spotting differs from the inheritance of most color patterns in that it is much more quantitative in character. Success in studying this quantitative character, therefore, depends on developing genetically homogeneous lines. The relative amounts of eumelanin and phaeomelanin present in the hair shafts, determined by the agouti series of alleles of the mouse, can also be greatly influenced by the cumulative expression of other genes with which these alleles are associated. For this reason there exist certain modified strains of genotypically yellow mice, usually described as being of sable color, which contain a variable amount of black pigmenta- tion down the middle of the back or even the entire dorsum. Some of these animals contain so much dark pigment that, although genetically yellow, phenotypically they TACTICS IN PIGMENT-CELL RESEARCH 325 resemble the black-and-tan pattern resulting from the agouti allele, a1-. The darken- ing of the fur of these animals is attributable to the cumulative action of a number of factors which are independent of the agouti locus.300, 1067 Because of this extreme variation in the amount of eumelanin and phaeomelanin which can and does occur in heterogeneous stocks of agouti-locus genotypes, it should be recognized that in such studies as those concerned with possible enzymatic differences between these genotypes, it is extremely important to control the background on which each of these agouti alleles expresses itself. Studies on nongenetic aspects of mammalian pigmentation. — Inbred strains are almost obligatory for determining the significance of nongenetic factors in producing coat- color variations. In the guinea pig Wright and Chase 1454 showed that white spotting depends primarily on a recessive factor, s, with many minor genetic factors having additive effects. Because of the availability of highly inbred strains of white spotted guinea pigs Wright1420 was able to demonstrate that, in addition, nongenetic factors also play a part in determining this character. Variation in the amount of white spotting even within highly inbred lines of these animals was found to be related to the age of the dam, the amount of white spotting of her offspring increasing with her age. A similar situation is also found in mice. In the C57BR/cd strain a white patch is present on the ventral surface in a majority of the animals. The extent of this non- pigmented area is extremely variable, ranging in size up to 10 per cent of the ventrum. Although, as Murray and Green 925 demonstrated, the occurrence and extent of these markings appear to have a hereditary basis ; the total effect of heredity is relatively slight, most of the variation being nongenetic in origin. Here, unlike the guinea pig, however, the age of the female apparently has only a very slight influence on the amount of white spotting in her offspring so that most of the nongenetic variation has still to be accounted for. DEVELOPMENT OF COISOGENIC COLOR LINES While the establishment of inbred strains of color stocks made available an un- limited supply of genetically uniform animals of specific coat-color phenotypes, it still had to be recognized that the differences between the coat-color patterns of these stocks might be attributable not only to the major color factors with which these stocks differed but also to the different genetic backgrounds on which these major factors were incorporated in each of the established strains. Although with some of the major genes concerned with pigmentation these variations are almost certainly negligible, with others, as noted above, they are certainly not. For this reason the only genetic material in which the effects of different alleles and different genetic loci can effectively be compared are coisogenic strains — strains which differ from each other in respect of a single locus concerned with melanin formation or, as in the case of white spotting, with its absence. Production of coisogenic lines. — The production of such color stocks is, indeed, a very 326 BIOCHEMICAL GENETICS tedious process. The incorporation of a dominant or semidominant color factor on a uniform genetic background requires repeated backcrossing of the mutant phenotype into the desired isogenic background. To replace a dominant gene with a recessive gene on the same background it is necessary to alternate backcrossing with inbreeding in order to recover the desired recessive character. This system of mating is described by Snell1238 for the production of isogenic resistant lines. For example, if one wishes to establish albino (cc) animals coisogenic with C57 Black (CC) this is accomplished as follows: First of all, the albino is mated with a C57 Black. All the offspring from such a mating will be black, of course, although all will be heterozygous at the C locus (Cc) . The albino phenotype is then recovered by inbreeding these heterozygous animals (one- fourth will be albino) which are then backcrossed to C57 Black and so forth. With each backcross generation the number of foreign factors (that is, not C57 Black) is reduced by half, so that after about ten such backcrosses, albino animals are produced which are essentially identical or isogenic with strain C57 Black except with respect to the C locus. It must be emphasized that the rigorous breeding procedures essential for the production of coisogenic stocks cannot be relaxed once the goal of isogenicity with the desired strain has been attained. In order to maintain these stocks coisogenic with each other and to eliminate all risk of the occurrence of genetic heterogeneity, it is necessary to continue the same procedures employed in the establishment of these stocks initially. Utilization of coisogenic lines in studying genie interactions. — In the mouse the in- corporation of specific genes concerned with melanin formation or white spotting on a C57BL/6 background was initiated some years ago by Dr. Elizabeth Russell at the Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. From foundation stocks of animals differing from each other with respect to only a single locus concerned with pigmentation, animals can be produced in which the interactions of two or more loci can be studied on a uniform genetic constitution. These are especially useful in studies to elucidate the effects of alleles at the agouti locus (described in the next section) on the amount of white spotting.! Little799 observed a reduction in the amount of white spotting in the coats of yellow mice from that in nonyellow animals when genes responsible for the production of white spotting were incorporated into the same yellow (Aya) and nonyellow (aa) genotypes. Since Ay is lethal when homozygous, all inbred strains of yellow animals must be maintained heterozygous at this locus and are, therefore, coisogenic in nature. Subsequently Dunn, Macdowell, and Lebedeff 307 demonstrated that yellow acts as a modifier of one form of spotting, Ww (dominant spotting,507) but has no corresponding effect on other forms. Although it has been shown that the agouti allelomorphs yellow-bellied agouti (Aw-) and black-and-tan (a1-), have no effect on piebald spotting (ss) or its modifiers, 307 their effects on the modifiers of dominant spotting has yet to be f A good method of measuring quantitatively the amount of white spotting is described by Russell, Lawson, and Schabtach.1106 TACTICS IN PIGMENT-CELL RESEARCH 327 determined. The availability of coisogenic ala, Ww, Aya, MiwhMi + , and aa stocks now makes such an investigation possible, since AyaWwMiwhMi+ genotypes! exhibit more pigmentation than aaWwMiwhMi+ animals on the C57BL/6 isogenic background (Russell, unpublished). It would be very interesting to determine whether ataWwMiwhMi+ genotypes phenotypically resemble the corresponding aa genotype on their dorsum and the corresponding Aya genotype on their ventrum, or whether there is no effect or a general effect of this allele on the amount of white spotting normally present in aa\VwMiwhMi+ animals. Although the former result is not anticipated, its possible occurrence would certainly have to be taken into consideration in explaining two of the most perplexing problems of mammalian pigmentation — the etiology of white spotting and how eumelanin formation differs from phaeomelanin formation. APPLICATIONS OF TISSUE TRANSPLANTATION IN STUDYING MAMMALIAN PIGMENTATION Since the acceptance or rejection of a vascularized graft depends upon the presence or absence in the donor tissue of genetically determined transplantation antigens that are foreign to the host, it was not until inbred and coisogenic color stocks became available that the technique of graft transfer could be added to the armamentarium of the mammalian pigment-cell worker. Because of the accessibility of avian embryos and their almost uniform acceptance of grafts of homologous or even heterologous origin, at least until hatching, embryologists, ably led by Willier and his associates, had been making important discoveries bearing upon the nature and origin of melano- blasts, their differentiation and the physiologic factors which influence it. The work did not depend upon the use of inbred animals, which were certainly not available. In retrospect, in the light of the work of Billingham, Brent, and Medawar92 and others, it now seems certain that the success of these early homologous transfer experiments leading to the production of transient, or even permanent, chimeras depended upon the fact that early exposure of the embryos to the foreign antigens induced a state of partial or complete specific nonreactivity usually referred to as immunologic tolerance. These embryos never developed the ability to reject their foreign grafts. Application of immunologic tolerance. — Techniques are now available91 to render adult mice and other mammals tolerant of homologous tissue grafts by inoculating them either directly as fetuses or intravenously at birth or shortly thereafter (that is, before their immunologic mechanism of defense has become functionally mature) with suspensions of living homologous cells. Such animals, when adult, may then accept tissue transplants having the same genetic constitution as the perinatally inoculated cells. f Miwh, white, is a semidominant associated with white spotting, and inasmuch as spotting genes tend to interact synergistically with each other, WwMiwhMi+ genotypes may be predominantly white. 32H BIOCHEMICAL GENETICS Although such immunologically tolerant mammals may have some utility in studies concerned with mammalian pigmentation, it is doubtful whether they will ever prove to be as useful as the tolerant avian embryos. This appears evident for a number of reasons. Mammalian embryos cannot be manipulated experimentally as can avian embryos, so that the transplantation of embryonic tissues must of necessity be made to newborn or older hosts. In mammals the tolerance-responsive period, determined by the genetic disparity between donor and host, usually requires exposure of the fetus to homologous cells. This is certainly the case in the guinea pig and appears to apply to many strain combinations in the mouse. Furthermore, in most species fetal inoculations are attended by a high mortality rate. The availability of coisogenic color stocks and inbred lines of mice has made it unnecessary to resort to the production of immunologically tolerant animals since these animals offer genetically tolerant combinations for transplantation studies. The existence of these stocks, therefore, not only makes it possible to perform similar trans- plantation studies in mammals hitherto restricted to avian embryos but, in addition, offers the experimentalist genetically uniform animals which have no parallel in any avian species. The facility with which these animals lend themselves to transplantation studies has already been utilized in a number of investigations to determine the mode of action of some of the genes concerned with pigmentation or its absence. An account of some of these studies, dealing with the action of genes at the agouti locus and with those concerned with white spotting and albinism, follows. Analysis of action of the genes at the agouti locus. — The six alleles at the agouti locus of the mouse determine the nature of the melanin produced by the melanocytes of the hair bulbs, that is, whether it is eumelanin (black, brown), phaeomelanin (yellow), or both. By substituting different alleles at this one locus the phenotype can be made to vary from all black (extreme nonagouti, aeae) to all yellow {Ay-). Between these extremes are the agouti (A-), yellow-bellied agouti {Aw-), black-and-tan (a1-) and nonagouti (aa) types which exhibit varying proportions of eumelanin and phaeomelanin. The agouti phenotype is characterized by a yellow banding of the otherwise black or brown hairs over most of the body, that is, the main eumelanin coloration is interrupted by a phaeomelanotic band ; the yellow-bellied agouti is identical with the above on its dorsum, but like the black-and-tan phenotype, has a yellowish ventrum which, depending upon its genetic background and regional variations, may contain either completely yellow hairs or yellow hairs with dark bases. The dorsum of the nonagouti animal is similar to that of the black-and-tan, containing only eumelanin pigment except in the region of the ear where some yellow-containing hairs are also found. Although the ventrum of the nonagouti animal is also predominantly eumelanotic, yellow pigment is found in some of the hairs of certain regions, for example, around the mammae and perineum. The extreme nonagouti, the last mutant described in the series,595 is completely black. The dominance relationships between these various alleles differ between the TACTICS IN PIGMENT-CELL RESEARCH 329 dorsal and ventral sides of the animal, ventrality appearing to favor yellow pigment formation. On the dorsum the order of dominance is Ay > Aw = A > a1 = a > ae, whereas on the ventrum Aw and a1 are dominant to A. Thus the phenotype in Aal animals is indistinguishable from that produced in the yellow-bellied agouti, Aw— There are two possible mechanisms by means of which the alleles at the agouti locus produce their effect: (1) by acting autonomously within the hair-bulb melano- blasts themselves, in which case each agouti-series genotype would differ from the others at the level of its melanoblasts, or (2) by producing their effects by altering the follicular environment in some manner which, in turn, affects the expression of what are essen- tially equally susceptible and similar melanoblasts common to all genotypes. Availability of the appropriate isogenic color stocks and their F1 hybrids has made it possible to discriminate between these two alternative mechanisms. This was done by transplanting histocompatible skin from near-term or newborn donors to neonatal recipients which differed in respect to the nature (agouti-locus constitution) and intensity (other loci) of their future pigmentation. Thus the analysis depended on the mode of expression of potentially intensely pigmented melanoblasts of one agouti-locus constitution when incorporated, following their migration, into the developing follicles of a graft which was not only of a different agouti-locus genotype or of a different tract origin (for example, ventral to dorsal), but with hairs (when pigmented by the melanoblasts indigenous in the graft) either light in color or white. For example, an experiment was conducted with an inbred stock of yellow animals consisting of two genotypes, Ayacece and aacece. Animals of the Ayacece genotype appear as black-eyed whites, and aacece animals are light gray in color. Newborn animals from this light-colored stock provided skin grafts, while the hosts for these grafts were intensely pigmented, histocompatible yellow (AyaCce) and black (aaCce) animals obtained by crossing Ayacece animals with those of the C57BL/6 (aaCC) inbred strain. Under these conditions it was found that when genotypically black melano- blasts, aaCce, migrated into the genetically yellow but phenotypically non-pigmented Ayacece hair bulbs of the grafted skin, intensely pigmented yellow hairs were produced. Conversely, when yellow melanoblasts, AyaCce, migrated into normally lightly pig- mented aacece hair bulbs, black hairs resulted. Similar studies have been conducted with other host-donor combinations at the agouti locus.1202- 1203' 1208 All the results are consistent with the hypothesis that the agouti-locus genotype of the receiving hair follicle determines whether eumelanin or phaeomelanin or both (the agouti pattern) will be produced by its melanocytes. Furthermore, the results also indicate that this expression of genie activity is dependent not only upon the genotype of the follicular environment but also upon the location of this environment on the integument, for example, whether it is on the dorsum or ventrum of the animal. These experiments, made possible only by the availability of the proper isogenic strains, show that at least some of the genes affecting hair color act indirectly upon the melanocytes via the milieu of the hair follicle. It is hoped that .350 BIOCHEMICAL GENETICS the mode of expression of other genes concerned with melanin formation will be investigated along similar lines.1044 Transplantation to mammalian eye or spleen and to chick-embryo celom. — Other methods for discriminating between those factors which act intrinsically within the melanocyte and those which act via the follicular environment involves the transplantation of embryonic melanoblast-containing tissues to the adult spleen,1205, 1206 the anterior chamber of the eye,853 or to the celom of the chick embryo.284, 881 The granular and morphologic attributes of melanocytes which have differentiated in the foreign environ- ment following their migration out of the grafts as melanoblasts, are compared with those of the melanocytes which occur either within the implant or in adult animals of the donor genotype. For example, it has been found that neural-crest cells from genotypically yellow embryos of the mouse not only differentiate into eumelanin- secreting melanocytes in the celom of the chick embryo or in the spleens of pheno- typically different murine hosts,1205 but also in those regions of yellow mice where melanin pigment is normally found outside of the hair, for example, in the Harderian gland or eye.854 These results established that the intrinsically determined capacity of melanoblasts of all genotypes is to produce eumelanin, and only in the local milieu of physiologically appropriate hair bulbs do they produce phaeomelanin. Melanoblasts appear to migrate more extensively from grafts placed in the anterior chamber of the eye or in the celom of the chick embryo than from those placed in the spleen.1205 In addition, the two former sites have the advantage in that grafts residing within them do not stimulate the host immunologically and procure their own destruc- tion. In the case of heterografts in chick embryos their acceptance is, of course, due to the immaturity of the recipient. Homografts in the anterior chamber of the eye are exempted from the consequences of any reaction because they usually remain without vascularization and, therefore, cannot succumb to an immune reaction even if they could elicit one. Grafts in the anterior chamber are rejected if they do become vascularized. The conditions of an unvascularized graft in the anterior chamber of the eye are similar to those within cell-impermeable diffusion chambers which make use of membrane filters to separate grafted tissue from host cells.5, 6 It must be stressed that it is of utmost importance not to overlook nongenetic influences in these transplantation studies, stemming, for example, from the purely mechanical or other properties of the unnatural environment into which the donor melanoblasts are introduced, which may affect the differentiation of the latter. That these must sometimes be taken into consideration in interpreting the results has been demonstrated in transplants to the anterior chamber of the eye853 and in transplants to the chick celom.944 White spotting and albinism. — Using appropriate genotypes these transplantation methods have been employed to investigate problems of white spotting and albinism, characters which have been recorded in a great variety of mammals792 including man. While it is beyond the scope of this paper to discuss these investigations in detail 10° some information is required in order to illustrate the methodology. TACTICS IN PIGMENT-CELL RESEARCH 331 Whereas in all species so far investigated (for example, mouse, rat, guinea pig, and rabbit) the hair bulbs of white spots are characterized by matrices consisting of regularly arranged cells of equal size, albino hair follicles contain, in addition, many large clear cells in their upper bulb region. Histologic observations suggested that these hyaline cells are amelanotic melanocytes, that is, melanocytes which are in every respect normal except for their inability to synthesize melanin, since lightly pigmented geno- types exhibit the same type of cell, except that there are pigment granules present in the cytoplasm. Furthermore, Quevedo1033 found that the melanocytes of intensely pigmented mice, artificially depigmented by biotin deficiency, are similar in morpho- logy to the follicular clear cells of the albino. To establish, beyond doubt, the identity of these cells a determination of their embryologic origin was required. In the mouse, melanoblasts originate from the neural crest and migrate to their definitive positions in the skin during the eighth to twelfth day of embryonic develop- ment. This was established by Rawles1040' 1041 who transplanted various portions of potentially pigmented embryos to the celomic cavity of embryonic chicks and observed the development of melanocytes from them. By this procedure she demonstrated that there was an anterior to posterior mediolateral spread of neural-crest cells. For example, melanocyte differentiation from 8.5- to 9-day-old embryonic transplants to the chick celom depended on the presence of the neural tube, whereas with 10.5-day-old embryos, skin ectoderm and adhering mesoderm from any level of the trunk, but not from the limb bud, gave rise to pigmented hairs. The limb buds receive their neural crest component during the eleventh day of development. Using an essentially similar procedure with the exception that the spleen of the adult mouse instead of the chick embryo celom was used as the site for explants, it was possible to prove conclusively that clear cells, like melanocytes, arise from the neural crest. The absence of neural crest cells in the transplanted embryonic tissue was associated with the failure of clear cells to appear in the hair bulbs of the developing grafts. In the design of these experiments care was taken to utilize histocompatible donor-host combinations which were readily available.1206 Whereas albinism appears to stem from an inherited metabolic defect — almost certainly a failure of tyrosinase synthesis — in melanocytes which are otherwise present in normal numbers and distribution, the basis of white spotting is still unknown. It is obvious that white spotting could not have the same etiology as albinism, since in white- spotted animals the ability to synthesize tyrosinase is present in some of the cells of the body. This becomes even more apparent from the fact that hair bulbs of white- spotted areas appear to lack clear cells and are indistinguishable from those which develop in skin experimentally deprived of neural-crest cells. Consequently, it can be assumed that a white spot results either from the absence of melanoblasts or from their failure to differentiate in a particular area. If white spotting does result from a localized environmental effect which inhibits the differentiation of melanoblasts present in the spotted area, one would expect to obtain functional melanocytes from these arrested cells if they are artificially introduced into an environment known to be 332 BIOCHEMICAL GENETICS favorable for melanocyte differentiation. It was with this idea in mind that the experiments described below were undertaken by Markert851 and Markert and Silvers.100 In the mouse there are at least 14 different loci associated with white spotting.499 Some of these genes, for example, IV, Wv, Wj (dominant spotting) and Miwh (white), when homozygous, produce animals which are completely white with melanin pigment occurring only in the cells of the retina. Indeed, the fact that the retina is pigmented in these otherwise all white mice indicates that the genetic capacity for tyrosinase synthesis certainly must exist. It is pertinent that retinal melanocytes originate from the outer wall of the optic cup instead of from the neural crest. Since the hair follicles of these white animals contain no demonstrable clear cells, they are essentially one large spot and are, therefore, extremely useful subjects for investigating the etiology of white spotting. To test the hypothesis that these animals are white because of an environmental effect which prevents a step in the differentiation of neural-crest cells into melanocytes, various embryonic tissues containing neural-crest derivatives, from both potentially pigmented and potentially black-eyed white animals, were implanted intraocularly into albino hosts. While such implants from potentially pigmented genotypes usually gave rise to numerous melanocytes that migrated over the inner surface of the host eye,853 in no case did this occur with implants derived from potentially black-eyed white animals. Thus, if these animals are white because of an arrest in the differen- tiation of melanoblasts, then this genetic suppression may be the result of a locus which acts autonomously within the melanoblasts themselves rather than through the environment in which these cells occur. While such a basis for white spotting is attractive in explaining the all-white phenotypes which are produced when such genes as those of the W series are homo- zygous, it does not adequately explain the localized white spotting produced in the otherwise pigmented animals heterozygous for these factors. Further investigations are required to elucidate the etiology of white spotting, which may very well result from a multiplicity of causes inasmuch as there are so many different loci associated with this character. OTHER INVESTIGATIONS UTILIZING INBRED AND COISOGENIC COLOR STOCKS OF MICE Other studies concerned with genetic aspects of pigment function in which inbred and coisogenic strains of color stocks have proved invaluable include : those concerned with the capacity of hair bulbs of different genotypes to bring about the oxidative blackening of dopa1119; those concerned with the ability of homogenates prepared from skin of different genotypes to oxidize tyrosine, tryptophane, or other amino acids;395 autoradiographic studies of the incorporation of C14-labeled tyrosine into melanin;381- 852 and microscopic analyses of the size, shape, number, and arrangement TACTICS IN PIGMENT-CELL RESEARCH 333 of the pigment granules present in the hair septules in various genie substitutions to determine the effects of individual loci on these attributes.1092, 1093, 1094, 1095 Studies utilizing tissue-culture methods such as those described by Cohen210 and Reams, Nichols, and Hager1042 in the chick might also be profitable with these coisogenic strains. These stocks also provide genetically uniform material required for investigating the pleiotropic effects which many of the genes associated with pigmentation have in other tissues. Among these effects are those which involve hematopoietic tissues, absorption of bone, eye size, formation and function of the nervous system, and number of germ cells.499 PROBLEMS OF PIGMENTATION IN SPECIES OTHER THAN MICE