Synergistic interactions between two skeletal mutations in mice: individual and combined effects of the semidominants cleidocranial dysplasia (Ccd) and short digits (Dsh).

Heterozygotes for cleidocranial dysplasia (Ccd) and short digits (Dsh) were crossed to test whether synergistic interactions occur between different dominant mutations whose individual pleiotropic phenotypic effects exhibit a common feature. These unlinked mutations are homozygous lethal, and they are congenic on the C57BL/10 background. Each mutation caused more than 10 different anomalies and showed variable expressivity. Each mutation produced several malformations that were present in every heterozygote. Seven different synergistic interactions were found, including one that yielded an entirely new abnormality not predicted from any abnormalities found in either of the single heterozygotes. Although synergistic interactions between dominant mutations have not, to our knowledge, been described in humans, these findings in mice increase the probability that they occur in humans. Under certain circumstances in human populations, the segregation of mutations causing synergistic interactions of the type demonstrated might be confused with recessive inheritance. It will be important to learn whether synergistic interactions can occur between other mutations. If they can, it will probably become important to take synergistic interactions into account when estimating the genetic hazards to humans from mutagens. Three antagonistic interactions were also found.

Specific-locus experiments show that female mice exposed near the time of birth to low-LET ionizing radiation exhibit both a low mutational response and a dose-rate effect.

Female mice were exposed to 300 R of 73-93 R/min X-radiation either as fetuses at 18.5 d post conception (p.c.) or within 9 h after birth. Combining the similar results from these two groups yielded a specific-locus mutation frequency of 9.4 X 10(-8) mutation/locus/R, which is statistically significantly higher than the historical-control mutation frequency, but much lower than the rate obtained by irradiating mature and maturing oocytes in adults. Other females, exposed at 18.5 days p.c. to 300 R of 0.79 R/min gamma-radiation, yielded a mutation frequency that was statistically significantly lower than the frequency at high dose rates. The low-dose-rate group also had markedly higher fertility. It appears that the dose-rate effect for mutations induced near the time of birth may be more pronounced than that reported for mature and maturing oocytes of adults. A hypothesis sometimes advanced to explain low mutation frequencies recovered from cell populations that experience considerable radiation-induced cell killing is that there is selection against mutant cells. The reason for the relatively low mutational response following acute irradiation in our experiments is unknown; however, the finding of a dose-rate effect in these oocytes in the presence of only minor radiation-induced cell killing (as judged from fertility) makes it seem unlikely that selection was responsible for the low mutational response following acute exposure. Had selection been an important factor, the mutation frequency should have increased when oocyte killing was markedly reduced.

Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia.

The extreme mutagenic effectiveness of N-ethyl-N-nitrosourea in the mouse has permitted the accumulation of the most extensive dose--response data yet obtained for chemical induction of specific-locus mutations in spermatogonia. In the lower portion of the curve, below a dose of 100 mg/kg, the data fall statistically significantly below a maximum likelihood fit to a straight line. Independent evidence indicates that, over this dose range, ethylnitrosourea reaches the testis in amounts directly proportional to the injected dose. It is concluded that, despite the mutagenic effectiveness of ethylnitrosourea, the spermatogonia are apparently capable of repairing at least a major part of the mutational damage when the repair process is not swamped by a high dose. This finding is important both in basic studied on the mutagenic action of chemicals in mammals and in risk estimation.

Spermatogenic stage sensitivity to 6-mercaptopurine in the mouse.

Hybrid (101 X C3H)F1 male mice were given [3H]thymidine intraperitoneally, and 1 h later 150 mg/kg 6-mercaptopurine in 0.03 N NaOH. Autoradiography of testis sections showed that the rate of spermatogenesis was not altered, and the time of appearance of labeled spermatozoa in the ejaculate indicated that 6-mercaptopurine also had no effect on minimum sperm transport time. Labeled spermatozoa persisted in the ejaculate for a longer interval in 6-mercaptopurine-treated males than in controls, most likely as a result of oligospermia. Combined treatment with 150 R X-rays and 150 mg/kg 6-mercaptopurine gave an additive effect and demonstrated conclusively that the peak incidence of dead implants observed at 30.5 - 35.5 days can be attributed to cells treated as preleptotene spermatocytes and must result from genetic damage that is not cytologically detectable; previous work has shown that chromatic and isochromatid breaks at diakinesis-metaphase I occurred only on days 14 and 15 after 150 mg/kg 6-mercaptopurine. From the present experiments it is clear that these aberrations are not related to the dominant lethality at 30.5 - 35.5 days.

Analysis of the albino-locus region of the mouse: IV. Characterization of 34 deficiencies.

Thirty-four independent nonviable c-locus mutations (types cal, albino lethal and cas, albino subvital), derived from radiation experiments, were tested for involvement of nearby markers tp, Mod-2, sh-1, and Hbb: 10, 22, and 2 involved, respectively, none of these markers, Mod-2 alone, and Mod-2 plus sh-1. When classified on this basis, as well as according to developmental stage at which homozygotes die, and by limited complementation results, the 34 independent mutations fell into 12 groups. From results of a full-scale complementation grid of all 435 possible crosses among 30 of the mutations, we were able to postulate an alignment of eight functional units by which the 12 groups fit a linear pattern. Abnormal phenotypes utilized in the complementation study were deaths at various stages of prenatal or postnatal development, body weight, and reduction or absence of various enzymes. Some of these phenotypes can be separated by complementation e.g., there is no evidence that mitochondrial malic enzyme influences survival at any age); others cannot thus be separated (e.g., glucose-6-phosphatase deficiency and neonatal death).--We conclude that all of the nonviable albino mutations are deficiencies overlapping at c, and ranging in size from less than 2cM to 6-11 cM. The characterization of this array of deficiencies should provide useful tools for gene-dosage studies, recombinant-DNA fine-structure analyses, etc. Since many of the combinations of lethals produce viable albino animals that resemble the standard c/c type, we conclude (a) that the c locus contains no sites essential for survival, and (b) that viable nonalbino c-locus mutations (cxv) are the result of mutations within the c cistron. Viable albinos (cav, the majority of radiation-induced c-locus mutations) may be intracistronic mutations or very small deficiencies.

Analysis of the albino-locus region of the mouse. III. Time of death of prenatal lethals.

The stage at which homozygotes die was determined for 28 mutations (general symbol c*) at the albino (c) locus, of which 26 had earlier been found to be probably prenatally lethal. Within each of the mutant stocks, the uterine contents of c*/c(ch) females, made pregnant either by c*/c(ch) males ("Ex" series) or by c(ch)/c(ch) males ("Co" series), were examined between 13 and 17 days postconception. Altogether, 743 females were dissected and 7197 corpora lutea (representing ovulations) counted. In selected stocks, an additional 40 and 13 females were dissected on days seven or nine, respectively.-In each of the 26 presumed prenatally lethal mutants, there was a deficiency of living fetuses in the Ex, as compared with the Co, group. Overall, this deficiency was 23.6% (expectation, 25% c*/c*). All meaningful excesses were in numbers either of moles (death shortly before, during, or just after implantation), or of early preimplantation losses. Homozygotes in none of the mutant stocks die between days nine and 19 postconception. Of 24 c-locus mutants known to be deficiencies since they lack the closely linked Mod-2, 13 clearly kill before implantation, ten around implantation, and one neonatally. The c and Mod-2 loci and the region between them are not needed for intrauterine survival.-There are indications that the distinction between early-preimplantation death and implantation death may, in a general way, be related to length of the deficiency.