Genetic Analysis of Three Dominant Female-Sterile Mutations Located on the X Chromosome of DROSOPHILA MELANOGASTER

Three dominant female-sterile mutations were isolated following ethyl methanesulfonate (EMS) mutagenesis. Females heterozygous for two of these mutations show atrophy of the ovaries and produce no eggs (ovo ^D1) or few eggs (ovo^D2); females heterozygous for the third mutation, ovo^D3, lay flaccid eggs. All three mutations are germ line-dependent and map to the cytological region 4D-E on the X chromosome; they represent a single allelic series. Two doses of the wild-type allele restore fertility to females carrying ovo^D3 and ovo^D2, but females carrying ovo^D1 and three doses of the wild-type allele remain sterile. The three mutations are stable in males but are capable of reversion in females; reversion of the dominant mutations is accompanied by the appearance, in the same region, of a recessive mutation causing female sterility. We discuss the utility of these mutations as markers of clones induced in the female germ line by mitotic recombination as well as the nature of the mutations.     

X-Linked Female-Sterile Loci in DROSOPHILA MELANOGASTER

We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed.     

Dominant Maternal-Effect Mutations of DROSOPHILA MELANOGASTER Causing the Production of Double-Abdomen Embryos

Dominant mutations at two loci, BicaudalC (BicC ) and BicaudalD (BicD), cause heterozygous females to produce double-abdomen embryos. These mutations cause the production of embryos with a range of defects extending from the anterior end of the differentiated embryo. The same array of defective embryos is caused by mutations at either locus and is similar to that produced by the original mutation at bicaudal (bic). The array of defective embryos suggests that these mutations cause the loss of positional values from the anterior end of the embryo, associated with a duplication of the posterior end if too few positional values remain. BicaudalD mutations appear to be antimorphic, gain-of-function mutations, whereas BicaudalC mutations are likely to be hypomorphic or amorphic mutations. Mutations at all these loci (bic, BicC and BicD) act as mutual enhancers of each other, and a number of other maternal-effect mutations also act to either enhance or suppress the expression of these dominant bicaudal mutations.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER. IX. Dominant Cold-Sensitive Lethals on the Autosomes

Ethyl methanesulfonate-treated autosomes were screened for the presence of dominant cold-sensitive (DCS) lethal mutations in Drosophila melanogaster. None was found among 6,552 treated and 168 untreated third chromosomes. Twenty-three DCS-L chromosomes which caused death at 17 degrees C but survived at 22 degrees C and 29 degrees C were recovered from 5,046 mutagenized chromosome 2's.-The DCS-L mutations all mapped around dp and appeared to be functionally allelic. Lethality of heterozygotes for most of the DCS-L's occurred over a prolonged interval from the embryonic through the larval instars. Prolonged incubation at 17 degrees C did not demonstrate any maternal effect on zygotic survival.     

Analysis of Y-Linked Mutations to Male Sterility in DROSOPHILA MELANOGASTER

Mating type in haploid cells of the yeast Saccharomyces cerevisiae is determined by a pair of alleles MATa and MAT alpha. Under various conditions haploid mating types can be interconverted. It has been proposed that transpositions of silent cassettes of mating-type information from HML OR HMR to MAT are the source of mating type conversions. A mutation described in this work, designated AON1, has the following properties. (1) MAT alpha cells carring AON1 are defective in mating. (2) AON1 allows MAT alpha/MAT alpha but not MATa/MATa diploids to sporulate; thus, AON1 mimics the MATa requirement for sporulation. (3) mata-1 cells that carry AON1 are MATa phenocopies, i.e., MAT alpha/mata-1 AON1 diploids behave as standard MAT alpha/MATa cells; therefore, AON1 suppresses the defect of mata-1. (4) AON1 maps at or near HMRa. (5) Same-site revertants from AON1 lose the ability to convert mating type to MATa, indicating that reversion is associated with the loss of a functional HMRa locus. In addition, AON1 is a dominant mutation. We conclude that AON1 is a regulatory mutation, probably cis-acting, that leads to the constitutive expression of silent a mating-type information located at HMRa.     

Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

Induced Dominant Lethal Mutations and Cytotoxic Effects in Germ Cells of DROSOPHILA MELANOGASTER with Trenimon, Pdmt and Sodium Monofluorophosphate

Male and female Drosophila melanogaster with special sex chromosome or special autosome constitutions were fed with the mutagenic chemicals Trenimon (2,3,5-trisethyleneimino-1,4-benzoquinone) and PDMT (1-phenyl-3, 3-dimethyltriazene) and with the toxic substance Na2PO3F (sodium monofluorophosphate). The frequency of dominant lethality was recorded among the progeny. The results clearly show that dominant lethality is dose dependent for Trenimon- or PDMT-treated chromosomes in mature sperm and mature oocytes, and an increased amount of chromosomal material per nucleus yields an enhanced lethality. In contrast, a pure toxic effect of Na2PO3F on mature oocytes was demonstrated with one type of female. --With the stocks of Drosophila used, it is possible to distinguish between mutagenic and toxic effects of chemicals on the germ cells. Therefore, dominant lethality can be used as a simple and quick screening test for chemical mutagens.     

Revertants of Dominant Mutations Associated with the Antennapedia Gene Complex of DROSOPHILA MELANOGASTER: Cytology and Genetics

Using X-ray mutagenesis we have induced and recovered phenotypic revertants of four dominant mutations thought to be associated with the Antennapedia complex of Drosophila melanogaster. These include seven revertants of Antennapedia-73b (Antp^73b), six of Extra Sex Combs of Wakimoto (Scx^w), three of Deformed (Dfd) and one of Humeral (Hu). Fifteen of the 17 revertants are associated with chromosomal aberrations and localize Antp^73b, Scx ^w and Hu to polytene chromosome bands 84B1,2. The Dfd lesion is apparently located in or adjacent to bands 84A4,5. Since all of the dominants are reverted by events that delete their respective chromosomal loci, we conclude that all four are the result of a gain-of-function lesions. Complementation analysis of the various revertant chromosomes has shown that Scx^w and Hu are dominant allelic variants of the Antp locus. The Dfd lesion represents a dominant mutation at a locus just proximal to Antp and previously only occupied by recessive lethal mutations. Characterization of the revertants of Scx^w and a comparison with the properties of the original mutation has revealed that the original lesion has effects on both the Antp and Sex Combs Reduced (Scr) loci and that these defects are in some cases separable by the reverting event.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER Xii. the Genetic and Developmental Effects of Dominant Lethals on Chromosome 3

Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.     

Partial Dominance of Ems-Induced Mutations Affecting Viability in DROSOPHILA MELANOGASTER

More than 700 EMS-treated second chromosomes marked with either cn (cinnabar) or bw (brown), and derived from long-inbred stocks, were measured for their heterozygous effects on viability in both isogenic (homozygous) and nonisogenic (heterozygous) backgrounds. Each test was replicated five times. When the background was homozygous, flies heterozygous for a treated chromosome were an average of 2.1% less viable, per 0.005 m EMS, than flies heterozygous for an untreated chromosome. Classified according to their homogous effect in an accompanying series of crosses, the lethal-bearing chromosomes (L), which carry genes of less drastic effects as well, reduced the viability of their heterozygous carriers by 3.3%, severe detrimentals (D(s)) by 2.2%, and mild detrimentals (D(m)) by 1.2% at this dose. In the heterozygous background, the mean heterozygous disadvantage for the entire group was 1%, or about half as large.--When computed separately for each count from a single mating, the heterozygous disadvantage was consistently greatest for the earliest counts (4.8%), next highest for the middle count (0.8%), and lowest in the latest count (0.5%), in the homozygous background, indicating that mutant heterozygotes were delayed in time of emergence. The figures in the heterozygous background were, again, reduced, but in the same direction.-The relative viability disadvantage of the cn marker was about 2(1/2) times greater in the homozygous than in the heterozygous background, further supporting the conclusion that the homozygous background can accentuate differences. The enhancement of treatment and marker effects could be a direct result of the level of background heterozygosity per se or attributable to the reduced vigor of the inbred strain.-Dominance, a measure of the heterozygous effect of a mutant relative to its homozygous effect, is greater for genes with small homozygous disadvantage than for more drastic genes. In the homozygous background the average dominance for lethals was 0.019 in contrast to 0.183 for mild detrimentals, supporting other published results suggesting that genes with milder effect, because they occur more frequently, have a greater impact on a population.-The homozygous D:L ratio of EMS mutations was 0.266 and the D(m): L ratio, 0.092, which are lower than comparable load ratios for spontaneous mutations, but greater than for X-ray induced mutations.     