Despite the availability of large numbers of genetically uniform coat-color types of mice and their obvious experimental advantages for pigment-cell genetics, there are certain pigmentary phenomena which can only be investigated in the particular species in which they occur. Among these is partial albinism found in Himalayan rabbits and certain other mammals of similar phenotype. In this condition the fur is predominantly white but pigmented at the extremities of the body. However, it has long been known that pigmented hair appears in other normally nonpigmented regions of the body after shaving the skin and maintaining the animal in a cold environment.1177 The intriguing tortoise-shell (epep) pattern in the guinea pig, which is characterized by an irregular intermingling of yellow with black hairs, and the effect which spotting has on this pattern is also without a counterpart in the mouse and certainly deserves investigation. In the presence of spotting factors, especially ss, not only is there an increase in the amount of yellow in tortoise-shell genotypes but, in addition, in epepss animals there is a tendency toward segregation of yellow and black so that a tri- colored yellow, black, and white spotted phenotype results.199, 1419, 1449 Pigment spread. — Finally the provocative phenomenon of pigment spread as it occurs in guinea pigs, Friesan cattle, and other mammals100 is of interest to those concerned with the potentialities of the melanoblast and melanocyte. If black skin from a black-and-white-spotted guinea pig is grafted to a white area on the same animal, there is a slow centrifugal encroachment of pigmentation into the white skin from the graft margins. Conversely, if white skin is grafted into a black area, it gradually blackens from its periphery inward. This spread of pigment occurs from black into yellow (red) or from yellow into white skin, and it also occurs naturally where skins of two different colors are juxtaposed on the same animal. Although this phenomenon has been investigated repeatedly, no completely satisfactory or generally acceptable explanation of the underlying mechanism has yet been forthcoming. The two most attractive hypotheses which have been advanced to explain this phenomenon are: (1) it results from a transformation of amelanotic melanocytes into melanin-producing cells or of phaeomelanin-producing into 334 BIOCHEMICAL GENETICS eumelanin-producing cells by a postulated infective agent derived from pigmented melanocytes, 94 • 95 • 96 • 97 • 98 and (2) pigment spread is simply due to the actual migration of melanoblasts or melanocytes. The fact that pigment spread can be initiated by seeding a white spot with cellular homografts of dissociated epidermal cells including melanocytes,96 but may sub- sequently bleach out spontaneously after a variable period or may be caused to bleach out promptly following an immunizing skin homograft from the donor of the melanocyte- containing suspension, has some important implications. The fact that albino mice will permanently accept coisogenic pigmented skin constitutes strong evidence that Fig. 46. Principle of method of determining origin of melanocytes in annulus of SPREAD SURROUNDING A BLACK-IN-WHITE GRAFT IN THE GUINEA PIG. STRAIN A (Donor) STRAIN B PRIMARY HOST Fi(AxB) Hybrid -Pure epidermal graft containing melanocytes from annulus of spread SECONDARY HOST 'Strain A sensitized against strain B by an Fj or B homograft melanin per se is not an isoantigen. Thus if the pigment spread initiated by homologous melanocytes is the result of a progressive infection, it follows that homologous melano- cytes must infect the postulated amelanotic melanocytes with transplantation antigen (s) as well as with a capacity to synthesize melanin, unless part of the melanogenic machinery happens to contain a transplantation antigen. The general biological implication of the infection theory suggests that differences between the various melanocytes of a single guinea pig are due to cytoplasmic rather than to nuclear differences. Furthermore, if proven, it would represent the first example of an autonomous cytoplasmic component that gives cytoplasmic inheritance in a mammal at the somatic-cell level. TACTICS IN PIGMENT-CELL RESEARCH 335 To establish whether the melanocytes in the annulus of pigment spread around a black-in-white graft are of graft origin or not, a crucial point for the migration hypo- thesis, Billingham and Silvers are currently utilizing two isogenic strains (as con- firmed by skin-grafting procedures") of guinea pigs and their F1 hybrid. These strains 2 and 1 3 are of the epepss genotype (described above) and, therefore, can each provide and receive black grafts to initiate pigment spread in initially white areas. The experimental procedure represented diagrammatically in figure 46 is as follows. Pigment spread is initiated in white spotted areas of Fx hybrid hosts by means of black grafts originating from histocompatible parental strain animals. The specific techniques for carrying out these experiments have been described by Billing- ham and Medawar.95, 96 After a period of not less than 100 days, when there is a sufficient annulus of pigment spread available, an attempt is made to determine whether the genotype of the melanocytes present in this annulus is of the donor strain or of the F1 hybrid. This is achieved by isolating pigmented epidermis from the annuli of spread and determining the ability of the melanocytes contained therein to survive in specifically sensitized animals (in respect to Fx hybrid homografts) of the donor strain. If pigment spread is simply the outcome of melanocytic or melanoblastic migration, then the melanocytes of the graft and those that surround it in the annulus of spread must be of the same genotype : that of the donor. They should, therefore, survive when transplanted back to the donor strain. If, however, an infection process is involved, then only the melanocytes from the graft should survive, since those from the annulus of spread would be of hybrid genotype and consequently should be des- troyed very soon after they are placed on the presensitized, secondary host. CONCLUSION An attempt has been made to discuss only some of the many methods which can be employed in studying the genetic aspects of mammalian pigmentation. Most of these methods have involved tissue transplantation, since this technique has proved to be of utmost importance in approaching experimentally many of the problems of pigment-cell workers, particularly those concerned with the physiologic genetics of pigmentation. It is obvious that the progress which has been made in understanding how genes control such processes as melanoblastic differentiation and synthesis of melanin has rested, first, upon the availability of color stocks uniform with respect to at least those hereditary factors concerned with pigmentation, second on the production of inbred color stocks, and finally on the existence of coisogenic color lines. Although almost all of these have been established in either the guinea pig or the mouse, mainly through the pioneering efforts of Drs. Sewall Wright and C. C. Little, they include such a wide range of different phenotypes that among these two species can be found suitable subjects with which to investigate almost all aspects of pigment-cell biology. There is 336 BIOCHEMICAL GENETICS no doubt that a systematic analysis of mammalian pigmentation still provides one of the best methods in elucidating the many diverse ways that genie action can influence the behavior of a single cell. DISCUSSION Dr. Burdette : Dr. Morris Foster will discuss Dr. Silvers' paper. Dr. Foster: I find myself in the quandary of finding something to say when Dr. Silvers' work is so clear that it drastically reduces the number of possible questions any fertile imagination could produce. Therefore I should like to defer any questions directed to Dr. Silvers in favor of anyone in the audience who has perhaps less favorable bias toward his work and to provide instead a very brief supplement describing some recent advances in methodology relating to the genetically controlled biochemistry of mammalian melanin pigmentation. At the subcellular level, we are confronted with processes which occur in at least three distinct echelons of organization in the sequence from tyrosine to melanin. At the first, small molecular level within differentiated and functioning melanocytes, we deal with substrates such as tyrosine. The precursor undergoes a series of trans- formations, and we then proceed to a second organizational level, that of the macro- molecule, when the late intermediates being to polymerize. We finally arrive at a third level of organization, that of a fully formed cytoplasmic organelle, when a poly- merizing product is conjugated with an already formed protein matrix to produce a mature melanin granule. It should be emphasized at the outset that it is quite naive to equate tyrosinase activity with the whole process of melanin formation ; it is asking too much of mam- malian material to expect that assayed tyrosinase activity and the amount of melanin would be modified either to the same degree or, indeed, in the same direction by given allelic substitutions. Thus, before trying to relate a given genotype to a given amino- acid sequence of the tyrosinase molecule, it might be useful first to find out which genie substitution affects which part or parts of the pigment-building system, and at least to obtain, for biochemical purposes, a tyrosinase-rich tissue. At least one such genotype was unexpectedly found, and it seems to offer now the greatest hope for extracting and solubilizing the enzyme and, ultimately, for determining its amino-acid sequence (perhaps also permitting analysis of secondary and tertiary structural features). Our own recent approach to objective and quantitative assays for genetically controlled, melanogenic attributes involves the use of lyophilized individual skins, from each of which we obtain four, equal, dry-weight subsamples. These subsamples are assayed respirometrically for tyrosinase and dopa-oxidase activity, and subsequently they are also assayed for melanin content by means of turbidimetric measurements of alkaline thioglycolate suspensions of unincubated tissue or of tissue which had been incubated in the Warburg vessels and removed after a standard period of incubation. The genetic materials we used involved separate and combined substitutions at the TACTICS IN PIGMENT-CELL RESEARCH 337 pink-eyed dilution (/»), maltese dilution (d), and brown (b) loci; giving us a total of eight color genotypes. We also used albinos as controls to provide baseline measure- ments.396 Fig. 47. Sample oxygen -consumption curves during the incubation in vitro of skin SUBSAMPLES OBTAINED FROM TWO DIFFERENT COLOR GENOTYPES. 180 160 140 120 O 100 o 80 60 40 20 _ THEORETICAL MAXIMUM NET 02 CONSUMPTION TYROSINE: 232 Jtl (5 ATOMS/MOLECULE) D0PA: 170 Mi (4 ATOMS/MOLECULE) 2 3 4 5 TIME (HOURS) Our first surprise was that a very pale animal (that is, pink-eyed, dilute, brown; genotype aappddbb) could have far more assayed oxidative enzymatic activity toward added substrate than a very dark one (that is, intense black, genotype aa). (See figure 47.) Our second, and most pleasant, surprise concerns the use of the thio- glycolate dermal suspensions which are easily prepared. I should like to compare our own data, obtained directly as readings of the Klett colorimeter scale, with those computed from the histologic data of Elizabeth S. Russell.1092, 1093 (See figure 48.) 338 BIOCHEMICAL GENETICS Except for possible differences of scale (Dr. Russell's scale, using an arbitrary unit called pigment volume, is based on the number and average dimensions of melanin granules at a given developmental stage), there is a very striking and gratifying paral- Fie 48 Comparison of turbidimetric estimates of natural melanin content ("un- * *&* *w* , 1flQ9 10Q3 incubated" subsamples) with estimates based on E. S. RUSSELL S HISTOLOGIC DATA1 I ("pigment volume"). 260 240 220 2 200 j: i8o LU * 160 S 140 < m => 120 o z §■ 100 80 900 - 800 _ to H § 700 >- < 600 CC K cd 500 cc < 400 LlI 2 3 300 > i- 200 z 111 § 100 4th -30th FIELDS DATA FROM ES. RUSSELL, 1946,1948 _ 4th -20th FIELDS 1 p 1 P 1 P 1 P 1 PP 1 PP 1 PP 1 PP 1 D dd D dd D D dd dd Albino B B bb bb B bb B bb lelism as to order of effect, with respect to natural melanin content (subsamples not incubated), as a function of genotype. This parallelism gives us a reasonable measure of confidence in the use of the thioglycolate suspensions for obtaining measurements of TACTICS IN PIGMENT-CELL RESEARCH ,35.9 melanin content. One final point should be made: on Dr. Russell's pigment- volume scale, albino skin would have a zero value. In our case, however, suspensions of albino skin give a residual turbidity of about 80 Klett units. (Since making these remarks originally, we have learned that there are important physiologic differences between the intense black homozygote (aaBB) and the slightly less pigmented, but enzymically heterotic, heterozygote (aaBb).398 These recently discovered differences do not, however, contradict our less detailed data summarized here.) When the data are subjected to an analysis of variance, it turns out that the greatest source of significant biological variation is due to differences in color genotype. The next major source of biological variation is attributable to differences between litters within genotypes. (See sample analysis in table 59 for tyrosinase activity.) Table 59 Sample analysis of variance for tyrosinase activity (Maximum rate of net oxygen consumption in microliters per hour) Source of Variation D.F. Sum of Squares Mean Square F P F' P' Between genotypes Within genotypes 1. Between litters 2. Within litters 8 216 50 166 7,079.85 3,248.29 2,400.72 847.57 884.98 15.04 48.01 5.11 173.19 9.40 1.00 < 0.001 < 0.001 58.84 1.00 < 0.001 Total 224 10,328.14 F, P stand for variance ratios and corresponding probabilities when "within litters" mean square is used as error mean square. F', P' stand for variance ratio and corresponding probability when "within genotypes" mean square is used as error mean square. The data analyzed here are more extensive than those previously summarized.396 Significant variance ratios of between : within genotypes permit ranking of genotypic means for all measured melanogenic attributes according to the methods developed by Duncan,293- 294 who also described an admirably compact way of summarizing the results of all possible tests of significant differences between means. (See examples by Foster.396) While many statements are implied in such compact summaries of numerical ranking, I wish to make only a few additional points. First, black skin, which is obviously much more heavily pigmented than albino skin, is hardly distinguishable from albino skin with respect to rate of respiration upon incubation with tyrosine. Second, the skins of pink-eyed, dilute-brown mice react very strongly with added substrates, despite their very low level of natural melanin content. (See figures 47 and 48.) Thus there is no simple correspondence between assayed enzymatic activity and natural content of melanin. Third, detailed comparisons regarding allelic substitu- tions on different genetic backgrounds indicate some regular pattern of effects. For example, in black versus brown contrasts, brown skin is more active than black toward 340 BIOCHEMICAL GENETICS added substrates. Black skin, however, is best able to form melanin when relying upon its own endogenous resources, that is, when incubated in the absence of sub- strates. We therefore conclude that the inherited pigmentary defect in brown skin is in part due to some form of endogenous limitation of substrate. This conclusion was "recently" independently "confirmed" by Sewall Wright in 1916 and 1942.1416- 1449 (Addendum: Recently we have noted an additional feature of the genetic affliction in brown skin; it cannot form as much total tissue-bound melanin as black skin upon in- cubation in vitro. Thus the genetic damage in the brown genotype may also involve decreased number or effectiveness of the melanin binding sites of the protein matrix.) Finally, if we make systematic comparisons to determine the effects of single and combined allelic substitutions at the p and d loci, we find that the amount of tissue- bound melanin formed upon incubation in vitro does not run parallel to the amount of oxidase activity. We are therefore led to suspect that the limiting factor in these color mutants is not one of oxidase activity but rather one or more defects in the terminal phases of the pigment-building process, phases which are not readily measured respiro- metrically. A useful by-product of this study is the discovery of an unsuspected, exceptionally rich source for enzymatic extraction procedures, namely, the pink-eyed, dilute, brown genotype. While the methodologic improvements discussed are encouraging, they have not yet helped us solve two important problems. The first concerns the nature of the process for forming yellow pigment ( phaeomelanin) . At the present time no evidence conclusively demonstrates tyrosine or any other likely chromogen as the natural precursor of phaeomelanin. (See, however, Foster395 and also Fitzpatrick and Kukita.382) A second apparent paradox is the striking albino-like behavior of deeply pigmented skins obtained from animals bearing intermediate alleles at the albinism locus. As long as allele C is lacking, that is, the chinchilla mouse (aacchcch), the skin behaves like the completely unpigmented albino skin toward added tyrosine or dopa. A similar situation in the guinea pig was previously reported.394 No evidence has yet been obtained to explain this apparent paradox. One possible approach involves the use of paper chromatographic methods for identifying diffusible endogenous melano- genic precursors and intermediates. This method was already proved successful in identifying the unstable, diffusible, intermediate dopa, in an incubation medium containing skin with high tyrosinase activity (Foster and Brown,397 work supported in part by NIH Grant C-4305 and NSF Grant G-6480). I regret that Dr. Silvers failed to mention the excellent studies performed by himself, in conjunction with Dr. Russell, concerning the role of the melanocyte environ- ment in instructing the cell to synthesize either eumelanin or phaeomelanin. At this point I should like at least to draw attention to the pertinent publications.1202- 1203, 1205. 1208 Dr. Barrett: I have a question that is not too profound, but I have usually wondered about it when thinking of this work. Regardless of which theoretical explan- ation applies, why do we not see in nature some explanation for the fact that in spotted, TACTICS IN PIGMENT-CELL RESEARCH 341 banded, hooded, and otherwise multicolored animals the old animals are not all pigmented? Why in a multicolored animal is not the old animal characterized by all, or almost all, black pigment ? Dr. Silvers: Part of the answer may be due to the fact that in the general integu- ment of pigmented rats and mice, melanogenesis only occurs in the melanocytes of the hair bulb. The other melanocytes in the skin do not synthesize melanin. In the guinea pig there is a population of active melanocytes in the basal layer of the epidermis as well as in the hair bulbs. In this animal at the region of transition where black and white areas are juxtaposed, one does find a very narrow band of pigmented skin from which white hairs originate. This area has been secondarily pigmented by a migra- tion of melanocytes from the black area. However, why this process does not continue throughout the life of the animal, I do not know. It may at an imperceptible rate. The rate of spread around a black graft to a white area also falls off asymptotically. Dr. Nalbandov: I have two questions. First, what is the origin of the pigment cells in such structures as the testes in chickens, which may be completely black? Second, what is the origin of pigment cells in the uterine region? Neither the testes nor the uterus are of ectodermal origin. Could chickens which may have one black and one yellow testicle or in which each testicle may be two-toned serve as experimental animals for the study of questions in which you are interested, that is, the origin of the yellow and black pigments? Dr. Silvers: If the cells you refer to are dendritic in form, then it is almost certain that they originate from the neural crest. Melanocytes may occur in many regions of the body where one might not expect them, for example, in the spleen, parathyroid, and ovary of the mouse. It is possible that during that period of development when melanoblasts are making their way from the neural crest to their definitive positions, they migrate to and through many areas of the animal, but only persist, differentiate, and produce melanin in suitable environments. The existence of both black and yellow pigment in the testis of the chicken is very interesting. Unfortunately we do not have any inbred strains of appropriately pig- mented chickens to investigate this, although it might be possible to make appropriate grafts to chick embryos. In the mouse there are two types of melanin, pheomelanin and eumelanin. Although it has not been proved, I am of the opinion that in the agouti mouse a melanocyte can alternate its pigment production, producing eumelanin at one time and pheomelanin at another. In the chicken, however, all the evidence is consistent with the hypothesis that, although each melanocyte has the ability to syn- thesize either black or yellow pigment, once it begins to synthesize one type it cannot change and produce the other. This is a problem which deserves further investigation. Dr. Slatis: Dr. Silvers described a very good experiment for distinguishing between infective and migration theories, because a positive result would support the migration theory and rule out the infective theory. The quality that made the experi- ments on transplanting eye discs in Drosophila look so good was that they distinguished between effects due to the genotype of the host and the genotype of the transplanted 342 BIOCHEMICAL GENETICS material. If the infective theory is correct, then one might expect that, if one trans- plants into a host that cannot produce pigment of certain colors, infected cells would be obtained that would give pigment on a white background, but the pigment would be of the wrong color. It appears to be a more discriminating method than the one that you tried in which a positive result is required in order to give you something on which to base a conclusion. Dr. Owen : Have you followed this reaction colorimetrically as well as by oxygen consumption ? Dr. Foster: No, but I think we can now by dealing with material incubated inside dialysis membranes. Although we have not done it because of other work, it is certainly a very worthwhile thing to do in order to have some information about intermediate steps along the way. Dr. Coleman: We have used a different assay, in conjunction with those already described, in studying this problem.213 This assay involves the measurement of the amount of incorporation of radioactive tyrosine into the pigment granules. This assay has been found sensitive enough to distinguish clearly all the alleles of the C locus, a distinction not possible with the manometric method. Otherwise our results are essentially the same as those of Dr. Foster. Our studies with the pink eyed black mouse (BBCCpp) suggest that pink eye decreases the amount of tyrosine incorporated into actual pigment in contrast to the increased tyrosine oxidation associated with the pink eye gene as measured manometrically. These observations suggest that pink eye causes tyrosine to be oxidized faster but to some product which is not melanin. This demonstrates the value of using more than one assay method as suggested by Dr. Owen. As for the yellow mouse our work indicates that less tyrosine is incorporated into pigment in this genotype than either of the normal brown or black genotypes. Both studies in vitro and in vivo indicate that tyrosine is the normal precursor to yellow pig- ment. No evidence has been found which would implicate tryptophan or any other amino acid as a precursor to this kind of pigment. Dr. Herzenberg: The kind of experiments that Dr. Silvers carried out in animals which are inbred could be carried out in other animals as well, particularly chickens and other birds by making them tolerant to the donor of the skin with which they are willing to work. If Dr. Nalbandov is interested in carrying this out, I am sure he could do it. Dr. Heston: There are strains of chickens at the Regional Poultry Laboratory at East Lansing that may be inbred enough for your experiments. Dr. Silvers: Dr. Billingham and I have tested some of the inbred lines of chickens maintained at East Lansing, Michigan by skin graftings and have not found any histocompatible strains as yet." However, we are now testing some more of these lines. Dr. Schaible: With regard to transplantation experiments involving pigmented and unpigmented regions, the chicken has another drawback in that there are no mutants which produce adult spotting patterns like those found in mammals. In chickens, melanocytes seem to be able to proliferate until they occupy all feathered TACTICS IN PIGMENT-CELL RESEARCH 343 regions. The only types known to have restricted pigment areas are those carrying E in their genotypes; then only the extremities (wing tips, ventral regions, and feet) are white in the newly hatched chick. Even when migration of pigment cells appears to be retarded by teratogenic agents until only a small pigmented area remains on the back in the down pattern of £ types, the birds eventually become fully pigmented.747 IMMUNOGENETICS Ray D. Owen, Ph.D. METHODS in MAMMALIAN IMMUNOGENETICS Antigen-antibody reactions depend on mutual complementarity of structure between the antigen, which induced the antibody to be formed, and the antibody, which reacts only with the antigen that induced its formation and with other sub- stances having a sufficient degree of configurational similarity to it. Antibody re- actions therefore provide ways of studying individual similarities and differences on a molecular level, less precise but easier than detailed structural-chemical analysis. Because the molecular differences so defined are generally found to relate in rather direct ways to particular genie differences, in almost any species investigated im- munogenetics taps a reservoir of genetic characteristics amenable to straightforward genetic analysis. Thus, many of the best genetic markers for studies on either natural populations or laboratory mammals are those identified by serologic methods. This aspect of genetics is one of only a few to which mammals as experimental material have contributed in unique and basic ways. Even in studies of other kinds of animals and plants, it is usually the antibody response of a mammal, most often a rabbit, that is used in providing tools for immunogenetic study. And as a source of genetically regulated antigens, easily obtained and tested, probably nothing at present rivals the mammalian red blood cell. As a demonstration of individuality, probably nothing is so impressive as the regular incompatibility encountered when tissues are exchanged between individuals, to be rejected by the immunologic mechanisms of the host. Because the genetic qualities dealt with in immunogenetics are generally simple ones, the methodology of the genetics side of this kind of study is mainly simple, straight- forward, and traditional ; for the most part, these methods need not be exploited here. It will be assumed that it is the methodology of immunology that will be unfamiliar to 547 34H IMMUNOGENETICS