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The mouse spot test, developed 23 years ago, is an in vivo assay capable of detecting genetic effects of several kinds, including intragenic mutations, minute deficiencies, deletions (through breakage or nondisjunction) of various amounts of chromosomal material, and somatic crossing-over. The method involves exposing embryos that are heterozygous for a number of coat-color markers to the test agent, and, 3 weeks later, looking for clones of mutant cells, i.e., spots of color expressing the recessive marker in an otherwise black fur. Spots having other causes may also be induced, specifically white midventral spots due to cytotoxic effects, and certain spots resulting from misdifferentiation. Spot-test results have, to date, been reported from 7 laboratories. Because the control results for any one cross and solvent were found to be reasonably consistent between the laboratories, we pooled these to develop a "historical" control with which experimental results for the same cross and solvent were compared. Experimental results were classified as positive, negative, or inconclusive on the basis of a multiple-decision procedure produced by the testing of the following 2 hypotheses: (1) the mutation frequency (induced + spontaneous) in treated mice is not higher than the mutation frequency in the appropriate pooled control, and (2) the induced mutation frequency of the treated mice is no less than 4 times as high as the observed mutation frequency in the appropriate pooled control. Each hypothesis was tested at the 5% significance level. To date, 30 substances have been employed in the spot test, including 3 that are solvents for some of the others. Of the remaining 27 (26 compounds and 1 mixture), 16 were positive, 6 negative, and 5 inconclusive. The 26 compounds fell into 27 chemical classifications (using a system provided for use by the GENE-TOX program). The inadequacies in the design and reporting of some past experiments indicate a need for a carefully specified protocol. When properly done, the spot test will fulfill a useful role in mutagenicity testing programs because (1) it is an in vivo mammalian assay, (2) it detects genetic effects of many kinds, and (3) it is relatively rapid. Since the test appears well suited to the identification of potent mutagens, its main value should be in screening large numbers of substances and singling out the potentially worst offenders to be further studied in germ-line mutagenesis tests.

Future research in mammalian germ-line mutagenesis will benefit from the reciprocal relationship that can exist between the production of mutations and their analysis. Thus, mutagenesis experiments supply altered genes, and thereby tools for basic studies; while, in turn, information on gene structure and function helps in understanding mechanisms of mutagenesis. The nature of genetic alterations recovered in specific-locus tests is illustrated by an account of the analysis of 112 radiation-induced mutations involving the c locus on chromosome 7. Using phenotypic characterizations of various kinds, deficiency mapping with nearby markers, and complementation studies, the mutants could be classed into 13 groups, and 8 functional units could be identified in the c-locus region. Twelve different deficiencies overlapping at c range in length from less than 2 to 6-11 cM. Using the deficiencies, as well as a tandem duplication that involves the c region, and several T(X;7)'s in which c is inactivated in a mosaic fashion, it is possible to generate gene doses from 0 to 3, in steps of 0.5, not only for c but also for other genes included in various deficiencies. Cis and trans configurations can also be compared. This array of genetic materials is now being used for the isolation of DNA sequences from genetically-defined regions. Results from analyses of mutations can contribute answers to some of the pragmatic questions in germ-line mutagenesis and risk assessment. Areas in which contributions may be expected are: the relative roles of intracellular conditions (e.g., nature of chromatin, presence of repair enzymes) and secondary circumstances (e.g., selection) in determining the quantity and quality of transmitted mutations; the validity of quantitative extrapolations, such as projections to low doses and calculations of doubling dose; and the relation between measures of mutation rate and projections of phenotypic damage.

In its simplest form the hypothesis of the single-active-X chromosome does not explain variegated-type position effects in the mouse. Inactivity appears not to involve one entire X chromosome; furthermore, even those parts of the chromosome that can change to an inactive state spread inactivation not to the entire attached piece of autosome, but along a gradient to limited distances.

New data have clearly confirmed the earlier finding that specific locus mutation rates obtained with chronic gamma irradiation of spermatogonia are lower than those obtained with acute x-rays. Since this result is in contrast to classical findings for Drosophila spermatozoa, and apparently contradicts one of the basic tenets of radiation genetics, it was important to determine what factors were responsible for it. Experiments undertaken for this purpose reveal the following: (i) the lower mutation frequency is due mainly to difference in dose rate of radiation, rather than quality; (ii) a dose-rate effect is not obtained in experiments with mouse spermatozoa, confirming classical findings for spermatozoa, and indicating that the explanation for intensity dependence in spermatogonia resides in some characteristic of gametogenic stage; and (iii) a dose-rate effect is found not only in spermatogonia but also in oocytes, where cell selection is improbable, indicating that the radiation intensity effect is on the mutation process itself. A threshold response for all mutations in spermatogonia and oocytes is not a necessary consequence of the findings. Plausible hypotheses consistent with the present results can lead to other predictions. From a practical point of view, the results indicate that the genetic hazards, at least under some radiation conditions, may not be as great as those estimated from the mutation rates obtained with acute irradiation. However, it should not be forgotten that even the lower mutation rates obtained with the present intensity levels are still appreciable (16).