Mutations in Genes Encoding Essential Mitotic Functions in DROSOPHILA MELANOGASTER

Temperature-sensitive mutations at 15 loci that affect the fidelity of mitotic chromosome behavior have been isolated in Drosophila melanogaster. These mitotic mutants were detected in a collection of 168 EMS-induced X-linked temperature-sensitive (ts) lethal and semilethal mutants. Our screen for mutations with mitotic effects was based upon the reasoning that under semirestrictive conditions such mutations could cause an elevated frequency of mitotic chromosome misbehavior and that such events would be detectable with somatic cell genetic techniques. Males hemizygous for each ts lethal and heterozygous for the recessive autosomal cell marker mwh were reared under semirestrictive conditions, and the wings of those individuals surviving to adulthood were examined for an increased frequency of mwh clones. Those mutations producing elevated levels of chromosome instability during growth of the wing imaginal disc were also examined for their effects on chromosome behavior in the cell lineages producing the abdominal cuticle. Fifteen mutations affect chromosome behavior in both wing and abdominal cells and thus identify loci generally required for the fidelity of mitotic chromosome transmission. Mapping and complementation tests show that these mutations represent 15 loci. One mutant is an allele of a locus (mus-101) previously identified by mutagen-sensitive mutants and a second mutant is an allele of the lethal locus zw 10.--The 15 mutants were also examined cytologically for their effects on chromosomes in larval neuroblasts. Taken together, the results of our cytological and genetical studies show that these mutants identify loci with wild-type functions necessary for either maintenance of chromosome integrity or regular disjunction of chromosomes or chromosome condensation. Thus, these mutations define a broad spectrum of genes required for the normal execution of the mitotic chromosome cycle.     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER with Special Attention to Eggshell Mutants

To study genes that function mainly or exclusively during oogenesis, we have isolated and analyzed female-sterile mutations, with special emphasis on those that affect eggshell formation. Following treatment that induced 61 to 66% lethals, 8.1% of the 1071 X chromosomes tested carried recessive female sterility mutations (87 isolates), and 8.0% carried partial female-sterile mutations (86 isolates), respectively. In addition, three dominant female steriles were recovered. Some of the mutants had very low fecundity, and others laid morphologically normal eggs that failed to develop. A third category included 29 mutants that laid eggs with morphological abnormalities: 26 were female steriles, two were partial female steriles and one was fertile. Mutants of this third category were characterized in some detail and compared with 40 previously isolated mutants that laid similarly abnormal eggs. Approximately 28–31 complementation groups with morphological abnormalities were detected, some of which were large allelic series (11, 9, 7, 6 and 5 alleles). Twenty-four groups were mapped genetically or cytogenetically, and 21 were partially characterized by ultrastructural and biochemical procedures. Of the latter, one group showed clear deficiency of yolk proteins, and nine showed prominent ultrastructural defects in the chorion (at least eight accompanied by deficiencies in characterized chorion proteins). At least six groups with clear-cut effects were found at loci not previously identified with known chorion structural genes.     

Pleiotropic Effects on Fitness of Mutations Affecting Viability in DROSOPHILA MELANOGASTER

The relative viabilities and fitnesses of wild-type second chromosomes in heterozygous condition were determined. Joint analysis of these permitted an estimation of a parameter that relates the viability effect of a mutation to its effect on fitness as a whole. For newly arisen mutations, the estimate was slightly greater than one, indicating that the reductions in viability caused by these mutations are associated with reductions in other components of fitness. For mutations from an equilibrium population, the estimate of the parameter was near zero, implying that the deleterious viability effects of these mutations are compensated by improvements in other aspects of fitness.