the users of this paper, and it will be toward that part of our subject that attention will mainly be directed. A few of the general terms and concepts of immunology should be introduced at the start: Antigens are molecules with two properties: first, they will induce an antibody response when injected into an animal, and second, they will react specifically with the antibodies they have induced. Often, the term antigen is not defined on a molecular basis; for example, a red-cell suspension injected into an animal may be described as the "antigen" injected. In general under such circumstances, one should recognize that this gross "antigen" is composed of a number of discrete molecular antigens, each involved in its own antigen-antibody reaction systems. Sometimes, a molecular antigen is found to be unable to induce antibody formation although it is able to react with antibodies evoked by a related material. For example, a fragment of a protein may be able to react with the antibody induced by the intact protein, although the fragment itself when injected may not give rise to a detectable antibody response. In general, in order to be a complete antigen in the sense of both inducing and reacting with antibodies, a molecule needs to be relatively large; most of them are in excess of 40,000 molecular weight, and many are in the millions. A small molecule, however, such as a substituted benzene ring, may react specifically with antibodies; the reactive sites on antibody molecules are themselves only small parts of the molecules.672 Thus, a complete, macromolecular antigen is conceived as being composed of numerous individually small sites on sections of its surface, each site capable of reacting with antibodies specific for it. These sites are spoken of as valence sites; complete antigens are polyvalent and complete antibodies have two valence sites per antibody molecule. The two valence sites on an antibody molecule seem to be identical with each other. Very small reactive compounds may appear to act as antigens, as in idiosyncrasies for particular drugs like aspirin, or dermal hypersensitivities to small compounds such as picryl chloride. In such cases, it is generally assumed that the small molecule com- plexes with larger ones in the recipient's system; it is the macromolecular complex that acts as a complete antigen, the strange small added grouping being responsible for the foreignness of the total molecule and directing the specificity of the antibody response and reactions. Antibodies are modified serum globulins, secreted into the circulation by cells of the antibody-forming series as a consequence of the injection of a foreign material and specifically reactive with the material that caused their formation and release. The basis for this remarkable adaptive response on the part of the injected animal is a fascinating problem in somatic-cell genetics, currently under scrutiny and debate. The easiest antibodies to discern are those that result in a visible reaction with the anti- gen in ordinary media in test tubes, such as the agglutination of red cells in saline or the precipitation of a soluble antigen from saline solution. Such reactions can be formally conceived as involving two processes: first, the specific combination of antibody molecules with sites on the antigen, and second, a derivative reaction in which a visible METHODS IN MAMMALIAN IMMUNOGENETICS 349 change occurs in the system. The discrimination of two phases came about largely as a result of observations of systems in which the first step occurred without the second, and in which the second could be promoted by making some nonspecific addition to or change in the system. For example, in some systems, although antigen-antibody unions occur at low salt concentrations or in the cold, visible changes do not follow until salt is added or the system is warmed. Red cells combine specifically with their corresponding antibodies at low temperatures, but they fix complement and lyse in relatively short times in the presence of complement only when the system is warmed. Such observations tend to distinguish between a first stage of antigen-antibody com- binations and a derivative second phase which may or may not follow depending on the conditions of the test; nevertheless, in many systems the ultimate effect is generally believed to be the result of the continued operation of the same mechanisms as are involved in the initial antigen-antibody unions. For example, a visible precipitate is generally believed to form as bivalent antibodies combined with polyvalent antigens in a continuous process, the antibody molecules providing bridges in a lattice forming the developing aggregate. The degree to which nonspecific secondary interactions may enter into serologic reactions is often debatable and probably depends on the particular system under test. For example, in some systems of cellular agglutination there is reason to believe that changes in cellular membranes consequent upon the initial antibody attachments at isolated sites may reduce the tendency of cells to repel each other, and therefore lead secondarily to aggregation. Hemolysis clearly depends on attacks on the antibody-sensitized cell by nonspecific components of normal serum, leading only secondarily to cell lysis. Many reactions in vivo, such as anaphylaxis, also depend on secondary consequences of the initial antigen-antibody combinations occurring in tissues. When a system has been defined for a particular test (such as saline agglutination) , it is often found that some of the antibodies capable of uniting specifically with the antigen are unable to promote the second, visible stage of the test reaction. Such antibodies are often described as incomplete antibodies. A number of methods are available for their detection; for example, they may block the antigen by combining with it, so that access to the relevant sites on the antigen is prevented for complete antibodies later added to the system. Or they may promote aggregation in other media, though they fail to do so in saline. They may sensitize cells for hemolysis but not promote agglutination, or they may combine with cells and render them subject to aggregation by other antibodies directed against the antibody globulin molecules themselves. Or they may provide a vehicle for passive transfer of tissue sensitization, so that, for example, if they are injected into the skin of an insensitive individual they confer a transient local sensitivity to their corresponding antigen at that skin site. In precipitating systems, multivalence of both antigen and antibody has been mentioned as a necessary condition for the appearance of a visible aggregate. Failure of an anti- body preparation to produce an aggregate is sometimes the occasion for the use of the term "univalent" with reference to the antibody. Often the term is an unfortunate one; 350 IMMUNOGENETICS antibodies may fail to produce a visible reaction for numerous reasons other than univalence, and the careless use of this term suggests an explanation for their behavior which should be applied in any particular case only when this explanation has been established in fact. Univalence in a proper sense can derive from several different causes: some antibody maybe univalent when it is made, an originally bivalent anti- body may be split into two univalent fractions, or one of the valence sites may be covered by aggregation, sometimes with quite different materials such as serum albumin. Aside from their heterogeneity in respect to visible effect produced on the antigen under test, antibodies are heterogeneous in numerous other respects as well. Of primary concern is their heterogeneity with regard to specificity ; some antibodies react essentially only with the antigen that induced their formation, whereas others cross-react with related antigens as well. The directions of these deviations in specificity vary within the heterogeneous antibody population, so that within the limits imposed by the general concept of serologic specificity there is a smear of subspecificities in any given antiserum. Recognition of this kind of heterogeneity is important to interpretation of antiserum reaction in immunogenetics.981, 985 Perhaps a related type of antibody heterogenity involves the avidity with which an antibody combines with its corre- sponding antigen. Variations in avidity can be conceived as variations in the closeness of fit and in the extent of mutual complementarity between the combining sites of the antigen and antibody. Operationally, avidity is generally expressed in the behavior of the system upon dilution. The equilibrium, Antigen + Antibody ^ (Antigen-antibody Complex) lies to the right with avid antibody, to the left with antibody of lesser avidity. Anti- body is also heterogeneous on physical-chemical grounds, as measured by solubility, electrophoretic behavior, chromatography, and ultracentrifugation. Changes in the character of antibody, with regard to specificity, avidity, and other characteristics, commonly occur during the course of an immune response.663 Some types of immune responses have not been shown to involve the participation of classical, plasma antibody. Among these are reactions that appear to be strictly cell borne and cell mediated, such as the delayed hypersensitivity reactions,770 and at least some types of tissue-transplant rejection.861 Except for some reference to tissue- transplant rejection, however, consideration here will be limited to plasma antibody responses and genetic test systems based on such antibody. IMMUNOGENETICS OF RED BLOOD CELLS Procurement of antisera. — In general, it is the immune response resulting from exposure to a foreign antigen that provides the most useful tools in immunogenetics. In many important instances, however, antibody-like materials which react specifically with the red blood cells of other individuals are normally present in the plasma of METHODS IN MAMMALIAN IMMUNOGENETICS 351 particular individuals. In such cases no injections are necessary, although injections may increase the titer or the reactivity or change somewhat the character of the anti- body normally present. Materials that will aggregate the red cells of mammals, with some degree of specificity, are not restricted to mammalian sera ; lower vertebrates and even many invertebrates have such hemagglutinating materials in their plasma. Even some plant-seed extracts provide useful reagents for distinguishing individual differences within mammalian species; practically all of the useful extracts of plant seed are from legumes.101 The plant-seed extracts have been used mainly for studies of human red cells, and to a lesser degree those of birds; they have as yet found relatively little application in the immunogenetics of mammals other than man. The preparation of the plant-seed extracts is generally simple; the pulverized seed is extracted with saline, and the extract clarified by filtration or centrifugation before it is used as a test fluid on red-cell suspensions. The classical and best-studied example of normal antibodies that make individual distinctions within a species is, of course, concerned with the ABO blood groups of man. Similar normal antibodies are useful in studies of cattle and sheep and occur in a number of other species as well. In rats, normal antibodies reacting with the cells of other individuals are rarely encountered; there is, however, at least one simply in- herited difference displayed by normal antibodies in the plasma of certain rather rare rats, which agglutinate in saline the red cells of almost all other rats.147, 980 Normal antibodies for murine cells also occur in certain sera of the mouse; they are, however, irregular and weak. Normal antibodies for the red cells of other species are very common. The techniques used to obtain test sera, either normal or immune, vary with the kind of animal being used. In such animals as cattle or sheep, blood can be collected in quantity by needle puncture of a large vein at the side of the neck. In rabbits, small quantities of blood are conveniently obtained by nicking the marginal ear vein with a razor blade, and permitting the blood to drop into dry test tubes. Warming the ear or rubbing a drop of xylol on its tip distends the vein and promotes more rapid bleeding. It is useful also to rub a light film of vaseline on the ear at the site of the incision to reduce blood clotting during bleeding. Bleeding can be stopped by pressing a bit of dry absorbent cotton over the incision and compressing the vein with the fingers distal to the incision for a minute or two. Larger quantities of blood are obtained from the rabbit by cardiac puncture, a procedure that in experienced hands is of little danger to the animal. The rabbit is tied down on a board, without anesthesia; anesthesia is a greater threat to the survival of the rabbit than is the operation itself. Bleeding is generally done with an 18-gauge 1-^-inch needle on a 50-ml. syringe. Quantities of 50 or 60 ml. can easily be taken at a time without killing the rabbit; he may be bled in this manner on at least two successive days. Several different ap- proaches to cardiac puncture are in use ; in one, the V formed by the lowest attached ribs and sternum provides the point of orientation. The needle, on the syringe, is then inserted through the second costal space, close to the sternum on the animal's left side, 352 IMMUNOGENETICS aimed slightly toward the animal's head and toward the midline. It is useful to wet the syringe before use with an isotonic citrate solution (2 per cent sodium citrate, 0.5 per cent NaCl). In mice and rats, small quantities of blood may be obtained by incision of a vein in the tail; particularly in mice, it is preferable to make a shallow cut with a razor blade across the ventral surface of the tail, rather than to use one of the lateral tail veins. Rapidity of bleeding can be promoted by warming animals under an ordinary study lamp, before bleeding. Hemolysis of the serum can be avoided if the dry tubes into which the blood is allowed to drip have been coated with silicone. Many believe that a more convenient technique than tail-vein incision or cardiac puncture in the mouse or rat is to obtain the blood by Halpern's method from the orbital sinus.1283 Hema- tocrit determinations can be made directly in the tubes used for bleeding, without transfer, if heparinized tubes are prepared. Cardiac puncture is also convenient in mice and rats; mice are bled under Nembutal anesthesia and rats under ether or urethan anesthesia. The approach to cardiac puncture described for the rabbit also works for the mouse and the rat; shorter and smaller needles are used (for the mouse | inch, 26 gauge; for the rat, \ inch, 24 gauge). Many find it easier to bleed a mouse by cardiac puncture, using an approach immediately under the tip of the sternum and directing the needle straight forward and almost horizontally. With this approach a 22-gauge, 1-inch needle is proper. Guinea pigs and other small mammals are also conveniently bled by cardiac puncture ; we have bled, for example, opossums, monkeys, and raccoons easily this way. When considerable quantities of blood are to be obtained from the mouse, particularly when blood from homogeneous populations is to be pooled, mice are often sacrificed, and blood is collected in a beaker after decapitation. If the plasma is to be used, or if test red-cell suspensions are the main objective of the bleeding, the blood is collected in an anticoagulant. Several of these are used: for example, heparin, versene (0.1 M, pH 6 to 7, in 0.85 per cent saline), oxalate, or citrate solutions. When, however, it is a test serum that is required, the blood is collected in a dry (preferably silicone-coated) tube, and is allowed to clot. The clot should be freed from the wall of the test tube after it has formed ; for most samples, it can simply be shaken free after it has been permitted to stand undisturbed for an hour after bleeding, or the clot may be rimmed with a wooden applicator or glass rod. In most bloods the clot then shrinks, squeezing out the serum; after an hour or two the serum is poured from the clot, the free cells are centrifuged out, and the clear serum is decanted or pipetted into storage bottles. Better yields of serum are obtained from mice if the blood is collected into a tilted petri dish, so that the clot forms as a film over half of the plate. The clot can then be freed from the glass surface over most of the area, and the plate tilted so that the serum as it is squeezed out of the clot runs down to the clear side of the plate. With the small quantity of blood obtained from an individual mouse it is very desirable to keep the plate covered during the process, to avoid drying. For most practical laboratory METHODS IN MAMMALIAN IMMUNOGENETICS 353 purposes, sterile technique is not used in processing sera; the fresh serum itself is effectively bactericidal, and the serum is generally stored frozen between periods of its use. Preservatives are frequently added; Merthiolate (0.05 per cent) or sodium azide (0.1 per cent) are most common. Mouse serum to be used for blood-typing tests sometimes changes in its specificity or loses its activity upon ordinary storage; routinely, mouse-typing reagents are therefore subdivided into small quantities in ampoules, and are then lyophilized and stored in sealed ampoules, if possible after evacuation of the ampoules. Most serologic typing reagents, however, are much less critical in their storage requirements than are those prepared from murine serum. Immunization. — Numerous routes of injection are used; the amount and the character of the antibody response is often influenced by the route adopted. Rabbits are generally injected intravenously into the marginal ear vein. For immunization with red-cell suspensions, we commonly inject 0.5 ml. of a 20 per cent suspension of washed cells intravenously three times a week for three weeks. The rabbits are then rested for 7-10 days before the antiserum is collected. The injection is made with a f-inch, 25-gauge needle on a 2-ml. syringe; the use of inexpensive disposable syringes and needles is becoming common and has several advantages. The amount injected, the frequency of injection, and the duration of the immunizing period are arbitrary, and other schedules are as effective as that described. Frequently, rabbits are kept after their first bleeding, and after several weeks are injected again to produce more antiserum. Very often the results of the later immunizations are better than those of the first series. Because of the possibility of an anaphylactic response in the im- munized rabbit when he is injected after an interval of rest, we usually make the first reimmunizing injection intraperitoneally rather than intravenously, injecting about four times as much material as is used in a single intravenous dose ; this has the effect of desensitizing the rabbit, and two additional intravenous injections can then be made as usual at two-day intervals. For some types of antigens, especially relatively small protein molecules of low antigenicity, it has been found desirable to use adjuvants in connection with the injections. Alum precipitation of the antigen before injection is often effective. Another type of adjuvant very frequently used is based on the studies of Dr. Jules Freund, and in its several modifications is generally referred to as Freund's adjuvant. Procedures are described in connection with antiglobulin techniques in a later section of this paper. Material in Freund's adjuvant is generally injected intramuscularly (in rabbits, into the large muscle of a rear leg or into the loin), or subcutaneously (under the skin of the back). Intense local reactivity is induced at the site of injection, and repulsive necrotic lesions may appear. If the outer surface of the needle is kept free of Freund's material, dry and sterile, such lesions are less frequent. An adjuvant often makes the difference between getting a useful antiserum and getting none at all. Adjuvant, however, in our hands generally has had little effect on circulating antibody responses to red cells and similar large materials ; it would appear that such preparations generally 354- IMMUNOGENETICS have sufficient "adjuvant" activity of their own. In some systems, such as the auto- sensitization of mice to red cells of their own type, Freund's adjuvant may play an irreplaceable role. Avoid young animals, or animals in poor physical condition, for the preparation of antisera. We commonly use a market strain of New Zealand white rabbits and require that each animal weigh at least 5 lb. before injections are begun. Mice achieve sufficient serologic maturity after they are about 6 weeks of age, but they are commonly used at 12 weeks. Mice are commonly immunized by way of intraperitoneal or subcutaneous injec- tions, although intravenous injections into the lateral tail veins are not difficult. Rats are somewhat more difficult to inject intravenously than are mice; intravenous injec- tions of rats are possible, but intracardiac injections or injections by cannulating a femoral or carotid vein are often easier.237 Mice do not produce good antisera to murine red cells in response to the injection of blood; instead, macerated tissues (particularly spleen) or tumor transplants are commonly used to sensitize them. We have evidence that better hemolytic antisera are likely to be produced in mice if they first reject a skin transplant from the donor line; shortly after the rejection of the sensitizing transplant we give the first of two intraperitoneal injections of splenic cell suspensions (1 donor spleen per 3 recipients) at weekly intervals, then bleed the reci- pients for test serum about ten days after the second injection. Preparation of antibody reagents. — Under the simplest and most fortunate circum- stances an antiserum, either normal or immune, may contain an antibody population recognizing only a single difference among the individuals tested — those having the specificity recognized by the antibodies, and those lacking it. Serologically, this simple situation is revealed when absorptions are conducted with the red cells of a panel of test individuals. Packed, washed, red cells of each test individual are mixed with a saline dilution of the test serum and thrown down in a centrifuge. The supernatant, absorbed serum is then used as a test reagent; in the simplest situation all positive cells will absorb the antibody for all other reactive cells, whereas negative cells have no effect on the antibody population. Genetically, this simple serologic situation is almost always found to reflect a single genetic alternative; positive individuals are found to be either homozygous or heterozygous for an allele producing the test specificity, and individuals lacking it are homozygous for an allele of the gene so defined. Much more commonly, an antiserum will be found to contain two or more anti- body populations of discrete specificity. Serologically, this situation is revealed by absorption analyses; not all reactive cells will remove the antibody for all others. Table 60 shows an absorption analysis leading to the recognition of two specificities, designated A and B, by means of anti-^4 and anti-Z? antibody populations in a particular antiserum. An analysis like that illustrated in table 60 should be followed by further absorptions on the reagents postulated from this set of absorptions to be of single specificities; the test fluid remaining after absorption with cell no. 2, for example, should now be further absorbed with each of the reactive cells separately, and it should METHODS IN MAMMALIAN IMMUNOGENETICS 355 Table 60 Absorption analysis leading to the recognition of two specificities Antiserum Unabsorbed Absorbed with cells of individual: Test cells from individual: 1 (A) + 0 + 0 + 0 0 + + + 2 (B) + + 0 0 + + 0 + 0 + 3 (AB) + + + 0 + + 0 + + + 4(-) 0 0 0 0 0 0 0 0 0 0 5(A) + 0 + 0 + 0 0 + + + 6 (AB) + + + 0 + + 0 + + + 7H 0 0 0 0 0 0 0 0 0 0 8(B) + + 0 0 + + 0 + 0 + 9H 0 0 0 0 0 0 0 0 0 0 The unabsorbed antiserum evidently contains two distinct antibody fractions discernible with these test cells, recognizing four cell types, A, B, AB, and (-). Absorbing with type A leaves anti-5; with B leaves z.nt\-A. be shown that each of these cells removes all of the antibody reactive with each of the others. Very frequently, an antiserum will require three or four or more sets of symbols for its consistent analysis (for example, antw4, anti-Z?, anti-C, anti-Z)). The analysis of such an antiserum provides an interesting exercise. The behavior of each cell in both the absorption columns and the test rows of the table, and relative to the behavior of all other cells in the test, must be consistent with the symbolic designations. In such complex situations, unit reagents are often achieved only after further absorp- tions with pools of two or more cells of different symbolic types. Realistically, we must observe here that frequently deviations from ideal behavior are observed in these complex systems, such that, for example, a given cell may appear to be removing anti- body in absorption with which it does not appear to react in tests. The published work in this field is generally based on selections of reagents that behave in logical and consistent fashion, and authors and teachers in this field often ignore a tangled hinter- land of poor reagents set aside, unused and unexplained, that violate the principles of straightforward symbolic absorption analyses. Genetically, reactions that require two sets of symbols (such as A versus non-yl; B versus non-5) are often found to depend on two independent pairs of alleles. Fre- quently, however, multiple allelic series are discerned ; some of the longest multiple allelic series in all genetics are noted in this field. When sets of specificities are found to depend on a series of multiple alleles, there are very frequently cross reactions among the products of related alleles. This situation, dependent mainly on the lack of complete specificity of serologic reactions, leads to uncertainties regarding the sig- nificance of the symbolic designation of antigens, in terms of their antibody reactions, 356 IMMUNOGENETICS and of genes, in terms of the antigens they control. This point of principle will not be discussed further here; it has been considered in detail elsewhere.981- 985> 1286 As to the methodology of absorptions themselves, the details will be found to depend to some degree on the particular system to be investigated. In many systems, such as rabbit antisera to cattle blood, or an antiserum produced by one cow against red cells of another, one simply mixes the packed, washed, absorbing cells with a dilution of the antiserum to be absorbed in nearly equal quantities, allows the well- mixed mixture to stand for a few minutes, then spins the cells down and repeats this procedure on the supernatant with fresh absorbing cells until all of the antibody reactive with the absorbing cells has been removed. This process may be complete in two successive absorptions (as with most cow-anti-cow sera), or may require four or five successive absorptions (as with rabbit-anti-cow serum). The objective here is get out all of the antibody with which the absorbing cells can react; further absorptions with these cells or with negative cells then have essentially no effect on the reagent. In many other systems, however, the amount of packed red cells used in an absorption, and the number of absorptions, must be cautiously controlled; a procedure described by Race and Sanger1036 as that in use at the Medical Research Council for the prepara- tion of anti-human M and N sera is a useful reference. The reason generally given for the necessity of proceeding with caution in situations like