The great strides made during the past two years in the whole field of mammalian cytogenetics have, in particular, enlarged our knowledge of the role of the mammalian sex chromosomes. The following summary briefly lists the most recent discoveries in the mouse, where genetic findings have played a relatively greater role than in the other species of mammals. The male-determining property of the mammalian Y chromosome, established earlier in mouse and man, has been further confirmed by the finding of an XXY mouse, which was detected by genetic means and has been studied cytologically. This animal is a fully viable, phenotypically normal, though sterile, male. Since various doubts concerning detectability of the XXY type have been removed by the discovery of this animal, it can be concluded that the occurrence of XXY in the mouse is extremely rare. It has been shown that the X chromosome of the mouse, when it is involved in certain chromosomal rearrangements, has the power to produce variegated-type position effects, a phenomenon formerly not observed in any animal except Drosophila. The fact that the X chromosome is involved in all four of the known cases of V-type position effect in the mouse indicates that it is strongly heterochromatic, while there may be little heterochromatin on the autosomes. Recent findings have shown that the presence of two X chromosomes is necessary for the expression of the position effect in one of them. This fact, when related to various cytological findings in other species, permits the hypothesis that, in mammals, genic balance requires the action of one X in a manner which precludes realization of its heterochromatic potentialities, so that only any additional X's present assume the properties characteristic of heterochromatin. A variety of different findings sheds light on the mechanisms that may lead to the occurrence of individuals with abnormal numbers of sex chromosomes. The XXY mouse proves, by virtue of its sex-linked marker genes, that nondisjunction can occur in the first meiotic division of a normal male (a proof not previously provided by human cases of XXY, which could have been of different origin). However, first-meiotic nondisjunction is apparently very rare in males, and there is not yet any evidence that it ever occurs in females. Data from numerous types of crosses involving five sex-linked markers yield the following results: no cases of X(M)X(M)Y or OX(P) have occurred to date; X(M)X(P)Y << X(M)O; OX(P) << X(M)O (where the superscripts M and P designate maternal and paternal derivation, respectively, of the X). The total frequency of XO individuals can be increased by irradiation shortly after fertilization. This treatment has yielded, in addition to X(M)O, several animals of the OX(P) constitution, a type that has not yet been found to occur spontaneously. The various findings on spontaneous and induced frequencies of mice with abnormal numbers of sex chromosomes lead to the conclusion that XO individuals are most often the result of events occurring after fertilization. Specifically, it is suggested that there exists a relatively high probability of loss of the paternally contributed sex chromosome some time between fertilization and the first cleavage(32).

Histological and cytological analyses of the testes were carried out in 42 sterile sons of males treated in the spermatozoal or spermatid stage with 250 mg/kg ethyl methanesulfonate (EMS) alone or after prefeeding with butylated hydroxytoluene (BHT); or treated with 200 R X-rays. Of the 42 sterile males, 17 had some mature spermatids, nine were blocked at diakinesis, 15 were blocked in pachytene, and one lacked spermatogenic cells altogether, having Sertoli cells only. Mitotic (spermatogonial) metaphases could therefore be analyzed in 41 of the males and meiotic configurations in 26.-(1) None of the males showed abnormalities in chromosome number, such as monosomy, trisomy, or mosaicism for either of these conditions. Certain classes of chromosome abnormalities that have been found associated with male sterility in other investigations, namely trisomies, XXY's, and X-autosome translocations, are not expected from treatment of 19A + Y cells when F(1) males are studied. (2) A very high percentage of the sterile males carried translocations. Direct meiotic evidence for this was found in 22 of the animals. In addition, 11 of the 16 that were blocked (or virtually blocked) in pachytene, and thus could be analyzed in mitosis only, consistently showed one abnormally short chromosome (or, one short plus one long), which presumably had resulted from unequal exchange (or sizable deficiency). Of the meiotically detected translocation males, 1 carried a T(A;Y), 17 had single autosomal translocations, and 4 had multiple autosomal rearrangements involving three, four, four, and six breaks, respectively. In addition, three males showed failure of X-Y pairing. (3) Translocations that cause sterility, rather than partial sterility, in males appear to be those in which at least one of the breaks occurs close to one end of a chromosome. The mitotic and meiotic evidences for this were found to be correlated. (4) It is proposed that many cases of induced F(1) male sterility may be the result of position effects produced when paracentromeric regions are translocated to euchromatic regions of certain other chromosomes. Since many translocations that produce partial sterility in the female cause complete sterility in the male, the male must be assumed to be more susceptible to disturbances of fertility by the postulated mechanism. (5) There is evidence that EMS, especially in the lower dose range, more often breaks chromosomes near one of their ends than does X-irradiation.

Mice of genotypes S1/S1(d), S1/+, and S1(d)/+ (but not S1(T)/S1(T) or S1(T)/+) were resistant to spleen focucs-forming virus. The "environment" in which hemopoietic target cells for this virus develop was not conducive for infection or focus formation, or both, but the target cells were not altered directly. The same genes appear to regulate hemopoiesis and leukemogenic transformation.