that for anti-human M and N is that the anti-M and anti-iV antibodies are not fully specific for the corresponding antigen, but will in fact unite with low avidity to the other antigen as well. Even the relatively rare cases in which human beings develop anti-A7 prove to be specific for N at only certain temperatures; they react with M cells at lower temperatures, or after enzymatic treatment of the cells.585 The plant anti-A7 can be adsorbed by M cells, although it is much more easily dissociated from them than it is from N cells.784 Similar situations are encountered with reagents for red-cell differences among mammals other than man. They lead, of course, to difficulties; for example, in such a situation a questionable test result can be checked only with difficulty by an absorption test because even a negative cell may remove or reduce the antibody in question. They also make it difficult to judge the specificities of cells by tests on eluted antibody after absorption. Techniques for elution of antibody will not be discussed in detail in this chapter ; one of the easiest involves the principle of adsorbing antibody to the surface of the cell at a low temperature, washing the cells gently in the cold, and then warming and shaking the system in a saline medium. The antibody may then elute from the cell surface into the saline, from which the cells can be removed by quick centrifugation at the higher temperature. Another basis for deviations from ideal behavior in absorptions has been sug- gested by the work of Jacquot-Armand et a/.659 It appears that the red cells of certain animals will remove a fraction of human anti-5 only when the cells are used in large excess, and that this adsorption is promoted by nonspecific factors in serum, such as added serum from persons of type AB, which of course lacks the anti-i? antibody itself. More work should probably be done along this line ; if it is true that nonspecific co- METHODS IN MAMMALIAN IMMUNOGENETICS 357 factors may sometimes be involved in the fixation of antibody in absorption, part of the logic upon which absorption analyses are based becomes doubtful. Most immuno- geneticists generally choose to believe that absorption tests are the last compelling resort for ascertaining the specificity of questionable test cells ; it is now clear, however, that even this resort is not unfailingly reliable. Nevertheless, in addition to their utility in providing simpler test fluids as reagents, antibody-absorption procedures are a useful and often irreplaceable source of detailed information about cell and antibody specificities. If one were to give a value estimate, he would probably conclude that more misleading conclusions have entered the literature through failure to conduct adequate absorption analyses than through the overzealous application of this technique. When one is working with so small a mammal as the mouse, and particularly in segregating generations when recourse cannot be had to pooled bloods from numerous representatives of an homogeneous group, shortage of absorbing cells and of antisera may become a limiting consideration. Under these conditions, in vivo absorptions, as conducted by Amos,19 may prove useful. For example, when 0.1 ml. of an antiserum produced in BALB/c mice against the C57 black leukosis EL4 was injected intravenously into C57 black mice and the recipient animals were bled at intervals, the titer of the serum of the recipient animal against ^4-cells had fallen below detectable levels after 30 minutes. Injected into the BALB/c mice, the same amount of serum remained at a titer of 1 /256. The passively administered antibodies persisted in the serum of the nonreactive mouse with a half-life of about two days or longer. Intraperitoneal rather than intravenous injections are now commonly used for this technique. Given an antiserum that can withstand the dilution factor observed (about 1 /20) , this tech- nique therefore provides a convenient method of absorption analyses in murine test systems. Red-cell test systems. — A variety of test systems have been elaborated for immuno- genetic studies of red cells. These have been described in detail in research papers and books on technique in the literature,241' 1290 and only a brief survey will be undertaken here. 1. Saline agglutination. Tests for saline agglutination of red cells are commonly performed in small test tubes (10 x 78 mm.), into which one or two drops of the diluted test reagent are dropped from a 1-ml. pipette equipped with a 1-ml. rubber bulb. A drop of a suspension of the washed test cells is then added in the same manner — usually, about a 2 per cent suspension. When a number of samples of cells are to be tested against a number of reagents, usually the reagents are arranged in rows in a series of racks, and the test cells are added in columns, to provide a complete test pattern. For some reagents, it may be necessary to allow the test to stand for an hour or two or more after shaking the cells into smooth suspension, allowing the cells to settle by gravity and then reading for agglutination either macroscopically or micro- scopically or both. Frequently, however, the test can be read only five minutes or so after the cells have been added, sedimenting the cells by brief centrifugation. The 838 IMMUNOGENETICS macroscopic reading is performed bv shaking the pellet of sedimented cells gently back into suspension, meanwhile checking for agglutination by examining the suspension for clumps against a light background, for these and similar tests, a special small centri- fuge called the Sero-fuge (Clay-Adams, Inc., N.Y.) provides a significant saving in time. Variants of this test system are numerous. For example, for particular reagents the cellular suspensions may need to be heavier than 2 per cent; the temperature may be optimally either higher or lower than room temperature; a slanting capillary tube technique, with examination for the sedimentation pattern on the side of the capillary tube, is a sensitive technique for some systems and conserves valuable test sera. For some tests, such as Rh tests with particular reagents, slide agglutination is used. Direc- tions for special techniques are provided with commercial reagents if these are to be purchased. 2. Other systems for agglutination. Antisera that fail to agglutinate red cells in saline, or that do so only weakly or unreliably, may produce strong and reliable agglutination if a medium other than saline is employed, or if the cells are treated in particular ways. One of the most common modifications is to use 20 per cent bovine albumin as a diluting fluid for the test serum and as a medium for the suspension for test red cells. Frequently, a somewhat heavier red-cell suspension is used in such systems — often of the order of 4 per cent. These tests are often incubated for an interval of two hours or so, at 37° C, before being read. Normal serum is sometime used as a diluting fluid ; if the normal serum contains antibody, this can be absorbed out with washed red cells before it is used. Dextran, gelatin, and polyvinylpyrrolidone are also sometimes used.380, 665, 829 Murine antibodies generally produce only irregular and uncertain agglutination of murine red blood cells in saline media. A major methodologic contribution, which made it possible to bring the mouse into productive use for investigations of red-cell immunogenetics, was the development by Gorer and Mikulska459 of the Dextran method lor hemagglutination. In this method, the test red cells are suspended in a 1/2 dilution with saline of normal human serum, which has been absorbed with washed, pooled, murine red blood cells in order to remove any normal, human-anti-mouse red- cell antibody. We ordinarily make up the test cell suspension in saline to proper concentration and just in the amount needed, then centrifuge down the cells, aspirate off the supernatant saline, and replace it with the absorbed and diluted human serum. The test reagents, mouse-anti-mouse antisera, are diluted with a saline dilution of Dextran, the latter at a concentration of 2 percent. Unfortunately, different Dextran preparations vary in their desirability for this purpose; some of them agglutinate cells themselves without the addition of antibody in nonspecific fashion, while others fail to promote the agglutination of antibody-sensitized cells. The Dextran of choice is Tntradex, produced by Glaxo Laboratories, Ltd. (Greenford, Middlesex, England) but this is difficult to obtain. After adding a drop oi' the normal, serum-suspended, test cells to a drop of the METHODS IN MAMMALIAN IMMUNOGENETICS 859 Dextran-diluted test reagent, the cells are allowed to settle for a period of an hour or longer, and most of the supernatant fluid is then pipetted off with a capillary pipette. The sedimcnted cells and a small amount of the remaining fluid are then streaked across a microscope slide, and the slide is rocked back and forth manually five or six times. In negative tests the cells may at first appear to be aggregated, but they spread smoothly during the rocking procedure. In positive tests they remain conspicuously clumped. The technique is very sensitive to handling; negative tests may be made to appear positive if they are insufficiently rocked, or positive tests may be made to appear negative if the fragile aggregates are broken up during the pipetting procedure or through overmanipulation. With experience, however, this test becomes entirely reliable and is stil! the technique of choice for routine typing and testing of red cell antigens in the mouse, f Two other techniques for red-cell typing in the mouse, agglutination in saline medium after enzyme treatment of the red cells and hemolysis, will be mentioned below. Treatment of red-cell preparations with particular enzymes sometimes renders them subject to agglutination by antibody that does not cause the reliable agglutina- tion of untreated cells. The technique for trypsin treatment recommended by Morton and Pickles900 is essentially as follows: A stock solution of 0.1 gram of crystalline Armour trypsin is prepared in 10 ml. of N/20 HC1; this can be kept for several months at 4° C. One part of this stock solution is diluted with nine parts of M/10 phosphate buffer (/>H 7.7) on the day it is to be used. Four volumes of the diluted trypsin are added to one volume of well-washed packed cells; the mixture is held at 37° G. for \ to 1 hour. After a single additional washing the cells are made up to 5 per cent concentra- tion in saline. In Rh testing, the test sera as well as the suspensions of cells are equili- brated to 37° C, and the test is incubated at that temperature in order to avoid false positive reactions caused by cold agglutinins. In applying enzymatic treatments to other mammalian red cells, adjustments frequently have to be made for the particular system under test, in order to avoid false positive reactions. Numerous other enzymes have also been used to treat red cells; in addition to trypsin, papain and ficin are most frequently used.1388 A rapid and simplified enzyme test has come into common use in human red-cell typing. Low's technique806 is as follows: a papain solution is made up by grinding 2 g. of papain (papayotin Merck 1 : 350) in a mortar with 100 ml. of M/15 phosphate buffer, />H 5.4. This is filtered, and 10 ml. of 0.5 M cystein is added to activate the enzyme. The solution is diluted to 200 ml. with the buffer, incubated for 1 hour at 37° C, and stored at -20° G. It retains its activity over long periods under these circumstances. Three parts of this enzyme solution are mixed with one part of the test serum. Then equal parts of the serum-enzyme mixture and a 3 per cent red-cell suspension are mixed in a test tube and incubated for 2 hours at 37° C. before being read for agglutination. Hekker et fSee, however, Stimpfling, J. H., Transplant. Bull. 27:109, 1961 for a technique using polyvinylpyrrolidone. 360 IMMUNOGENETICS a/.543 add a drop of a papain solution directly to a drop of test antiserum on a slide and then add a drop of the red-cell suspension. This is reported to give an accurate and rapid test system. These and other test systems are ably described by Race and Sanger.1036 Another method of setting up and reading tests is to examine the pattern of the sedimented cells after they have settled out by gravity. This may be done in ordinary round-bottomed test tubes, or through the use of plates containing round- or sloping- bottomed depressions. Antibody-sensitized cells often behave in a nonspecifically sticky fashion so that they do not sediment to the lowest point of the depression but remain attached to the sides and bottom of the tube or depression. Frequently, agglutination is easily read under low-power magnification or even macroscopically. In such receptacles, and in cases in which the agglutinates are very fragile, positive reactions can often be identified in the undisturbed plates much more easily than they can be read by ordinary tube and microscopic methods of examination. Hildemann (personal communication) reports good results through the application of such a system to murine red-cell typing. 3. Hemolysis. Agglutinating tests are sometimes convertible to hemolytic ones through the addition of complement to the system. The most common source of complement is fresh normal guinea-pig serum, a drop of which, usually at a dilution of 1/8, added to the saline tube-agglutinating system described in the preceding section, may cause sensitized red cells to lyse. Another common source of complement is fresh normal rabbit serum, which is generally used at a somewhat lower dilution, usually 1/2. The optimal source of complement in any new system is unpredictable; for example, certain antibody reagents will lyse positive cattle red cells far better with rabbit complement than with guinea pig, whereas others work much better with guinea pig complement. Rabbit complement is best for testing mouse cells with murine antibody.574 Rabbit, guinea-pig, or other normal sera sometimes contain antibodies against the test cells and may cause the cells to lyse without the addition of reagent antibody. In such a circumstance it is often advisable to screen individual rabbits or guinea pigs in advance and to select as complement sources those whose sera lack normal antibody against the red cells to be tested. Alternatively, the normal antibody can be removed from a serum by a quick absorption at 0° C. ; we generally use a volume of packed red cells for this absorption about equal to the volume of undiluted normal serum to be absorbed, and conduct a 5-minute absorption with prechilled cells and serum and with the tubes in an ice bath, followed by centrifugation in a refrigerated centrifuge. The antibody is removed by the absorbing cells, but the complement activity is little affected. Complement is a relatively labile material; it should never be permitted to sit out at room temperature for any extended interval before its use in the tests. It is inactivated by heating at 56° C. for 20 minutes. Hemolytic systems can often be rendered more sensitive by the use of an isotonic buffer containing magnesium and calcium for the diluting and suspending medium. METHODS IN MAMMALIAN IMMUNOGENETICS 361 The buffer favored for such purposes was reported by Pillemer1006 in his studies of complement and properdin. The recipe is as follows: 85.0 g. NaCl 5.75 g. 5,5-diethylbarbituric acid 3.75 g. 5,5-diethylbarbiturate 5.0 ml. M MgCl2 1 .5 ml. M CaCl2 Dissolve in about 1500 ml. hot distilled water. Cool, add distilled water to final volume of 2 liters. Store as stock at 1 ° C. ; add one part stock buffer to four parts dis- tilled water before use; discard diluted portions after 12 hours (pH 7.4). One laboratory reports that time can be saved in setting up extensive red-cell hemolytic tests if measured amounts of diluted complement are mixed with the test serum and dropped into the tubes in a single operation, before the cells are added. Another laboratory, however, reports that this technique in some systems leads to some inactivation of the complement, and that it is better to add the complement to the sensitized cells as a final step. Hemolytic tests are often read at an interval of one-half hour after the test has been set up and shaken, in order to detect the quick hemolysis that is often characteristic of cells homozygous for the test antigen, in contrast to the slower hemolysis detected with the cells of heterozygotes. In any case, the cells are then allowed to settle for a further hour and a half before a definitive reading is taken ; often an additional reading is taken after two more hours. Readings are generally recorded on a scale from 0 to 4, 0 representing no hemolysis, in which the supernatant is free of hemoglobin deriving from the test cells. With increasing degrees of hemolysis deeper color appears in the supernatant, and the size of the pellet of sedimented cells decreases. Complete hemolysis (4) leaves no unlysed cells; the shaken tube remains a sparkling clear red. Hemolytic tests are especially well adapted to evaluating the proportions of positive and negative cells in a mixture, such as the cellular population found in a chimera. The supernatant of the hemolytic test can be read for hemoglobin colori- metrically; and after subtraction of an appropriate blank prepared to measure the contribution in color, if any, from the non-red-cell components of the test (that is, the complement and the test serum), the degree of color in the test tube can be ex- pressed as a fraction of the color in the tube containing the same amount of red cells osmotically lysed. This technique has proved useful in following the course of re- population of the erythropoietic tissues of irradiated mice after homologous bone- marrow transplants.452, 981, 985 In other systems, it is reported that more reliable values may be obtained by washing the residual cells after the specific lysis of positive cells in a mixture, then lysing these residual cells osmotically and reading the hemo- globin colorimetrically.848 4. Antiglobulin test systems. A sensitive and versatile technique for the detection of incomplete antibodies was described by Coombs, Mourant, and Race in 1945.225 362 IMMUNOGENETICS The principle of the test is as follows: Antibody globulin combines firmly with its corresponding antigenic sites on cell surfaces and remains attached during washing of the positive cells. Unbound globulin will be removed from the system during the washing. The washed cells, having the antibody on their surface if they are positive, are then exposed to antibody to the globulin itself. Reaction between the cell-bound globulin and the antiglobulin results in the aggregation of positive test cells. To illustrate the technique, our procedure (entirely derivative from the experiences of others with other systems) for the detection of individual differences among rhesus monkeys may be used as an example. The tests are set up in small agglutination tubes, as for saline agglutination. The test fluids are rabbit-anti-rhesus red-cell antisera fractionated by antibody absorption to leave reagents that react with the red cells of some monkeys and not others. Two drops of the reagent diluted in buffered saline (130 g. NaCl, 12.3 g. Na2HP04, 3.6 g. KH2P04, 17 liters distilled water, pU 7.08) are first placed in the appropriate tubes. One drop of a 2 per cent suspension of washed test red cells is then added to each proper tube. The tubes are shaken and allowed to stand at room temperature for at least one-half hour, after which they are shaken, filled with buffered saline, and centrifuged ; the supernatant is poured off and the cells are washed again with saline. To the pellet of cells remaining after pouring off the last supernatant, one drop of a 1/10 dilution of goat-anti-rabbit-globulin serum, which has been absorbed with rhesus red cells in order to remove anti-rhesus red-cell antibody, is added. The procedure for preparing this anti-rabbit-globulin serum will be des- cribed below. The test is shaken again, allowed to stand for another hour, then centrifuged briefly and read for agglutination as the pellet of centrifuged cells is shaken back into suspension. With this test system, antibody reagents that produce little or no reliable agglutination of positive test cells in saline become very sharply discrimi- nating. The type of antiglobulin serum used in this kind of test depends, of course, on the source of the cell-sensitizing antibody. In the rhesus test described above, since the test antibody was rabbit, the Coombs antiserum was anti-rabbit globulin, in this case prepared in a goat. For test systems in which the cell-sensitizing antibody is other than rabbit, it is commonly the rabbit that is used as the source of the Coombs reagent. For example, sheep cells and rat-anti-sheep red-cell antibody can be used in a Coombs system if rabbit-anti-rat globulin is used as the developing reagent. Similarly, in human tests, where the Coombs system has become a very important test procedure, it is rabbit-anti-human globulin that is generally used as a Coombs reagent. In preparing the Coombs reagent, first a predominantly globulin fraction is salt- precipitated from an immune serum — for example, to prepare an anti-rabbit-globulin serum in a goat, we first take a rabbit antiserum to bovine serum albumin which we know to contain a good deal of antibody and precipitate this antibody by the addition, at room temperature, of an equal volume of saturated ammonium sulfate solution to a 1/2 dilution of the serum in saline. After adjustment to pH 7.8 with 10 M NaOH, the precipitated globulin is centrifuged out and redissolved in saline. For this purpose, METHODS IN MAMMALIAN IMMUNOGENETICS 363 we do not ordinarily purify it more than one further precipitation and resolution, and we recognize that we have more than y-globulin in this preparation. We then alum- precipitate the protein for injection according to the following procedure. Dissolve the globulin prepared as above from 40 ml. of rabbit serum, in 25 ml. buffered saline. Add 80 ml. distilled water, then 90 ml. of 10 per cent potash alum in water. Adjust with NaOH to pH 6.5. Let stand ten minutes, centrifuge and wash once with saline. Resuspend in 15 ml. buffered saline, and divide into three equal aliquots. Inject one (intramuscularly) ; freeze the other two aliquots to be injected (i.m.) respectively one and two weeks later. Bleed after an additional two-weeks' rest. The goat can be restimulated by injecting globulin in solution, or even by injecting whole rabbit serum. Alternatively, the globulin or whole serum can be injected in Freund's adjuvant. The procedure we use is essentially that described by Munoz.919 To 2 parts of paraffin oil (Drakeol 6-VR, obtained from the Pennsylvania Refining Company, Butler, Pennsylvania), containing 2 mg. of heat-killed and lyophilized M. tuberculosis (we use an avirulent strain H37Ra, obtained from Dr. Sidney Raffel of Stanford University), is added one part of Falba (obtained from Pfaltz and Bauer, Incorporated, Empire State Building, New York 1, New York). After autoclaving, this mixture is taken at 3 parts of the sterile adjuvant to 2 parts of the salt-precipitated globulin. If this is, for ex- ample, mouse globulin to be injected into a rabbit, we prepare three ampoules, each with 1 ml. of the adjuvant-globulin mixture, and inject the contents of one ampoule subcutaneously once each week for three weeks, then bleed after a rest of two weeks. There are other methods for making antiglobulin sera and conducting anti- globulin tests, some of them better for particular systems than others. References, and a quick and clear survey of the field, can be found in Race and Sanger's book.1036 Coombs and Roberts226 have published a brief review, including further applications and adaptations of the method. 5. Elimination rates of labeled cells. A useful serologic technique is based on the accelerated clearance of labeled antigen from the circulation of immune animals. With soluble antigens, this is one of the more sensitive and convenient indicators of the immune state.205, 278 The method has found little application in immunogenetics, although it offers promise. Some use has been made, however, of the clearance of labeled red cells, and the observations at hand should stimulate further study. The label of choice appears to be Cr51. Mollison,888 who has contributed actively to this area, has reviewed the methodology recently and has called attention to rather frequent evidence of incompatibility based on reduced survival of Cr51-labeled human cells injected into normal human recipients in whose serum no incompatible antibodies could be found. An example of fruitful application of a similar technique to problems of immunogenetic incompatibilities in mice is a paper by Goodman and Smith.453 6. Tests on mixtures of cells. As mentioned earlier, the saline-agglutination system, hemolysis, and the antiglobulin system (in the case of rhesus red-cell mixtures) 364 IMMUNOGENETICS have all been used for the quantitative identification of cellular mixtures in chimeras deriving from either natural or experimental transplants of erythropoietic tissues. In some mixtures, however, only minute quantities of positive cells may be present in an overwhelming predominance of negative cells, or, in the reverse situation, very small numbers of nonreactive cells may be present among very large numbers of positive ones. Several techniques have been developed to evaluate these minute populations. No detailed description of techniques will be attempted here, but reference should be made at least to an isotope-dilution method together with use of plant lectins for the selective removal of positive cells;36 to the "mixed agglutination" found with detector cells added to a suspension ; 666 and to the potential utility of antibody marked with a radio- active tracer. For a general discussion of this subject, see Cotterman.228 Preserving red cells. — Contamination of blood samples can be controlled by adding certain antibiotics. In a procedure described by Cahan,154 200,000 units of penicillin G (crystalline-potassium) is dissolved in 100 ml. of a citrate solution (6.0 g. sodium citrate, 7.0 g. sodium chloride, 1 liter distilled water). One gram of Streptomycin sulfate (Squibb) is dissolved in 100 ml. of a phosphate buffer (16.4 g. Na2P04-7H20, 5.36 g. NaH2P04H20, 1 liter distilled water). Stone and Beckstrom1284 report that, by using 0.25 ml. of the penicillin solution and 0.1 ml. of the streptomycin solution in the anticoagulant provided for 10 ml. of whole cattle blood, the blood can be shipped for long distances without refrigeration and can be stored under refrigeration for extended periods. Blood cells can also be stored frozen for years in proper media. A technique effective for cattle erythrocytes has been described by Stone et a/.1285 Whole blood warmed to 37° C. is centrifuged, and the plasma is removed and replaced with 40 per cent ethylene glycol at 37° C. made up in a 6 per cent sodium citrate solution. After mixing, the blood is put into plastic tubes, stoppered and stored at —20° C. When needed, the blood is thawed at room temperature or 37° C. A minimum of 4 ml. is centrifuged and the supernatant discarded. The cells are resuspended in an excess of 20 per cent ethylene glycol, centrifuged, and the procedure is repeated with solutions of 10, 5, and 2 per cent ethylene glycol in sequence. The cells are then suspended in 0.9 per cent saline. Although many of the cells may lyse during this procedure, enough remain for typing, and they type normally after periods of storage in excess of two years. For some species, glycerine or glycerine and plasma provide excellent media for freezing and preserving red cells and other tissues.195 For others, such as cattle, the ethylene-glycol method described above seems to work much better. Several papers on red-cell preservation were included at a meeting of the American Association of Blood Banks in San Francisco in August, 1960, and abstracts appear in the published proceedings of that meeting. Tests on contaminated blood samples should be regarded with caution, because particular types of bacterial contamination may change the test reactions with particular reagents.1287 METHODS IN MAMMALIAN IMMUNOGENETICS 365 OTHER CELLS IN SUSPENSION Although suspensions of red blood cells have provided the most convenient material for mammalian immunogenetics in the past, increasing attention is being given to the antigenic characterization of other types of cells as well. Some of the red- cell specificities seem to be limited to the erythrocytes; others are found in or on diverse types of cells and tissues. Particularly in man, a variety of white-cell antigens has become available for immunogenetic analysis. In some instances, these are of significance in systems of maternal-fetal incompatibility, comparable to those involving the Rh and ABO red-cell antigens.152- 586- 744- 1071 Terasaki1317 has described a procedure for obtaining rather pure lymphocyte suspensions from chicken blood, and for conducting agglutination tests with lymphocyte suspensions. Agglutinating test systems have also been applied to mammalian leucocyte suspensions; in the past, however, such tests have often given inconsistent results.242 Absorption tests with A and B human white cells have given straight- forward results, as have such tests with spermatozoa, though sperm are not agglutinated directly by anti-^4 or anit-i?.750 Race and Sanger1036 report that the ability of spermatozoa to absorb anti-^4 and anti-5 antibodies is not removed by as many as nine successive washings of the sperm, indicating that this property of the spermatozoa is probably not simply adsorbed from the seminal fluid. Kiddy and his colleagues706 have reported rather extensive tests on the antigenic qualities of rabbit sperm. The sensitive and versatile technique of mixed agglutination was reported by Coombs and Bedford in 1955.223 Essentially, it involves sensitizing test cells with anti- body, then adding red cells of known type. If the test cells adsorb the antibody to their surface, red cells of the corresponding type cluster on the surfaces of the sensitized cells, whereas failure of the test cells to combine with the antibody results in their failure to accumulate a shell of the corresponding red cells. This technique, with relatively minor modifications, has been used to type human epidermal cells,224 cells in tissue culture, and other cells. A basically similar procedure was reported by Gull- bring511 to fractionate sperm-cell suspensions from AB men; anti-^4 sensitized A red cells combined with part but not all of the sperm, leaving an unreactive fraction, and anti-5-sensitized red cells had a similar effect. The question of whether a sperm cell may express its own antigenic genotype, however, remains open at present. Another useful and versatile technique depends upon the cytotoxic effects of anti- body on cells, especially in the presence of complement.456 Several procedures are applied ; the one we use, based entirely on the experiences of others and most directly from Vos et a/.1351 is as follows: Murine spleen-cell suspensions are prepared by pressing the spleen through a fine stainless steel screen, and suspending in Tyrode's solution. Small clumps are suspended by flushing the suspension in and out of a syringe with a 22-gauge needle. One drop of the cellular suspension is mixed with one drop of murine antiserum or normal serum. One drop of rabbit complement, which has been absorbed with red cells of the mouse in the cold in order to remove normal rabbit-anti- 366 IMMUNOGENETICS mouse antibody, is then added. The mixture is incubated at 37° C. for 30 minutes. After incubation the tubes are kept at 4-6° C. until the ratio of viable to nonviable cells is determined. The suspension may be stored at this temperature for as long as six hours. Immediately before viability is determined, the cells are resuspended with a capillary pipette and the reaction mixture is allowed to come to room temperature. To one drop of the mixture is added one drop of 0.4 per cent eosin in Tyrode's solution and in a hemocytometer stained nucleated cells are counted as nonviable. Non- stained nucleated cells are counted as viable. About 100-200 cells are counted from each tube ; the degree of cytotoxicity of the antiserum is expressed in terms of the fraction of nonviable cells observed, in comparison with controls treated only with normal serum and complement. Standardization of the procedure, particularly in terms of the numbers of cells in the suspension, the various dilution factors, and the number of units of hemolytic complement added, is essential for quantitatively repeat- able results. (Dr. Winn comments on this subject in more detail at the end of this chapter.) An interesting point has been made by Amos and Wakefield:21 although mouse complement is ineffective for cytotoxic action of murine antibody on murine cells in vitro, similar cells are lysed in diffusion chambers in vivo without the necessity of added complement. In some instances, the effects of antibodies on mobile cells provide a good index of serologic reaction. Unfortunately, we have not yet encountered in mammals a system as sensitive and productive in this regard as the antibody-immobilizing systems of the Ciliates. Mammalian sperm-cell suspensions, for example, do not generally appear to be sensitive to antibody immobilization. The ameboid motions of some of the leucocytes, however, are affected by antibody, and this has provided a test system of some utility. SOLUBLE ANTIGENS Molecules in free suspension or solution in the body fluids, or obtainable in extracts, should provide a rich source of material for the immunogeneticist. Until recently, however, techniques for working with the serology of soluble antigens could cope only with extreme difficulty and uncertainty with complex antigenic preparations. A rather high degree of purification by chemical or physical methods was a usual prerequisite. A number of powerful methods are now available for working with mixtures, and we can sample this active area only inadequately here. Inhibition systems. — Materials having a specificity related to one of the red-cell antigens can easily be tested, taking advantage of the red-cell reaction as an indicator. The general procedure is as follows: A determined amount of antiserum, say antwl, is mixed with a measured amount of the solution under test, such as saliva or a saline extract. After an interval, A cells are added to the mixture. If the test material had A specificity, it will have combined with the anti-yl antibodies, and thus will inhibit METHODS IN MAMMALIAN IMMUNOGENETICS 367 them from reacting with the A test cells. If, on the other hand, the test material lacked A specificity, the antibodies will not be inhibited and will therefore promote the agglutination of the test cells. This is the kind of system used for the definition of the blood-group substances in animal plasmas and other fluids. The techniques have been discussed in detail recently by Boyd,121 and will not be further elaborated here. Enzymatic inhibition. — Enzymes under genetic control are often excellent antigens and the effects of antibodies on enzymatic preparations are sometimes useful in dis- tinguishing genetic differences. This approach has been used more productively with microorganisms than with mammalian material in the past, but there is no reason why it should not be useful in mammalian genetics as well. An antiserum is prepared by injecting an enzymatically active material into an animal, generally a rabbit. The antibodies that are formed may precipitate the enzyme from solution; the enzymatic activity provides a sensitive and convenient tag for the removal of the enzyme from the supernatant by such precipitation. Precipitin tests will be discussed below; we will only note here that the complications of complexity in mixtures are to a consider- able degree avoided in enzyme serology, because of the easy identification of the particular antigen, the enzyme, in the mixture by means of its activity. In many but by no means all enzyme-anti-enzyme systems, the antibody may inactivate the enzyme without precipitating it. Under such circumstances, tests for enzymatic activity in mixtures can be made immediately after the addition of antiserum and substrate to an enzyme preparation; the results are quickly and easily read if convenient measures of enzymatic activity are at hand and if there is no interference by nonantibody serum factors in the assay. Coupling antigens to red cells. — Erythrocytes to which soluble antigens have been coupled are often endowed with the property of agglutinating or giving other visible reactions with antibody to the test antigens. A common technique is the use of "tanned" red cells; the procedure we use is based on a report by Stavitski,1270 and is derived from the original report by Boyden.123 Sheep blood in Alsever's solution is washed three times with saline, and 1 ml. of the packed cells is diluted with about 40 ml. of/>H 7.2 buffered saline, so that 1 ml. of this diluted cell suspension plus 5 ml. of dis- tilled water gives a reading of 400 with a no. 54 filter in the Klett colorimeter. The buffered saline, pH 7.2, is prepared by diluting 100 ml. of a buffer containing 23.9 ml. of 0.15 M KH2P04 and 76.0 ml. of 0.15 M Na2HP04, with 100 ml. saline. A stock solution of tannic acid (Merck or Mallinckrodt reagent grade) is diluted with saline, 1/100. A further dilution, to 1/20,000 of the acid, is prepared daily. One ml. of the cell suspension is incubated in a water bath at 37° C. for 10 minutes with 1 ml. of the 1 /20,000 dilution of tannic acid. The cells are then centrifuged gently and washed with 1 ml. of pH 7.2 buffered saline, resuspended in 1 ml. saline. The treated cells cannot be kept more than 1 8 hours before use. The antigen is prepared by mixing 4 ml. of/?H 6.4 buffered saline with 1 ml. of the antigen solution in saline and 1 ml. of the tannic-acid-treated suspension of cells, in this order, and allowing the mixture to stand at room temperature for ten minutes. 368 IMMUNOGENETICS The cells are then centrifuged, washed once with 2 ml. 1/100 normal rabbit serum, and then resuspended in 1 ml. of 1/100 normal rabbit serum. The saline at pH 6.4 is prepared by adding 100 ml. of saline to 100 ml. of a buffer composed of 32.2 ml. of 0.15 M Na2HP04 and 67.7 ml. of 0.15 M KH2P04. ThepH should be checked on a pH meter and adjusted if necessary with either phosphate solution, 0.15 M. Many antigens work in this preparation, in mixtures as well as in pure solution. Optimal amounts for sensitizing cells vary around 0.25 mg. of protein or other antigen per ml. treated red-cell suspension. Compounds other than tannic acid are used as coupling agents. For formaldehyde methods, see Ingraham631 and Czimas.235 The hemagglutination of antigen-coupled red cells is a very sensitive technique, acceptably specific if proper controls are run. Precipitation system. — Classical precipitation systems have been described for single, relatively pure, proteins in solution, in terms of reactions with their corresponding antibodies. Typically, a constant amount of antiserum is placed in each of a series of tubes, and increasing amounts of antigen are added to the tubes in sequence. The classical precipitin curve rises to a maximum at some intermediate antigen quantity, then decreases in antigen excess. Optimal relative concentrations are expressed in various ways — in terms of the rapidity of appearance of a visible precipitate, in terms of a maximum quantity of precipitate formed per mg. antibody nitrogen, and in terms of an equivalence zone within which all of the detectable antigen and antibody are included in the precipitate, none remaining in the supernate. These measures of central tendency do not usually coincide; on either side of a rather broad central zone, however, flocculation times increase, the quantity of precipitate decreases, and either antigen or antibody begins to be detectable in the supernatant fluid after precipitation is complete. Precipitin tests of this sort are subject to quantitative treatment through measure- ment of the amount of precipitate by nitrogen determinations or other methods. A chromatographic technique for the quantitative study of the precipitin reaction, especially adaptable to very small amounts of serum, has recently been described by Miquel et a/.875 Absorptions can be conducted, in simple systems, by reacting the test antigen with a given antiserum at relative concentrations within the equivalence zone, removing the precipitate and using the partially absorbed supernatant as a further test reagent. Somewhat different results are sometimes obtained if, instead, the antigen is added in small increments, the precipitate being removed after each addition, until precipitation no longer occurs. In the past, there has been a tendency on the part of some geneticists familiar with only the elements of immunology to assume the properties of a simple antigen-antibody system for complex mixtures of indeterminate numbers of related and unrelated antigens, and correspondingly complex antisera. Unfortu- nately, the system becomes very uncertain under such circumstances; two or more systems, precipitating in a single tube, are not often at equivalence within the same zone, so that antibodies to one may remain in the supernatant after the other has passed into a region of antigen excess. Soluble complexes for the second system are METHODS IN MAMMALIAN IMMUNOGENETTCS 369 therefore not removed from the absorbed "reagent." The uncertainties of the system probably increase exponentially with the number of components. Unless one is able to purify his antigenic preparation to an essentially single component basis, therefore, or unless he is working with an antigen that is certainly tagged, by something like enzymatic activity, a unique absorption spectrum, or a tracer label that it does not share with other molecules in the solution, or serologic specificities detectable by red- cell tests, there is nowadays little justification for conducting immunogenetic work on soluble antigens in mixtures by means of ordinary saline precipitation methods. The simple precipitin system, however, has probably contributed more to our basic know- ledge of serology than has any other. Readers are referred to discussions of this system from a methodologic viewpoint, for example, by Kabat and Mayer673 and Boyd.122 Gel-diffusion serology. — A new era in the immunogenetics of soluble antigens was ushered in by the development of gel diffusion methods by Oudin and Ouchterlony. These methods make use of the diffusion of antigen or antibody or both into a gel medium, usually agar. Since the different components of an antigenic mixture generally diffuse at different rates, this technique permits the separation of com- ponents of a mixture in a system comparable in some ways to chromatography. In the Oudin method974, 975 agar containing a known serum is allowed to solidify in a small tube, and then an aqueous solution of the antigen is poured over the solid agar. As the antigen diffuses into the underlying antibody gel, it forms a zone of precipitate for each antigen-antibody pair present in adequate quantity. The Ouchterlony method is a system of double diffusion ; the antigens diffuse through an intervening agar zone, to meet a front of diffusing antibody. Where corresponding antibody and antigen meet, a narrow line of precipitate is formed; and, when the antigenic preparation is a mixture, a number of bands form, each corresponding to a particular antibody-antigen system at positions related to the diffusion rates of the antigenic components and to the relative concentrations of the antigens and antibodies. This system is subject to quantitative analysis.14, 349 The method we use, adopted directly from G. J. Ridgway of the Bureau of Com- mercial Fisheries, Seattle, is as follows. An agar base is prepared by mixing 1.5 per cent Difco agar, 0.72 per cent NaCl, 0.6 per cent sodium citrate, and 0.01 per cent Merthiolate, in distilled water. This solution is adjusted to pH 6.7 with HC1 before the addition of trypan blue (to 0.01 per cent) while the agar is still hot. It is then poured into test tubes, 8 ml. per tube. In setting up the tests, one tube of the hot agar base (kept in a boiling water bath) is poured into a flat-bottomed petri plate. The plate should not be swirled to distri- bute the agar. When the plate is cool, penicylinders (obtained from Fisher Scientific Co.) are arranged in a pattern on the plate. We commonly use a porcelain peni- cylinder at the center and stainless steel penicylinders arranged at the points of a regular hexagon so that each is equidistant from the center. The distance from the center of the central penicylinder to the center of each peripheral one is 2 cm. Shreffler, 370 IMMUNOGENETICS in our laboratory, has prepared lucite guides which fit on the petri plate, with holes bored in the proper position just large enough to drop the penicylinders through to the agar surface. After the penicylinders are in position, another tube of 8 ml. of the hot agar base is poured around them. Again, the plates should not be swirled. The antiserum is then placed in the central penicylinder, filling it, and the test antigens are placed in sequence in the peripheral ones. Plates are sealed with rubber tape and placed at 37° C. for about a week (the time necessary will vary with the anti- gens and the serum) . The precipitin lines that form are then photographed ; a modified dark-field type of illuminator producing good photographs has been described by Klontz et al.72e The trypan blue in the agar, and the porcelain central penicylinder, improve the quality of photographs. Numerous variations of both the Oudin and Ouchterlony procedures have been described. Preer1021 has described a method adaptable to the use of very small quantities of material. Many investigators prefer to use molds of various design and to pour the agar medium for gel diffusion tests around the molds. When the molds are removed, wells remain in the solidified agar. Sets of agar-gel cutters for various purposes, together with descriptions of their uses and references, can be obtained from Shandon Scientific Co. Ltd., 6 Cromwell Place, London. Gel-diffusion methods have made possible the first extensive and definitive work with the immunogenetics of mammalian soluble antigens in precipitating systems, for example, genetically controlled variations in the specificity of rabbit serum globu- lins974, 975 and the sharp inherited differences in the quantity of a serum protein distinguishing particular inbred lines of mice. Immunoelectrophoresis. — Another powerful technique for dealing with the serology of mixtures has been developed mainly by Grabar472 and extended by many others. Essentially, the procedure first subjects the antigenic mixture to electrophoresis. After the electrical field has separated the components of the mixture along a linear axis as a function of their charge at the particular pH and ionic strength of the medium, the components of the mixture are permitted to diffuse through the agar to meet a front of diffusing antibody, so that a band of precipitate forms where antigens and their corre- sponding antibodies meet. The preliminary electrophoretic step provides an additional dimension of separation of the antigenic components ; the technique has been proved especially effective for the discernment of many components in very complex mixtures, such as whole serum or plasma. Bussard150 has described a useful modification, in which the precipitation occurs during the course of electrophoretic migration. A combination of starch-gel electrophoresis with gel-diffusion serology has been reported by Schwartz.1178 Starch gels, after the method of Smithies,1229 generally make cleaner electrophoretic separations than do agar gels, and the addition of anti- body precipitation to this system is a powerful technique indeed. Complement fixation. — A classical serologic procedure of high sensitivity and specifi- city is the system of complement fixation. In principle, the system is simple: an antigen is allowed to react with its corresponding antibody in the presence of comple- METHODS IN MAMMALIAN IMMUNOGENETICS 371 ment. In many instances, such reactions "fix" complement if it is present, even though the complement may not be necessary for the reaction to occur. After this phase of the test, in which the antigen-antibody reactions occur, a second indicator system is added in order to determine whether complement has been fixed. The indicator system is usually sheep cells which have been sensitized with rabbit-anti- sheep antibody. If complement is present, the cells will hemolyze; if it has been fixed by a preceding antigen-antibody reaction system, it will not be available to the hemo- lytic system and the cells will fail to lyse. The sensitized sheep cells, therefore, provide a measure of whether or not an antigen-antibody reaction has occurred in the test system. Techniques of complement fixation are highly sensitive, and are subject to modifi- cation by many external factors. Careful controls must be run ; the proper amount of complement must be added; the indicator cells must be properly sensitized, and so on. Descriptions of complement fixation procedures are to be found in Kabat and Mayer673 and Boyd.122 In general, although this system is a useful one in serology, it has found relatively little application in immunogenetics, particularly of mammalian materials. TISSUE TRANSPLANTATION Consideration of tissue transplantation will be limited to studies of normal rather than neoplastic tissues and to only a small selection from this active area. The method- ology of tumor-transplantation is considered elsewhere in this volume. Skin grafting. — The standard method for skin grafting, especially in mice, was developed by Medawar and his group. 98a The following technique, which we use in our laboratory, is essentially Medawar's. Donor mice are usually killed by the intraperitoneal injection of 0.1 ml. of Nem- butal sodium, 50 mg. per ml., which is commercially available. Alternatively, if not too many grafts are to be removed, the donor mice may be anesthetized by the injection of 0.1 ml. per 10 g. body weight of a 1/10 dilution of the above Nembutal preparation. Different lines of mice vary somewhat in their sensitivity to the anesthetic, and the first mice to be injected should be carefully observed, with the idea that slightly less or more anesthetic may be desirable for the line in use. After the donor mouse has recovered from anesthesia, the sites from which the grafts are taken may be left open; they will heal rapidly. If the donors have been killed they can be pinned out on a board; if they have not been killed they can be tied or taped down. The back of the animal, from the ears to the tail, is clipped closely ; we use an Oster small-animal clipper, model 2, with a no. 40 head. The surface of the clipped skin is then swabbed thoroughly with Zephiran (1/1000 in 50 per cent alcohol). An area of the skin of the donor is elevated in a small tent, with a small, very fine pointed forceps, the tips of which have been curved toward each other to meet at a single sharp point. As the skin is held under tension, it is cut from beneath with a 372 IMMUNOGENETICS concave (no. 12) Bard-Parker scalpel, pulling the scalpel against the firmest skin attachment and curving the cut back up to create a circular graft about 1 cm. in diameter and with even margins. About a dozen grafts can be taken from a single donor, if he has been sacrificed. Each graft is then placed with its dermal side down- ward on sterile filter paper, moistened with sterile physiological saline in a sterile petri dish. Only the dorsal skin is used, but this can extend to the base of the tail and the ears and along the sides of the animal. After the grafts are all in the petri dish, spread face downward, the donor is dis- carded or placed aside to recover, and the grafts are one by one turned dermal side upward and the panniculus carnosus is carefully scraped away with a dull, straight scalpel. To do this, a pointed forceps is used to press down the graft at a point near its margin, and the panniculus can be pulled loose in a single piece. Care must be taken not to injure the underlying dermis, and the last evidences of fat should be scraped away, leaving a thin, even graft. This is again turned dermal side down. The recipient mouse is anesthetized, and an area of his dorsal surface on the right side in the chest region is clipped and disinfected with Zephiran solution. Use the lower margin of the dorsal rib cage and the scapula as markers. It is convenient to graft on the right side of the midline, reserving the left side for an autograft or a later graft. Using a fine, sharp, curved scissors carefully pinch off an area of the skin and clip it with the scissors. If this is done properly, the skin through the dermis will be removed in a narrow slit, exposing the recipient's panniculus carnosus. This will be evident by its glistening surface with intact blood vessels running through it. The recipient area is then cleared by cutting away the skin with the scissors at the margins until an area somewhat larger than the prospective graft is exposed. There should be a margin of free surface (1 or 2 mm.) around the graft when it is in place. A selected graft is then put in place, being careful that the dermal side is down, and it is then covered with a piece of vaseline-impregnated, rather fine gauze, about \ inch by | inch. The gauze should have been prepared in quantity and sterilized earlier. It is convenient to sterilize the gauze in large rectangles and then to cut it as required in the petri dish. All of these manipulations are with sterile forceps and scissors. Care must be taken to cover the entire recipient surface with the gauze and not to disturb the graft from this point forward. A piece of plaster-of-paris bandage (we use Gypsona) about 7 inches in length and -f inch wide is moistened and the excess moisture shaken free. These pieces have been rolled on small lengths of plastic tubing about an inch long, secured with a small rubber band and stored in jars. There is no need to sterilize them. The moist strip is first unrolled and placed over the graft area so that an inch or more projects to the animal's right. The main roll is then passed under the animal's body and returned to the right side ventrally, and the dorsal projecting strip is grasped along its full width with a forceps. Tension is exerted on the lower strip while the forceps holds the upper strip firmly in place so that it does not slide over the vaseline gauze. After the first complete loop, the forceps is carefully released and pulled out METHODS IN MAMMALIAN IMMUNOGENETICS 373 gently. Then the bandaging is continued under tension, so that the animal is tightly bandaged. At the end, the bandage is moistened with a finger and rubbed to give a smooth and finished surface. The animal can then be set aside for the bandage to dry and if necessary the bandages can be marked with dots of dye to distinguish individuals or experiments. It is helpful to keep the mouse warm until his recovery from the anesthetic is complete. The bandage should not be removed for six days, and unless one is looking for established immunity (a second-set response) it is better to leave the bandage in place for eight or ten days. If the bandage is removed at six days, it is often wise to re- bandage until the eighth day to prevent damage to the skin by scratching. After that time the grafted area can be left open and examined daily for the end point of complete graft destruction. Early evidences of reaction include an easy peeling of the epidermis to reveal the glistening surface of the dermis below.93 Total excisional biopsy and microscopic examination is desirable to confirm end points and immune processes. Several short techniques for skin grafting in mice have been described, such as the use of a tissue punch to take the skin from the donors and to prepare the graft bed. In our hands, and those of others, however, these techniques have not always proved successful, perhaps because of the loss of the panniculus as a graft bed, and in our opinion it is preferable to follow the established and fully successful methodology described above. Transplantation of other tissues. — A number of tissues and organs other than skin have been successfully transplanted in small laboratory mammals. Procedures for ovarian transplants were described a number of years ago by Robertson.1063 Thyroid and adrenal transplants in the rat have been adapted to convenient experimental study by Woodruff and Sparrow1403 using the localization of injected radioactive iodine as an index of thyroid graft survival and function. Techniques for the transplantation of other tissues will not be referred to here, except for a later consideration of the trans- plantation of hematopoietic tissues. The Transplantation Bulletin is a rich source of reference materials. Parabiosis. — Several important compatibility studies and techniques depend on the use of parabiotic animals, generally mice or rats. A successful technique for parabiosis of mice has been described by Eichwald et at.319 Rubin1079 has suggested that somewhat different results may be observed depending on the nature of the surgical union obtained through different techniques of parabiosis. Finerty362 gives extensive references to earlier work with parabiosis, especially in the rat. Tolerance, paralysis, and enhancement. — Various methods of suppressing immune responses have come to attention in recent years. These include the tolerance to homologous tissue transplants induced in an animal through the injection of viable homologous cells when he is newborn or an embryo; the suppression of immune responsiveness in an adult by injection of large amounts of the antigen, especially effective with polysaccharides (paralysis) ; and the diversion of the immune response in the direction of a type of antibody that suppresses rather than promotes graft rejection 374 IMMUNOGENETICS (enhancement). Techniques for these essentially immunologic systems, of considerable genetic interest, will not be elaborated here; references to most of the current pro- cedures will be found in an earlier review by Owen.986 Bone-marrow transplantation. — There is a great deal of activity currently in the area of hematopoietic transplants into irradiated adult mammals, especially mice.393 The genetically controlled incompatibilities between host and graft in this system are of particular interest and importance. Brief mention will be made here of the essential and most-used techniques. Donor mice are generally killed by bleeding them to death, often by decapitation; there is reason to believe451 that exsanguination of the donor may reduce the number of a type of cell in peripheral blood that may have an unfavorable effect on the host into which the bone marrow is to be injected. The mice are pinned or taped securely on a board and each femur is exposed, dissected out, and transferred to a petri dish where the femurs are scraped free of adhering tissue and rinsed with Locke's solution. Generally, the dosage of bone marrow to be used will approximate one or two femurs per recipient. Both ends of the femur are cut off with a scissors, and the marrow plug is forced out, using Locke's or Tyrode's solution in small quantity to flush out the marrow canal with a 22-gauge needle and a 2 ml. syringe. The marrow is then suspended evenly by filling and emptying the syringe, forcing the suspension through the needle several times, and the nucleated cell count is taken in a hemocytometer using crystal violet in 1 per cent acetic acid as a stain. The suspension is adjusted so that it will contain the desired count of nucleated cells per \ ml., the quantity injected intravenously into each recipient. Radiation conditions for the recipients are specified in publications in the field.393 Several techniques have been applied to follow the course of repopulation of the erythropoietic tissues.983 Embryonal hematopoietic tissue, especially embryonic liver, is often substituted for adult bone marrow. It seems to have some advantages, in terms of its lesser tendency to produce a delayed incompatibility reaction and in terms of its ability to transplant, at least in genetically anemic recipient mice, even without irradiation of the host unless strong histocompatibility barriers are present.1099 The main genetic tools for studies of transplant incompatibility have been the in- bred lines of mice, their hybrids and segregating F2 and backcross generations.983 The development of coisogenic lines differing only at specified loci affecting histo- compatibility, by George Snell at the Jackson Laboratory, has provided the most precise information about immunogenetics in this important area, and the most useful materials for its advancement. DISCUSSION Dr. Burdette: Dr. Henry J. Winn of the Jackson Laboratory will open the discussion of Dr. Owen's paper. METHODS IN MAMMALIAN IMMUNOGENETICS 375 Dr. Winn: Dr. Owen's paper is a very impressive manuscript in which he has skillfully interwoven the complex terminology and methodology of immunology with what I consider the equally complex terminology and conceptions of genetics. As most of you know, the methodology of immunogenetics would have been a great deal simpler to discuss about ten to fifteen years ago. At that time it consisted largely of the study of the interactions of serum antibodies with intact red blood cells. Now the very rapid growth and development of the field of tissue transplantation has changed all this, and we find ourselves studying a very large variety of reactions in- volving not only serum antibodies but cellular or cell-bound antibodies. For the most part the work that has been done with the cell-bound or cellular antibodies does not really lend itself to any particular experiments that I know of in immunogenetics. Generally, this work has been directed to some understanding of the mechanism of graft rejection or the relationship, metabolic or otherwise, between soluble and cell- bound antibodies. Our interest in the cytotoxic technique came about because of the limitations of the red-cell agglutination test. In the mouse one finds that the red-cell agglutination technique devised by Gorer and colleagues makes a really fine tool for the study of antigens controlled by the genes at the H-2 locus, but there are some fourteen or fifteen or maybe twice that number of loci which control the acceptance or rejection of grafts. We had hoped that by studying the effects of antibodies on white cells we might be able to analyze the antigens controlled by the genes at these other loci. The technique is relatively simple. One mixes antiserum with white cells from either lymph nodes or spleen (for some reason we do not completely understand, thymus is not suitable). To this mixture is added measured amounts of complement, and after a suitable period of incubation some vital dye to determine how many of the cells are dead. Gorer has done some work on this and Schrek has also. For the most part they have added enormous quantities of complement to mixtures of undiluted or only slightly diluted serum and cells. We wanted to use the technique in a more quantita- tive fashion, and we decided to standardize the requirement for complement and anti- bodies. The longer we worked at standardizing the technique, the more difficult we made it, and we soon reached the point where in one day we could analyze only a single serum. (With the red-cell agglutination technique I estimate something over a hundred serums could be analyzed in one day.) It occurred to us that one of the problems we had was the fact that this system required an enormous amount of complement in terms of the amounts that are normally used to lyse red blood cells, described by Dr. Owen. This suggested that instead of going through this very laborious procedure of incubating the cells and obtaining differential counts for each tube, we might actually add a very large excess of complement and measure the amount that was used up. This has worked out very nicely and we are now using a test which is patterned after that used by Osier and his colleagues for the study of soluble proteins and carbohydrates. 376 IMMUNOGENETICS I would like to mention just one of the things we have been able to do with this test. Earlier I had mentioned that thymic cells are not suitable for cytotoxic tests, because the cells are not killed when one adds antibody and complement. Based on some preliminary absorption tests, we had postulated that this was because there were not enough antigen sites on the thymus cells and they were not fixing enough comple- ment. Using the quantitative complement-fixation test, we have shown that this is indeed the case; the thymic cells mixed with antibodies (even when the antibodies were made against the thymic cells) fixed far less complement than preparations of cells taken from either bone marrow, lymph nodes, or spleen. The cytotoxic technique and the complement fixation test may actually be applied to peripheral blood, and if this could be done with humans, it could con- ceivably open up the possibility, brought out by Dr. Russell, of typing human blood for histocompatibility factors. Dr. Snell : Dr. Owen and Dr. Winn have given us a very adequate summary of some of the methods of immunogenetics. There is little I could add. I am not an immunologist, but I have acquired some knowledge secondhand from other people working here. It has interested me to see, over the past ten to fifteen years, how the mouse has finally come to be used as a tool in immunology. Twenty-five years ago about all one could find in the literature were a few unsuccessful attempts to find blood groups in mice. The results were always negative. Now the mouse is very much in business. Of course one presumed the defect of the mouse originally was its small size. Here at the Jackson Laboratory we circumvent that now by using large numbers of mice. I mention one additional technique to illustrate some of the tricks of the trade. Dr. Owen mentioned absorption as a method of obtaining an antiserum with a single specificity. That is a somewhat messy and time-consuming technique, and rather particularly so in mice, in which the available amounts of serum and tissue are usually small. Also the resulting antiserum may be heavily contaminated with tissue proteins. A very simple and satisfactory alternative is absorption in vivo. One simply injects the mice intraperitoneally with antiserum and then bleeds them perhaps an hour later; the antibodies reactive with the recipient tissues are absorbed in vivo. The antiserum may be diluted about one in ten, reducing the titer from about one in a thousand to one in a hundred, but the antiserum is still usable for most purposes. Dr. Dray: Dr. Owen referred to some of the newer methods of immunochemistry pertinent to mammalian genetics which I believe merit further emphasis. During the past fifteen years, two very highly significant advances, agar-gel immunochemical analysis and allotypy, have developed from the work of Dr. Jacques Oudin at the Pasteur Institute. In 1946, Oudin discovered that when a mixture of antigens in solution diffuses into a gel containing a mixture of precipitating antibodies, the multiple bands of precipitate which result may be explained as due to the different antigen- antibody systems present rather than to the Liesegang phenomenon as thought pre- viously.976 Through his work and others, agar-gel immunochemical analysis has METHODS IN MAMMALIAN IMMUNOGENETICS 377 evolved rapidly into a variety of techniques suitable for the analysis of complex mixtures of soluble antigens; for their identification and interrelationships and for the determina- tion of the relative and absolute concentrations of the antigens, their diffusion co- efficients, molecular weights, and electrophoretic mobilities. In 1956, Oudin immunized rabbits with immune precipitates (ovalbumin-rabbit- anti-ovalbumin precipitate) plus Freund's adjuvants and demonstrated production of isoantibodies which would precipitate serum components in some rabbits but not in others, thus resulting in serum groups analogous to red blood cell groups.977 The term "allotypy" was proposed to designate the variation of the antigenic specificity of these serum antigens. This work has opened a new approach to the genetic study of proteins, one which we also have undertaken.290 It has been demonstrated that these allotypes may have electrophoretic mobilities of a-, (3-, and y-globulins; and that for the y-globulins there are at least two genetic systems with at least three alleles at each locus. In contrast to the polysaccharide antigens attached to red blood cells, which have been studied by agglutination reactions combined with absorption methods as cited by Dr. Owen, these soluble protein allotypes may also be studied by means of the precipitin reaction utilizing the agar-gel immunochemical methods. Since the anti- bodies are produced in the same species, specific antibodies are obtained without the necessity of absorption techniques. It would not be unreasonable to expect that other animal species would show the phenomenon of allotypy. Just as with the blood groups based on differences of red blood cells, serum groups based on antigenic differences of proteins should open new opportunities for work in mammalian genetics. I would like to make three additional comments on methodology in immuno- genetics. The introduction of paraffin-oil adjuvants with Mycobacteria by Dr. Jules Freund provided the most powerful immunization method known, at least for protein antigens.406 Such adjuvants have made it easier to produce antibodies to weak antigens. One must keep in mind that this method of immunization introduces the complication that very small amounts of antigen or contaminants will produce anti- body whereas ordinarily they might not. Also, the antibodies produced with the use of adjuvants may be more heterogeneous. When a mixture of several antigens are used for immunization, antibodies to each antigen are not necessarily produced. In fact, competition may develop among the antigens so that antibody production to one of the antigens, perhaps the one of interest, may be suppressed.2 Therefore, purified antigens should be used whenever possible. Cellulose ion-exchange chromatography devised by Peterson and Sober is one of the best methods now available for the fractionation of protein antigens.999 Finally, I would like to comment on the choice of species for antibody production. Most commonly the rabbits or horses are used as recipients for preparation of anti- bodies. However, a closely related species might be more revealing and perhaps more sensitive to minor differences of antigenic properties.289 This principle of immuno- logic perspective was pointed out many years ago by Landsteiner.749 Dr. Klein : I would like to ask Dr. Winn whether the cytotoxic method is applicable 378 IMMUNOGENETICS to antigens other than H-2, and, as far as H-2 is concerned, whether he has anyway of visualizing the cell-to-cell reaction between the lymph node cells and the target cells. Dr. Winn: I would like to answer the last question first. No, we do not have any techniques at all for visualizing the action between the specifically activated cells and the target cells. As a matter of fact, we have considerable evidence that the cells normally taken from draining nodes or spleen actually are immature in the sense that they have not yet developed the capacity to react with the target cell. This is because these cells are much more active on a cellular basis if they are transferred several days before the test graft is applied. In a series of studies in which we mixed the target cells with the immune, lymph-node cells and incubated them for very long periods of time and then selectively destroyed the lymph-node cells with antiserum and complement, the tumor cells grew as if they had never been in contact with the tissues at all. They grew just as if they had been incubated with serum alone. I think that if you want to visualize this reaction you have to find a source of the more mature cells, possibly in the peripheral blood, or alternatively, one might provide some system of incubating these cells from draining nodes and spleens in vivo in chambers or in tissue cultures and testing them several days later. I think I would have to ask Dr. Snell to comment on the use of cytotoxic tests. There is one system involving coisogenic strains in which we do have excellent com- plement fixation. So far as I know, all the evidence indicates that a non-//-2 difference is involved there. We also obtained a complement fixation with C3HK and C3H serum which I believe has an H-\ difference. As far as using other cells is concerned, lymph-node cells give far better reactions than any other tumor cells and any other normal tissue cells. The cytotoxic test, I think you may be aware, is not equally applicable to cells other than those from the lymph node. When the cellular prepara- tion is made, a high percentage of the cells are already dead ; but this is the advantage of complement fixation tests as they can be used on cells whether they are living or dead. Dr. Snell: I might merely comment as to whether two of the systems which Dr. Winn has employed are established as non-//-2 systems. The H-\ system is very definitely, there is no question about it; but Dr. Winn did not get quite as clear results with it as with the other system. Dr. Winn : It does not fix nearly as much complement. Dr. Snell: The other system, which does fix complement well, turned out to be a new locus, H-5, but there is one additional test to run. Dr. Herzenberg : Dr. Owen, would you comment on the uses of labeled anti- bodies? Dr. Owen: There have been many uses of labeled antibodies, for example in localizing antigen, but only scattered applications of these techniques that could be described as specifically immunogenetic. One example is the work of Masouredis,857 who was able to distinguish a dosage effect in the Rh complex by the use of I131-labeled antibody. METHODS IN MAMMALIAN IMMUNOGENETICS 379 Dr. Cohen: I have tried the techniques of Masouredis857 in studying gene- dosage effects on rabbit red-cell antigen and had no success. There appeared to be too much nonspecific absorption of the tagged antibody so that the small differences were difficult to distinguish. I used, as in my test system, tagged antibody which had been absorbed on the specific cell and then eluted. The tagged eluate was then used on cells of known genotypes. Masouredis did it this way in his human work and was successful. It did not work in the rabbit red-cell system. Dr. Reed: Fluorescent antibodies are, of course, the other major type of labeled antibody. Also, several tritium-labeled antibodies have been prepared to date.1037- 1045 Tritium-labeling is easily done by the Wilzbach method,1274 by sending the lyophilized antiserum to one of several firms to be exposed to tritium gas. Dr. Cohen: The fluorescent antibody technique has been used for the identifica- tion of blood-group antigens by Cohen and her co-workers209 as a means of investigating problems in immunologic genetics and hematology. They used human anti-^4, anti-5, anti-Z), and anti-C to identify minor cell populations occurring in a mother which might be derived from the transplacental transfer of cells from the fetus to the mother. This technique could also detect minor cell populations arising through mutations or through induction of chimeras. Dr. Gowen: It has not been mentioned, but there are real possibilities for isolating animal strains that will be good producers of immune serum. In my own experience there are great differences in serum titers in random-bred animals, indicating dif- ferences in their genetic capacity to make immune antibodies. The isolation of strains with high production of immune sera could be of both practical importance and contributory to better understanding of immune phenomena. Dr. Barrett: It has been said by several, on what I believe to be uncontrolled observations of race III rabbits produced by Dr. Sawin, that they are better- than- average antibody producers. I have used some of these rabbits myself. I would not say whether they are better or not; they certainly are quite good. Dr. Dray: Dr. Barrett raises an interesting problem concerning the individual variation of rabbits in their capacity to produce antibodies. Gamma-globulin groups of rabbits may offer an approach to this problem, since at least 36 genotypes based on three alleles at two loci are now known. 290 Since antibodies are found among the y- globulins, the possibility exists that the genetic control of y-globulin allotypes may be correlated with the genetic control of the capacity to produce antibodies. Such a result would also have interesting implications concerning theories of antibody formation. HOST-PARASITE RELATIONSHIPS John W. Gowen, Ph.D. GENETICS of INFECTIOUS DISEASES For every disease there is a host species in which the disease is recognized. Since individuals within species vary genetically in many ways, they, as expected, also vary in expressing the syndrome for any particular disease. Similarly if the disease is infectious, another species, pathogenic to the host species, is generally responsible for initiating and carrying on the disease. The organisms within the pathogenic species likewise vary genetically in virulence and other characteristics and, in consequence, also lead to variations in their invasive power to individuals within the host species. Some diseases further complicate the results by requiring other species, as mosquitoes for malaria, for vectors to maintain and carry the disease to the host, thus adding further genetic variables to the already complex situation. Different environmental agents of many kinds likewise strongly affect the expression of disease in numerous ways. These factors tend to confuse the data on causation of disease by inducing variation in disease expression so that individuals may vary all the way from no expression through various grades of morbidity to those that die. So far as is known, there are no problems of genetics that offer more complications in their solutions than those pertaining to disease resistance. The problems are, however, of interest because of the complexity, the genetic implications and discussions brought about thereby, and the methodology developed for their solution. Discussion of these problems will be limited largely to mice and only to certain aspects of the host-pathogen relations as our investigations fall almost entirely within this field. The research considered is largely that of associates and students who have made extensive contributions to the problems. Papers giving a broader coverage of the subject are listed in the bibliography. 383 384 HOST-PARASITE RELATIONSHIPS Because of the multifactorial nature of the causation of infectious disease, investi- gators without exception have limited their quantitative studies to specific pathogens in fixed amounts and under pre-established environmental conditions. For new diseases the limits within which these variables were controlled have been determined by trial and error. Search then has been made for ways and means by which the geno- typic variability of the host could be broken up into pure breeding strains so that the factors could be studied within a more limited range. Most workers have limited their attention to one or two of these strains generally derived by repeated inbreeding. Such limitations seriously restrict the generality of any disease studies as they may be applied to the species or to the more important transfer of the information gained to man. Our own viewpoint has been that the host strains for study should be specific samples, each covering a limited range of the whole species' susceptibility to the disease but that when all inbred strains are considered they should cover the full range of susceptibility or resistance to the disease as represented by the species genotypes. The same considerations have been given to the genotypes of the lines which may be developed from the pathogenic species, although in much of the work herein reported only one line of the organism (one that is remarkably consistent so far as virulence is concerned) has been utilized. Other observations have not been reported since this program is designed to cover methodology within mammalian hosts. Initial attention may be turned to the variations in resistance to murine typhoid due to Salmonella typhimurium 1 1 C that may be established for mice as a whole and fixed in inbred lines. The procedures by which these strains were established were diverse, but all have been inbred brother x sister over many generations with selection for the desired disease resistance in fixing the levels of mortality. The results are shown in table 61. Table 61 Resistance of inbred strains of mice to murine typhoid Salmonella typhimurium, 200,000 11C Strains of Per cent mice survived S 83.9 + 0.6 RI 67.7 + 1.6 K 50.6 + 1.3 Z 34.2 ± 0.8 C 29.6 + 1.4 E 23.5 + 1.0 N 16.9 + 1.2 L 9.6 + 0.9 a 1.7 + 0.4 Ba 0.2 ± 0.1 These strains have characteristic reactions to this disease. They range from the most resistant to the most susceptible. Binomial standard deviations of the means are GENETICS OF INFECTIOUS DISEASES 385 calculated for each strain. These data are for the period 1944 to 1956. The repre- sentation of the expected performance of each strain is on the whole good, but the data themselves do not tell the full story. The S mice are now more resistant to the invasion and growth of this pathogen than the figures show. For this strain there is a pronounced difference in the reaction of the sexes, the males having less resistance than the females, 74 to 95 per cent, respectively. No other strain shows such a difference between the sexes. Tests on the males show a much higher frequency of deaths, 1 to 4 days following inoculation, than those for any other strain or even for the females of the S strain. It is further found that the males are quite susceptible to large doses of killed S. typhimurium 11C. The deaths also occur within the period of 1 to 4 days following inoculation. These facts are interpreted as pointing to two causes of deaths for the males of this particular strain. The deaths which come early in the disease are attri- buted to rapid release of endotoxin from the digested bacteria. The deaths which occur later are attributed to the common cause of death for all strains, the growth of the organisms in the host. The genetic techniques which have been used to separate the differences in resistance to disease have varied, but in each case have included inbreeding to fix the particular genetic resistance observed within the strain. Throughout, the breeding stock which has been the source of animals for test has been free from S. typhimurium for a period of more than twenty years. Tests for resistance of some twenty different strains (genotypes) have been made over this period. From the years 1944 to 1955, 21,669 mice were observed in these tests. These animals had their first contact with the disease at ages from 45 to 700 days. All tests were made with a single line of S. typhimurium 11C at a dose of 200,000 organisms injected intraperitoneally. This line of the pathogen has shown a consistent virulence for over twenty-five years. In tests with this organism, survival ratios include nearly completely resistant to nearly completely susceptible, S and Ba strains respectively; the other strains show intermediate values for resistance. Although these values have fluctuated during the 1 1 -year period included in these data, they have kept their relative positions on the whole. The observed stratifications may be interpreted on the basis of a number of genes affecting different physiologic functions of the host responsible for the resistances or susceptibilities to the bacteria. These resistances or susceptibilities could scarcely be analyzed in a random-breeding population. In any such population, successive mice, when utilized for disease tests, would show hit-or-miss resistance or susceptibility to the disease and thus vitiate results coming from such experiments. Mortality represents the accumulated dysfunction of the various organ systems which may be attacked by the invading organism. Different organs may be susceptible in different strains. The temporal sequences in the progress of the disease may also differ between inbred strains. The severity of the sickness (morbidity) expresses some of the differences in the patterns of resistance. The S mice are so resistant that they show almost no effects of the pathogenic organisms. The Ba mice are extremely sick almost from the first contact with the disease. The other strains have characteristic 386 HOST-PARASITE RELATIONSHIPS patterns of morbidity which are apparent at different stages in the disease cycle. The pattern of morbidity for each strain is characteristic, indicating the specificity of ex- pression of the disease. TRANSMISSION OF DISEASE RESISTANCE IN CROSSES Convincing evidence for the dependence of resistance and suceptibility to infectious disease on the strain genotypes comes in repetition of the test results for the different strains generation after generation despite varying environments characteristic of changing seasons and years. However, this is not all the evidence desired. To collect the evidence properly would require an experiment comprising 1 ,600 + cells and possibly 100,000 mice. The time, energy, and space required for this analysis together with the three generations of breeding required have prevented us from doing the experiment up to this time. However, valid data covering critical crosses are avail- able through the work of Hetzer,573 Zelle,1467 Weir,1376 and Gowen and Stadler (unpublished). The combined data for the first generation are given in table 62. The S strain was under intense selection for resistance at that time. Table 62 Resistance of parents and crosses of inbred strains of mice to murine typhoid Salmonella typhimurium 11C, 200,000 organisms Parents Progeny Per cent Mating Male Female tested survived types S x S 728 89 (Res x Res) Pt RI x RI 42 80 (Res x Res) Yx BrR x BrR 851 78 (Res x Res) Px Ba x Ba 346 2 (Sus x Sus) Px L x L 207 2 (Sus x Sus) Px Hybrids S x RI 54 78 (Res x Res) Fx RI x S 35 73 (Res x Res) Fx BR x S 756 75 (Res x Res) Fx S x L 88 89 (Res x Sus) Fj L x S 133 79 (Sus x Res) Fx S x Ba 53 85 (Res x Sus) F1 Ba x S 40 80 (Sus x Res) Fi L x Ba 198 18 (Sus x Sus) Fx Ba x L 68 37 (Sus x Sus) Fx Res = resistant. Sus = susceptible. PL = parental purebreds. Fi = hybrids. Table 62 gives data on 3 resistant inbred lines and 2 that are susceptible. Com- parison with table 61 shows that the S mice of this period in the genetic development of the strain were 5 per cent more resistant than in the 1944-1956 period. The same was true for the mice of the RI strain, 12 per cent, and for the Ba mice, 1.8 per cent. GENETICS OF INFECTIOUS DISEASES 387 The L mice on the other hand were less resistant, 7.6 per cent. This difference is seemingly important, although numbers of mice are not large and other factors besides binomial variations contribute to the real variance of data dealing with problems of disease. The differences between the resistant and susceptible strains for either period were pronounced. Confining attention to the first three hybrids, S x RI, RI x S and BR x S, the results showed the hybrid resistances to be closely similar to those of their inbred parents, although they were, in each case, a little less resistant, 9 per cent. This was convincing evidence for the similarity of the genes making up the relatively resistant strains. The results did not indicate heterosis. Rather they suggested partial domi- nance for some of the genes controlling resistance. The hybrids SxL, LxS, SxBa, and Ba x S represent extreme crosses of resistant x susceptible. They were only 6 per cent less resistant than the resistant parental strain. Dominance of most genes for resistance was indicated. There was no significant evidence for the reciprocal crosses being different from each other. In mice the chromosomal arrangement is XY for the male and XX for the female. The male progeny received all AMinked genes from the mother, whereas the females had an X chromosome from each parent. Any major factors for resistance on the X chromo- somes should make corresponding changes in the survival ratios of the sexes in the reciprocal crosses. As Hetzer showed this was not the case. However, there was a ten- dency for the females to have a higher survival than the males^ but the sex differences in survival extended to all matings and not just those which were diagnostic of sex linkage. The equality of the resistances of the hybrids further emphasized that resistance or susceptibility to this disease was not due to a maternal effect but to inheritance being transmitted by both sexes. Passive immunity carried through the egg cytoplasm, the fetal circulation, the colostrum, or other maternal environmental effects was ruled out as a factor materially influencing resistance. The crosses of mice of the L strain mated with those of the Ba strain represented the crosses of two highly susceptible strains. The resulting hybrids' resistances were greater than either parent's and by a noticeable amount. This result indicated that in- bred strains L and Ba possessed genes for resistance even though they themselves were quite susceptible. These genes complement each other to give increased resistance to the F1 progeny. This is in contrast to the results for the crosses of the resistant strains. These crosses dropped slightly in resistance. The comparison between the two classes of hybrids could be used against the argument that the results of the L x Ba crosses were due to heterosis. However, that reasoning may not hold, for data have been collected which indicated that heterosis may be genotype specific. The backcross data gathered by Hetzer573 while in this laboratory further sup- ported these views. The main feature of these tested backcross animals was the further regression of their S. typhimurium resistances toward those of the susceptible strains. This places their sensitivity to typhoid as intermediate between those of their resistant and susceptible grandparents. Segregation of factors for resistance and susceptibility 388 HOST-PARASITE RELATIONSHIPS probably took place, several genes in different loci being important to resistance. The average mortalities for the crosses with L as the inbred parent were nearly the same as those with Ba as the parent, 53 and 48 per cent, respectively. Survival among the backcrosses ranged from 67 to 40 per cent. Examination of these crosses showed no established reason for the variability. Higher susceptibility came in the progeny of the F1 (S$ x L^) for the L backcross, whereas the higher susceptibility was evident in the offspring of the Fx (L$ x S^) for the Ba backcross. The resistances of these two crosses separately contradict hypotheses often used to explain resistance to disease other than on inheritance grounds. In the first case the data paralleled those of the two hybrid generations in suggesting differences in the reciprocal hybrids. This hypothesis is refuted, however, by the second cross. The second cross suggested that the Ba female parent was responsible for lowering the survival of her progeny, a type of maternal effect ; but comparison with the other backcrosses and all the crosses in general denies this conclusion. Tests on the inbred parents, hybrids between them, and backcrosses to the suscep- tible strains consequently support the dependence of resistance to disease on genetic factors transmitted along customary paths. The mode of action of this inheritance is capable of wide modification through changes in numbers of the infecting organisms of different lines of the bacterial species. Results of this type are generally sufficient to establish the genetic dependence of disease susceptibility. However, there is another hypothesis, contraindicated by many features of the above data, which often has been cited in the past. Evidence on the validity of these hypotheses, consisting of even more critical data on the problem, may be had by adopting the following approach. GENETIC ASPECTS OF DISEASE AS INDICATED BY PROGENY OF DOUBLE MATINGS The other hypothesis is that the progeny acquire an immunity either by active contact with the low-grade disease or passive immunization and thus develop trans- mitted resistance to the challenge tests. This hypothesis is based on the fact that ani- mals which survive a disease develop an active immunity of irregular duration. Some survivors retain a latent infection which is thought to protect them by keeping up an active immunity. These carriers may spread subclinical infection to their progeny, thereby inducing active immunity in them. Evidence indicates that passive transfer of immunity from dam to progeny may take place, but males are not able to transmit passive immunity to their offspring. A method for evaluating the weight to be given the factors of genetic versus acquired immunity in resistance to disease was designed by Gowen and Schott.467 This was applied to studies on typhoid in the mouse but has general applicability. The method consists in mating two males of different strains to a single female in the same estrus period so that litters will contain young of two known separable types. In our case S mice differ from L mice in their genetics of coat color, so that it is possible to distinguish between S mice as albinos, L mice as silver black, and hybrids as black mice. An L female mated in the same estrus period to GENETICS OF INFECTIOUS DISEASES 389 two males, one L and one S, may have pure L and hybrid progeny in the same litter. Environmental conditions are common to all animals of a litter, each having the same opportunity to receive immune bodies from the mother and to receive latent infection from the parents. According to the genetic hypothesis, the L progeny should be nearly 1 00 per cent susceptible and the hybrids should show resistance as may be seen in table 62. According to the hypothesis of acquired immunity, the L and hybrid littermates should be equally susceptible. The two types of progeny tested showed that the L mice of the litter were all susceptible, whereas the hybrid mice of the same litter were resistant as expected of this hybrid cross. The differences were highly significant. This technique offered a means of showing that the concentration of genetic factors for resistance in the selected strain, S, were truly significant whereas any factors for acquired immunity were without effect when the disease was murine typhoid (S. typhimurium) . The results further substantiate the earlier results as well as indicate a desirable technique for separating the genetic basis for natural resistance to a disease from that due to factors for immunity harbored by the mothers. EFFECT OF NUMBERS OF PATHOGENS ON SURVIVAL The effects of sheer numbers of organisms on the characteristics of a disease are of interest particularly as such data help to understand the pathogenesis whereby genotypes alter the phenotypic expressions of the disease. Schott1167 utilized a host population composed of a random selection of albino Mus musculus. They were infected by intraperitoneal injections of different numbers of the same line of bacteria. The survival for each dosage is shown in figure 49. Figure 49 shows that the survivals of the mice were greatly influenced by the num- bers of organisms which they received. Animals which received the low dosage (1 x 104 organisms) displayed a long incubation period followed by increased and then decreased mortality. An appreciable number of animals survived the disease attack. At the high exposures (1 x 107 organisms), the organisms rapidly overwhelmed the hosts. There was practically no incubation period. The course of the disease ended days earlier than those for the smaller dosages. Between these two extremes inter- mediate effects were observed. The pathogen was S. typhimurium in mice but the data are illustrative of what may be observed for all types of pathogens— chemical poisons, viruses, bacteria, protozoa, and multicellular pathogens. The scales of dosage will change with the material used, that is, with ricin a dose less than 0.001 mg. caused sickness but few deaths, whereas a dose of 0.004 mg. resulted in deaths of nearly all the treated mice, but the principle of mortality dependent on dose was maintained throughout this range. In genetic studies of host-disease syndromes, the investigator must stay within the limits of dosage set by the test curves for the particular material. Experience shows that the preferable limits for resistance studies are those set by .3.90 HOST-PARASITE RELATIONSHIPS Fig. 49. Survival of mice following acute exposure to different quantities of the pathogen, Salmonella typhimurium, and during the course of the disease. \5 x 10 \ V\2xiOb 2* I06\ S 5 10 15 DAYS SURVIVED 20 10 to 90 per cent survival of the treated population, as it is within these limits that most information on disease processes may be obtained. Possibly the most satisfying evidence for progress in selecting for resistance to disease, beginning with an unselected population, comes in the changes which appear in days survived in survival curves for different dosages. Figure 50 illustrates these curves as obtained by Schott1167 and Hetzer.573 The selected mice were unquestionably more resistant to the S. typhimurium when tested by each of these three different dosages than the unselected population from which they came. These tests were made in 1936. The S strain was further selected to the nineteenth generation. Selections were discontinued at that time and the strain maintained by sibling matings. Today the mice are as resistant as those shown by the data of figure 50. The lapsed time over which the S mice have retained their resistance is 2 1 years. This period represents a turnover of 63 ± generations, or in terms of generation time in man about 1 ,900 years. The syndrome by which the disease was first described also changed during the 1 1 generations of selection. The relative dosage effects of the different quantities of the bacteria have remained in similar positions in both the unselected and selected mice, but fewer mice from the eleventh-generation population die. In the unselected mice the lower dose causes somewhat less mortality than the intermediate dose. The high dose is much more lethal than the two lower dosages. The selected mice show greatly increased resistance. The low-dose treatment now has 87 per cent survivors, whereas GENETICS OF INFECTIOUS DISEASES 391 Fig. 50. Relationship between survivals and days survived for unselected and ELEVENTH GENERATION S MICE SELECTED FOR RESISTANCE. Unselected Mice \V2xl0 5 7 vX i V i >— Mice Selected for Resistance Nth Generation J L 5 10 15 20 DAYS SURVIVED 5 10 15 20 DAYS SURVIVED The mice were treated with the pathogen, S. typhimurium, in three dosages, 2 x 10s 2 x 106, and 1 x 107 organisms. only 4 per cent of the unselected mice survived. The median dose definitely caused median mortality. In the eleventh generation, 76 per cent of selected mice survived; whereas only 2 per cent survived in the unselected population. The high dose of ten million organisms was rapidly and completely lethal to the unselected mice, but 60 per cent survived during the eleventh generation of selection. Not only did the selected mice have fewer deaths than those of the unselected group from which they came, but also the distributions of the deaths took different forms. The frequency curves in per cent of total deaths for those mice which died in each group are given in figure 51. At the highest dose (1 x 107), the deaths occurred early in the unselected group, the modal value being between 4 and 5 days, whereas in the selected group the modal deaths were one or two days later. The mode for the unselected group was more than twice as high as that for the resistant cohort. The range over which deaths in the unselected group occurred was reduced to half that for the mice of the eleventh generation. The syndrome obviously changed. The intermediate dose acted on the selected and unselected mice in a similar manner. The modal day for death was 9 days after inoculation. The proportions of deaths on that day were about equal. Deaths occurred later in the unselected mice. It is difficult to compare the results for the lowest dose. The main effect observed was an increase in the 392 HOST-PARASITE RELATIONSHIPS Fig. 51. Mortality of mice from Salmonella 20r 20r UJ a LU o 40r ™ 20 ix 10 l i 5 10 15 20 DAYS SURVIVED V\ 5 ^ 2x10 5 10 15 20 DAYS SURVIVED Mortality (percentage frequency) from S. typhimnrium for mice of the unselected population (left) and mice selected eleventh generations for resistance (right). numbers of survivors. In fact the numbers of mice dying in the selected cohort are so few as to give them the appearance of random deaths. Analysis of pathogenesis in the resistant mice of the eleventh generation would re- quire a notably higher number of invading organisms than when mice of the original population were under study. In fact it is doubtful if the investigator could obtain the same syndrome by only adjusting the initial numbers of the invading S. typhimurium 11C organisms. Heavy reliance must be placed on data of this kind in the methodologic design of all infectious disease experiments. The information, however, has greater significance in that it is indicative of how at least some of the different pathogen genotypes may induce their effects. Increase in numbers of inoculated organisms of the same line would increase the dosages of any substance generated by these organisms that may be inimical to the hosts. Increased dosages lead to more severe morbid reactions. Pure lines of the pathogen, when selected, often as apparent mutations, occurring within the same original parental line, are often characterized by differences in virulence of entirely similar patterns to those discussed above, even though a fixed number of organ- isms are inoculated into each host. This suggests that, at least in some instances, the genotypic differences in the pathogen act as quantity controls on the same substance or capacity within the given mutant lines rather than in necessarily creating entirely new products detrimental to the host genotypes. GENETICS OF INFECTIOUS DISEASES 393 ROUTE OF INFECTION AND SEVERITY OF DISEASE Between 1920 and 1940 great emphasis was laid on studying disease through utilizing only natural routes of infection. The advocates produced little evidence for their contention that use of other routes could not lead to comparable resistances in the hosts. Attempts to cause natural infections often produced trauma as great as that of experimenters who openly avowed that the routes of infection they were using were not natural. Further difficulties arose in that attempts to introduce the pathogens by natural routes also introduced dosage variations to increase variance in the results. However, the real problems involved, while often overlooked, were of interest and have been pursued by several investigators. The methodologic problem really turns on whether natural resistance or suscepti- bility to a disease is dependent on localized differences in resistance of tissue at some customary portal of entry for the disease organism, or is a property of all cells of the body. Roberts and Card,1059 Irwin,642 Lambert and Knox,746 Schott,1167 Gowen and Schott,467 Hetzer,573 and others collected data showing that the normal portal of entry could be bypassed and genetic resistance to any one of several diseases could be established in the host. Webster1363 collected data for different routes of entry on mice which he had previously selected for resistance and susceptibility to Salmonella enteritidis introduced through the so-called natural route, the stomach. The selections were based on intrastomachal instillation of 5,000,000 organisms through a silver tube inserted into the stomach. The relative resistances of these susceptible and resistant strains of mice are indicated in figure 52. The most striking result of these tests is that each route of instillation of the S. Fig. 52. Relationship between route of entry and mortality of selected mice DUE TO BACTERIAL INFECTION. Intrastomachal intravenous Subcutaneous Intraperitoneal Dose 5,000,000 Dose 50,000 Dose 50,000 Dose 50,000 Relative resistance of mice selected for susceptibility (Sus) and resistance (Res) to Salmonella enteritidis when the bacteria are introduced into the stomach through the esophagus by silver tube (dose of 5,000,000 organisms), or injected intraperitoneally, subcutaneously, or intravenously (dose of 50,000 organisms). 394 HOST-PARASITE RELATIONSHIPS enteritidis picks out the mice of the susceptible strain from those of the resistant. In this sense, each of the tests is accomplishing its purpose. The second conclusion is that the intrastomachal instillation of the bacteria requires 100 times the dose of the pathogen to cause the disease than the intraperitoneal, subcutaneous, or intravenous routes. Conclusions from this fact may be opposed. (1) It may be supposed that the intra- stomachal route is the natural route of infection and as such has had most of the natural resistance mechanisms built up around it. If this were true, the numbers of bacteria immobilized would be large and a higher initial dose be required to cause the typhoid disease. (2) On the other hand, the stomach and contents furnish much material in which large numbers of bacteria may be lost through chance and thus never get an opportunity to reach the vital centers and cause death. The inoculation routes are closer to these centers so there is less chance of loss and fewer organisms are required. Of all the inoculation routes, it is a little surprising perhaps that the intravenous is least efficient, the subcutaneous is next, and the intraperitoneal is the most effective. S. enteritidis parasitizes the liver and spleen. In that sense, intraperitoneal inoculation places the bacteria near these organs and so could facilitate immediate infection possibly at the vital organ. On the other hand, at one stage the disease is a septicemia with large numbers of bacteria in the bloodstream. Should the septicemia be a funda- mental part of the disease, it would also seem that the intravenous injection should be more helpful in reaching the vital organs than subcutaneous inoculations which dis- tribute the bacteria more widely and also give them more chance for loss before their effects may reach the vital center. However, the data show that this is not the result. In any case, the large dose required by the intrastomachal routes is subject to at least two interpretations having opposite significance. Another test of this matter came in our own research some 30 years ago while studying genetic resistance to poisons as distinct from reproducing pathogens. The dose of ricin for the mice was 0.002 mg. The mice were of a single inbred strain and were 60 days of age. The routes of instillation were subcutaneous, intraperitoneal, and intravenous (into the tail vein) . One hundred and sixteen mice were used in the tests, figure 53. The data of figure 53 are in order of the susceptibilities of the mice, the subcu- taneous route being least toxic, the intraperitoneal route noticeably more lethal, and the intravenous being completely lethal. The order of effect is that which our pre- conceptions might lead us to expect. There should be more wastage of poison when introduced subcutaneously than when placed near the vital organs in the peritoneal cavity. Introduction into the circulation would seem to give the means to carry the toxic chemical most directly to the organs vulnerable to its effects. The order of effectiveness of the routes of entry into the body is not the same as that for disease due to S. enteritidis. It appears quite likely it will not be the same for other pathogens. In any case the evidence is equivocal on whether or not a given route for introducing a pathogen in testing for natural resistance of a host is better than another. GENETICS OF INFECTIOUS DISEASES 395 Fig. 53. Relationship between route of entry and survival of mice inoculated with 0.002 MG. OF RICIN. 60r 9§ 40 CO 20 LU Q_ 0 vm\ Subcutaneous Intraperitoneal Intravenous Direct comparisons of the products of the tests furnishes a better comparison. Data on this point are not entirely satisfactory since they were collected by different observers working with different populations of albino mice. Schott1167 and later Hetzer573 selected for natural resistance to S. typhimurium by selecting for resistance on the basis of survival to test dosages of 50,000 organisms introduced into the peritoneal cavity. The foundation population was rather susceptible since only 17.7 per cent survived the test. The first- and second-generation tests showed the families quite heterogeneous for resistance. Selection for resistance was continued for six successive generations when resistance of these animals gave them 75.3 per cent survival. Hetzer continued the selection for two more generations with the same test dose. The sur- vivors of that dose were 84 per cent. The data show that selection for resistance is an efficient method for establishing natural resistance to S. typhimurium when the disease is induced by intraperitoneal injection of the organisms. Webster1362 selected for a susceptible and a resistant strain of mice by testing previous siblings by intrastomachal inoculation of 5,000,000 organisms and then later unexposed siblings for breeding on the basis of these susceptibility tests. By this test, the initial population of mice was not as susceptible as that of Schott's as judged by their respective survivals — 62.6 per cent on Webster's test versus 17.7 per cent on Schott's. In four generations of selection Webster's selected, susceptible mice were susceptible to the point where only 15 per cent survived. The selection dose was then reduced to 100,000 organisms. On this lower dose the survival percentage became 17.6 per cent. Selection toward resistance for two generations gave a population of mice which, when tested with 5,000,000 organisms, survived 88.7 per cent. The test dose was then increased to 50,000,000 organisms. The survival of the fourth-generation mice treated with this dose was 82.5 per cent, but the degree of resistance had reached at least a temporary plateau for this method. In conclusion it may be said that the intra- stomachal route proves to be a successful but slower route for selection of mice for resistance and susceptibility. 396 HOST-PARASITE RELATIONSHIPS A test for how successful selections by the different routes have been is made by comparing the resistances of the resulting strains. As Webster showed, the strains of mice resistant to S. enteritidis administered by the gastric route were likewise resistant to the organisms introduced subcutaneously, intraperitoneally, and intravenously. Derived susceptible strains by the intrastomachal test route were likewise even more susceptible to the organisms introduced by the other three routes. ^The degree of progress toward resistance, although apparently less than that for susceptibility, was in fact more significant since resistant, unselected strains of mice were difficult to find in natural populations, whereas those which were as susceptible as or more so than the selected strains were relatively easy to find. Sample mice of the strains derived by Webster have been bred in our laboratory for some years. They may be compared directly with our S and RI lines developed here at Ames. The S mice are the most resistant and were established by selections based on the results of intraperitoneal inoculations of the test organism (see table 61). The RI mice take their origin from the Webster resistant mice but have had their resist- ance greatly increased through our selections based upon the intraperitoneal route of inoculation. The K and C mice are two branches of the Webster selections for resistance which were split from the main line on the basis of their resistance and suceptibility to St. Louis encephalitis and louping ill. Similarly the N and Q lines represent strains similarly separated from this established susceptible strain. There is some variation between his two resistant lines and between his two susceptible lines, but the resistant group are definitely more resistant than the susceptible group on the basis of the intraperitoneal tests as they were on the intrastomachal tests. But these resistant strains are not so highly resistant as those selected by the intraperitoneal route in the first place. These results are important from the methodologic viewpoint in pointing the way toward developing resistant and susceptible hosts to different diseases so that these diseases may be analyzed for the host, gene-based characters which are significant to the given diseases. They are also significant in indicating that, until a natural barrier can be differentiated from the diluting effect of distance from the vital region attacked by the particular disease, it is proper to assume that every cell in the body is endowed with properties which are contributory to making the animal resistant or susceptible to a disease. Further support for the significant part played by the cells of the whole body in resistance to a specific disease comes in a study of X-ray irradiation effects on resistance mechanisms of genetically differentiated strains of mice exposed to S. typhimurium. The experimental design, although straightforward, is at the same time a little complex. Details are found in three papers of Stadler and Gowen.1264, 1265' 1265a X rays were chosen as a means of altering the resistance of the cells in different regions of the body. The experiment was designed as a factorial having four elements. Five inbred strains of mice having known differences in X-ray and typhoid sensitivities were utilized in comparable numbers. The numbers for the two sexes were balanced for each strain. GENETICS OF INFECTIOUS DISEASES 397 There were four X-ray exposure doses : 0, 320, 480 and 640 roentgens. The levels of X-ray dosages were chosen to span the range from no effect to nearly complete lethality when the mice were exposed to whole-body irradiation. There were eight combinations of exposures to the X-ray effects: none, head, mid, rear, head-mid, head-rear, mid-rear, and whole-body exposure. The head region or anterior third of the body extended into the thorax. The middle third of the body (mid region) included the lower thorax and abdominal cavity containing stomach and upper intestinal tract, liver, spleen, adrenals, ovaries, and kidneys. The posterior third of the body (rear region) included the lower intestinal tract, bladder, and urinary system and testes of the males. The eight groups of different regional exposures were treated with 320 r, 480 r, and 640 r, making a total of 24 different X-ray treatment groups. In addition, the mice of one group were put in tubes and completely lead-covered for a time comparable to that of the longest dose, 640 r, but were not exposed. This group acted as a control on handling as well as for the unirradiated, 0 r group. There were 25 treatment groups with 5 strains and 2 sexes making up 250 cells in the experiment. Each cell represented a different strain, sex, and treatment. A minimum of 25 mice were treated in each cell. Some cells contained a few extra animals. The completed experiment involved a total of 6,904 mice. The immediate effects of X ray were largely completed 12 days following irradia- tion. Although there were marked differences in survival times between the different strains, few deaths occurred after the twelfth day. A 1 5-day interval following irradia- tion was allowed to cover the direct effects of exposure. The mice were then inoculated to test for their resistance to murine typhoid. Different lines of the bacterial species S. typhimurium cover the full range in virulence or pathogenicity due to differences in their genetic constitutions.468' 1467 This factor has been controlled in this work by limiting all the disease tests to one of our lines, 11C, of S. typhimurium. The data showed that the sexes were not significantly different in this experiment in their reactions to this disease. The physiologic sex differences introduced by the chromosomal differences played little part in the outcome of disease. Strain differences were obvious. The high innate resistance of the S strain prevailed over the latent effects from the X-ray exposures except in the severest treatments, those of whole-body exposure to 480 r and 640 r. The S strain was more than twice as resistant to the disease as were the Z and K strains. The survival curves of the S mice showed less marked reductions in survival due to X-ray treatments than those of the other strains. The differences in the disease-resistance levels of the Z and K strains and the level of the Q strain was even greater. Because of our previous knowledge of the susceptibilities of the Q and Ba strains, the Q strain received only one-hundredth the dose and the Ba only one-thousandth the dose of S. typhimurium 1 1C which was given to the Z, K, and S mice. The extreme susceptibility of the Ba mice under normal conditions allows no opportunity to 398 HOST-PARASITE RELATIONSHIPS observe effects like those of irradiation unless very small dosages are administered. When analyzed for X irradiation alone or typhoid resistance alone, the strain effects were highly significant. For typhoid resistance, differences between strains were more marked in those groups where the irradiation had been more detrimental to sur- vival. In only a few of the exposure groups did the radiation lower the resistance of the S strain to murine typhoid. For Z mice, the previous irradiations lowered survival to typhoid infection in all treatment groups. The Q strain, with its greater ability to withstand radiation, showed somewhat more reduction in survival from typhoid following the exposures of 320 r than of 480 r or 640 r, except in the whole-body treat- ment groups. Murine typhoid disease following 640- r, whole-body exposures was lethal to all the strains. Similarly typhoid following 480-r, whole-body treatments was lethal to all strains except S. The 480-r, regional exposures were effective in lowering the resistance levels of the Z and K mice. The average values of the regional effects on the four strains show that resistance to S. typhimurium was decreased as the exposure doses were increased. These observations support those of Gowen and Zelle469 who found that X irradiation 15 days before contact with typhoid resulted in reduced survival for the 6 inbred strains of mice tested. The high resistance level of the S strain to murine typhoid made the S strain the better indicator for differences between the treatment groups. The full range of survival from 100 per cent at 0-r to 0 per cent at the 640-r, whole-body exposure was covered by the S strain after typhoid infection. The range of survival was limited for the other strains by their greater susceptibility to typhoid even without X irradiation. The natural resistance of the S mice was most severely affected by the whole-body exposures followed by the head-mid exposures. The mid-rear exposure treatments at the three dosage levels were next in order of decreasing survival following typhoid infection. The other regional combinations were, in order of decreasing effects: the mid, head-rear, rear, and head exposures. The latter two exposure treatments had little effect on the S mice. The three other strains, Z, K, and Q,, reacted in a similar manner for the different regional irradiations. The Ba strain was so susceptible even to 200 S. typhimurium organisms that it contributed no useful information on the effects of previous X-ray treatments. The typhoid-resistance response was affected most severely by the previous irradiations to the whole body. Of the three body regions taken separately, resistance was lowered most by irradiation to the mid region. Irradiation to the head region reduced resistance somewhat more than irradiation to the rear region. The typhoid response following the X-ray treatments showed the head-mid exposure as somewhat more detrimental to natural resistance than the mid-rear exposure. Full expression of the effects of body cells in the different regions is exhibited by the S mice. Survival decreased with exposure of any part of the body to X rays at the 640-r dose. Exposure of the head and rear regions decreased survival by 4 per cent; exposure of the mid region decreased survival by 1 3 per cent. Treatment of any two of the regions still further decreased survival proportionate to the regions exposed. GENETICS OF INFECTIOUS DISEASES 399 The really big decrease in survival came when all the cells of the body were exposed ; but 20 per cent of this decrease was due to direct effects on the mid, head, or rear regions. The 80 per cent was accounted for by the absence of any unexposed cells. It mattered little which of the three body regions was left unexposed. Each third contributed much more than its share to typhoid resistance if it escaped exposure to the irradiation. These results are taken to mean that, when exposed to a disease of this type, all cells of the body retain much of their initial genetically controlled ability to resist disease invasion even when they differentiate into highly specialized tissues. From the methodologic viewpoint, high-energy irradiation offers a valuable means of specifically depressing the functions of individual organs so far as their properties of resistance to disease are concerned. Greater changes may be accomplished by whole- body irradiation when all cells are irradiated. The effects are genotype specific to some degree but are of similar kind for all genotypes. Methodologic problems pertaining to the basis of genetic resistance to disease such as differences in leucocytes and serum-proteins ; growth of heart, kidney, spleen, and other organs; active- and passive-immunity phenomena; mutation and other similar changes in hosts or pathogen; and diverse environmental effects are all part of this complex pattern of disease. Techniques for discriminating between various parameters are available, and introductory references to this material may be found in the bibliography. SUMMARY The expression of infectious disease is dependent on complex multivariate forces of host and pathogen genotypes as well as on a wide variety of environmental influences. Quantitative studies of disease have largely depended on restricting the range of varia- tion exhibited by two of these three variables. The methodology by which these restrictions have been imposed and some of their consequences have been discussed for host genotypes and breeding behavior. These include the numbers of pathogens and their route of invasion during initiation of the disease as well as radiation as a means of altering qualities of disease resistance of the host. DISCUSSION Dr. Burdette: Dr. J. A. Weir of the University of Kansas will open the discussion of Dr. Gowen's paper. Dr. Weir: Mice are expensive to maintain and require considerable space; consequently, workers employing mice exclusively are committed in a way that leads to specialization and narrowness in research interests. The role of the discussant, as I see it, is to relate the material presented to the larger picture. I find this a difficult task because Dr. Gowen paints with a broad brush, and he follows the axiom of genetics that problems are important and materials only secondary. Although he has limited 400 HOST-PARASITE RELATIONSHIPS his discussion largely to mice, he has also worked with large animals, chickens, viruses, Drosophila, bees, plants, and other living forms. At this point, I might be tempted to cite some obscure references from the literature, but, if I were to do so, I would likely use one of Dr. Gowen's papers as a general source. His reviews have been both thorough and penetrating.462' 463, 464, 465 Before proceeding with my own observations, it is appropriate to point out the similarity between Dr. Gowen's researches on infectious disease and the cancer-research program of the Jackson Laboratory. In both cases, the procedure is to investigate thoroughly a limited number of forms of the disease. On the one hand, several inbred strains differentiated on a basis of genetic resistance to murine typhoid are employed along with a specific, pure strain of the pathogen, Salmonella typhimurium. On the other hand, a few, specific, neoplastic diseases have been selected, along with special inbred lines of mice; for example, C3H as an object for study of mammary tumors and hepatomas. It also seems appropriate to give a short historical sketch. The year 1919 is of particular significance as a starting point (work with plants started somewhat earlier, with Biffen's demonstration in 190590 that resistance of certain varieties of wheat to striped stem rust fungus depended upon a single gene). In 1919 Dr. Sewall Wright began sending his surplus guinea pigs from Washington to Philadelphia, there to be inoculated intraperitoneally or subcutaneously with human-type, tubercle bacillus. The importance of host resistance was clearly demonstrated. Over 30 per cent of the variation in survival time after inoculation in crossbreds was determined by relationship to the best inbred family.1457 The year 1919 was also the time of the great influenza epidemic and this stimulated, on both sides of the Atlantic, work in experi- mental epidemiology. W. W. C. Topley and associates in England, supported by the Medical Research Council and L. T. Webster and associates at the Rockefeller Institute in the United States, studied the natural history of typhoid in populations of mice.1365 Host resistance, or susceptibility, was not emphasized at first but it was frequently mentioned.1325, 1364 This factor obviously could not be ignored and it became a subject of major emphasis. The scene now shifts to the midwest where Professor W. V. Lambert, in 1924, started his work on the inheritance of resistance to fowl typhoid in chickens.746 Under Lambert's direction similar work was initiated, using mouse strains obtained from E. C. MacDowell, L. G. Dunn, L. C. Strong, and M. R. Irwin. A culture of the organism, then named Salmonella aertrycke, was supplied in 1926 by W. W. C. Topley, Public Health Laboratory, Manchester, England. R. G. Schott and H. O. Hetzer completed their Ph.D. dissertations in 1931 and 1936 respectively.573, 1167 When Dr. Lambert was called to administrative duties elsewhere, Dr. Lindstrom, with characteristic wisdom and foresight, induced Dr. Gowen to join the department at Ames as Professor of Genetics. In Ames, Dr. Gowen, already a pioneer in the field in his own right, picked up Dr. Lambert's work and also brought his own special strains of mice from Princeton. Later he added the surviving lines from L. T. GENETICS OF INFECTIOUS DISEASES 401 Webster's selection experiments. With this great wealth of materials he initiated the intensive investigations that are still in progress. The type of selection experiment used to produce resistant and susceptible lines of mice bears comment. Parenthetically, a number of strains have been produced by inbreeding without selection, much as the cancer strains have been developed. Pro- ponents of selection theory choose their characters for ease of measurement, and this introduces elements of artificiality. Even selection for DDT resistance falls in a special category, because the host is subjected to an artificial insult unlike anything it has faced during its long evolutionary history. Selection for resistance to infection, on the other hand, duplicates many of the features encountered in naturally occurring epizootics. The disease produced by intraperitoneal inoculation with known numbers of organisms of murine typhoid is identical in all its essential features to the disease as it occurs in an epizootic, because in mice the acute form of the disease is a septicemia and not primarily a gastrointestinal infection. As in work with malignant neoplasms, inbred lines provide the investigator with a tool of inestimable value for examining the nature of the syndrome, but there are certain pitfalls in a study of the mechanisms of genetic resistance. To look for strain differences is an important part of the methodology, but the number of degrees of freedom available for correlation studies depends on the number of inbred strains and is independent of the numbers of mice. Furthermore, the degrees of freedom may be depleted in the search for clues. It is noteworthy that Dr. Gowen has not been content merely to isolate mechanisms of resistance, but he has attempted to estab- lish physiologic relationships and to apply a variety of techniques. It should also be emphasized that he and his associates have taken elaborate precautions to separate the role of natural resistance from that of acquired immunity, to control dosage, and to apply genetic techniques of crossbreeding. The nature of some components of natural resistance should be commented on briefly. Gowen and Calhoun466 found that level of resistance and total leucocyte count were related. Subsequent studies using X irradiation have added strength to the conclusion that high leucocyte count and a high level of resistance are causally related. This stimulated some work of my own. I started with what now seems to have been a naive assumption, namely that resistance could be synthesized by selecting for single components. I selected for total leucocyte count and obtained a high line with total count of more than 1 5,000 cells per mm3 and a low line with less than 5,000 cells per mm3. The outbred line has a mean count of 9,000 cells per mm3. When mice were inoculated with standard doses of several strains of S. typhimurium, there were significant and consistent differences between strains in mortality and days to death. But the resistance levels were opposite to expectation on the assumption that the charac- ter selected is an additive component of resistance.1377 Selection for blood pH gave similar results.1377 Hill et.al.577 also obtained similar results in that mice selected for resistance to a partially purified, toxic fraction isolated from S. typhimurium proved more susceptible than the controls when they administered live organisms either per os or 402 HOST-PARASITE RELATIONSHIPS intraperitoneally. Returning to the role of leucocytes, the mere presence of large numbers may be a detriment unless the cells are effective in digesting bacteria. Otherwise they serve merely as a means of disseminating the pathogen throughout the host. These studies illustrate the wisdom of Dr. Gowen's approach, namely to deal with resistance as an entity. Work in his laboratory revealed that there are genetic differences in ability of leucocytes69 and macrophages949 to digest phagocytized bacteria. The Camp Detrick work with anthrax1375 has also demonstrated the importance of qualitative differences in leucocytes. The technique of double matings was mentioned by Dr. Gowen. This tool for investigation has not been fully exploited. We have found in our laboratory that about one litter out of 10 to 15 matings is of mixed parentage, when females are placed with pairs of males. All that is necessary to determine parentage is to have suitable color markers. When the seminal vesicles of one male are removed, to prevent plug forma- tion, the number of mixed litters is not increased as might be expected. We have had fewer mixed litters by this procedure. Direct observation has shown that mixed litters are usually the result of a rapid round robin, with one male providing sperm and no plug or only a partially formed plug. Dominance (in the behavioral sense) of one male over the other has no effect on the percentage of mixed litters. The experimental work with murine typhoid has a practical aspect. It is note- worthy that in Dr. Gowen's laboratory, even though the precautions are not elaborate, there has never been an outbreak of typhoid in the breeding colony. Mice and Sal- monella do not necessarily go together, like pie and cheese. In our own laboratory murine typhoid was introduced by mice from elsewhere. Until efforts to eliminate the pathogen were successful, the disease remained confined to the imports and their descendants. Except in the few cases in which crosses were made, our own laboratory stocks remained free from disease. At present there is a trend toward team research involving more or less intimate cooperation between workers in different disciplines. This is desirable and will no doubt increase. However, there will always be a place for an energetic, imaginative, and well-rounded independent investigator. May Dr. Gowen continue for many years to produce the type of research that he has described. Dr. Gordon: In considering methodology in this field, I would like to take just a minute or two to mention work of one of my colleagues at the Naval Medical Research Institute, Bethesda.621 Dr. Herbert S. Hurlbut's interest is in the transmission of arthropod-borne viruses, and he has determined the susceptibility of other arthropods than the ones naturally involved in transmission, using representative viruses of this arthropod-borne group. That is, he took arthropods such as house flies, a species of Lepidoptera, beetles, Hemiptera (all non-blood-sucking), and some hemophagous arthro- pods, and determined their susceptibility to the selected viruses by inoculation through the body wall with a capillary pipette. He found that many of these species were susceptible in the sense that they would propagate the virus, it could be recovered in fairly high titer in some cases, and could be transmitted from one individual to another GENETICS OF INFECTIOUS DISEASES 403 and maintained in such hosts indefinitely. At the present time we have seen no adverse effect on these hosts, and the demonstration of the virus is by subinoculation into mice. We have discussed these findings among ourselves as a method for investigating genetic factors in the ability of arthropods to propagate or transmit the viruses that infect vertebrates. With the system Dr. Hurlbut has used, the amount of virus going into the arthropod can be accurately controlled, and the amount present after a suitable incubation period can be satisfactorily measured. By choosing insect species in which genetic studies have already been made and in which various genetically described strains are available, suitable combinations of insect and virus could probably be found for a fruitful attack on this problem. Dr. Roderick: Dr. Gowen, did you find any difference between the selected and unselected lines with reference to qualitative manifestations of the diseases ? Differences in the symptoms between the lines might indicate that the selected lines were resistant because they had in some way changed their mode of reaction to certain specifications of the pathogen. Dr. Gowen : Symbiotic relations of an organism to one host, for which this host may supply a reservoir for nutrients, maturation, or multiplication, as well as acting as a vector for transmission of the organism and pathogenic relations of the same organism to a later ultimate host, constitute real challenges to genetic research. The two-step relations hold through a range of pathogenic organisms — viruses, bacteria, protozoa, helminths, fungi, and the like. The genetic controls for these diverse cases would seem to have been developed independently. Damage to the vector host may range from none observable, through moderate, to that which acts relatively slowly compared with the time necessary for infecting the ultimate host. The specificity of the host-parasite relations is so exact that basically the relations must be largely under genie control. For instance, some malarias and viruses seem to damage the mosquito but little, yet the mosquitoes offer conditions suitable for their multiplication. In retrospect it seems not unlikely that this long-standing relationship has an evolutionary history such that the virus or protozoan was initially pathogenic but that, with time and possibly through genetic modifications of both pathogen and host, they were able to live together in a symbiotic relationship. That genie changes are basic to this pattern is evident from studies on a number of related species displaying differences in reaction patterns. Huff608 some years ago found that Culex pipiens, to become a carrier of avian malaria, required the presence of the dominant gene of a pair of alleles, resistance being recessive. The second question concerns changes in virulence within particular lines of the pathogen. In our work Salmonella typhimurium 11C rarely changed its virulence. Zelle,1467 in a series of experiments covering three years, obtained convincing mutants in virulence only four times. The parental organisms were passed through successive transfer in mice of a resistant line or mice of a susceptible line. The mutants were always directional to greater virulence. Very rarely an avirulent mutant may also occur when planted on culture media. Salmonella gallinarum, on the other hand, 404 HOST-PARASITE RELATIONSHIPS I mutates either toward virulence or toward avirulence quite readily. A virulent culture placed on culture media favoring saprophytic growth for six months at refrigera- tor temperature will frequently have no virulence to chickens. On the other hand, avirulent gallinarium passed by blind passage through a series of as many as 20 chickens normally will mutate to full virulence at some point along the way. Dr. Roderick: Do your selected and unselected lines show any differences in symptomatic manifestations of the diseases? Dr. Gowen: In answer to Dr. Roderick's second question, our strains of mice do show differences in the manifestiation of disease when inoculated with the same line of Salmonella typhimurium 11C. There are distinct differences in morbidity. The S mice show scarcely any effects of the disease even when exposed to 2,000,000 organisms. The Ba mice are obviously sick when the exposure dose is 20 to 200 organisms. The other strains act as though they were between these extremes. Clear-cut differences in the survival curves are evident when they are analyzed for their different constants : mean, mode, standard deviations, skewness, kurtosis, and type of curve. Incidentally, there are no detectable immune bodies in the bloods of any of our strains before they have contact with the pathogen. One strain has a characteristic alpha-