Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

Genetic effects of cosmic radiation in Drosophila melanogaster]

To examine possible effects of space radiation on living organism, we have analyzedtwo types of mutations, sex-linked recessive lethal mutations and somatic mutations, in fruit fly of the species Drosophila melanogaster. Drosophila strains used were wild type strains and a radiation-sensitive strain mei-41. Two different developmental stages of samples were sent into space; young adult males to analyze sex-linked recessive lethal mutations and about 30hr-old larvae to detect somatic mutations in wing epidermal cells. For wild type and mei-41 strains each, about 200 adult male flies and about 6,000 larvae were loaded on space shuttle Endeavour. The male flies returned from space were mated to virgin female flies of a tester strain, and the presence of the lethal mutations was analyzed at F2 generation. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-4 1, respectively, than those in ground control groups. Most larvae sent to space emerged as adult flies within about 10 days after the landing. The presence of wing-hair somatic mutations, which give morphological change in hairs growing on the surface of wing epidermal cells, was analyzed under microscope. In wild type strain Muller-5, the frequency of wing hair mutant spots in flight group was about 1.5-fold higher than that in ground control, and in Canton-S-derived wild type strain the frequencies were similar between the two groups. By contrast, for mei-41 strain the mutation frequency was lower in flight group than in control group. The observed higher frequency of lethal mutations in the flight group might be due to a possibility that radiation effects on reproductive cells could be greatly enhanced under micro gravity. However, if this would be the case, we do not have appropriate explanation for the apparent absence of such synergistic effects on somatic wing-hair mutation system.     

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.     

Farm-grade chlorpyrifos (Durmet) is genotoxic in somatic and germ-line cells of Drosophila.

The mutagenic potential of Durmet, a farm-grade formulation of chlorpyrifos, was studied in the Drosophila wing mosaic and sex-linked recessive lethal tests. Larvae of the 2nd or 3rd instar carrying suitable recessive genetic markers on chromosome 3 were exposed to different concentrations of the insecticide and the frequency of induction of mutant mosaic spots on the wings was noted. The Basc technique was followed to study the induction of sex-linked recessive lethals. On the basis of the frequency of induction of mosaic wing spots and sex-linked recessive lethals, it is concluded that Durmet is genotoxic in somatic cells as well as germ cells of Drosophila.     

Mutation Induction in the Male Recombination Strains of DROSOPHILA MELANOGASTER

One group of the second chromosome lines isolated from a southern Texas population of Drosophila melanogaster, which has been known to show relatively high frequencies of male recombinations, was found to increase the frequency of sex-linked recessive lethal mutations from a control frequency of 0.18% to 1.63%. The second group, which showed a very much reduced frequency of male recombinations, was found to cause a slight increase to 0.48%, although it was not statistically significant. The first group was also tested for the recessive lethal mutation frequency in the second chromosome; the frequency increased from a control frequency of 0.28% to 2.82%. Mapping of a portion of the sex-linked lethals indicated a distribution along the entire X chromosome, although there was a tendency of clustering towards the tip of the X chromosome. One sex-linked lethal line so far tested was found to be associated with an inversion (approximate breakpoints, 14A-18A). It was suggested that the element causing male recombination might be similar to the hi mutator gene studied earlier by Ives (1950).     

Heterogeneity of Lethals in a "Simple" Lethal Complementation Group

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster, eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua 1 and pur 1, which yield auxotrophic mutations.--The eight new mutations have been characterized phenotypically in double heterozygotes with gua 1, pur 1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2. The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.--Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. I. Recessive Lethal Mutations

The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.     

The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.     

A screen to identify Drosophila genes required for integrin-mediated adhesion.

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function.     

Characterization of X-Linked Recessive Lethal Mutations Affecting Embryonic Morphogenesis in Drosophila Melanogaster

This study attempted to assay the zygotic contribution of X chromosome genes to the genetic control of embryonic morphogenesis in Drosophila melanogaster. A systematic screen for X-linked genes which affect the morphology of the embryo was undertaken, employing the phenotype of whole mount embryos as the major screening criterion. Of 800 EMS-induced lethal mutations analyzed, only 14% were embryonic lethal, and of these only a minority affected embryonic morphogenesis. By recombination and complementation analyses, the mutations that affected embryonic morphogenesis were sequestered into 26 complementation groups. Fourteen of the loci correspond to genes previously identified in a large-scale screen in which fixed cuticles were examined, and 12 new loci have been identified. Most of the mutations which disrupt embryonic morphology had specific and uniform mutant phenotypes. Mutations were recovered which disrupt major morphogenetic events such as gastrulation, germ band retraction and head involution. No mutations were found which arrest the embryos prior to blastoderm formation. However, a novel class was found, one comprised of mutations which interfere with the development of internal structures but not cuticular structures. Nevertheless, saturation of the X chromosome for genes important for embryonic morphogenesis is probably incomplete.     

Trenimon-induced sex chromosome loss in Drosophila: different dose-responses for ring-X loss as compared to rod-X loss and Y-marker loss

Drosophila melanogaster males carrying either a ring- or a rod-shaped X-chromosome were injected or fed with Trenimon (triaziquone) at concentrations ranging from 5 X 10(-5) to 2 X 10(-2) mM. The F1 generation was assayed for the occurrence of total sex chromosome loss and of Y-chromosome markers. Sex-linked recessive lethal tests were carried out simultaneously. The data show that significant induction of ring-X loss occurs already at very low treatment concentrations (5 X 10(-5) -10(-4) mM) whereas rod-X loss or Y-marker loss is only seen at 2-5 X 10(-3) mM and higher. Induction of sex-linked recessive lethals is observed from 10(-4) -10(-3) mM on. These results add to existing evidence that loss of ring-X chromosomes, induced by some chemicals, may proceed by a mechanism different from the kind of events leading to chromosome breakage, as measured by rod-X loss and Y-marker loss.     

Site-Specific Instability in DROSOPHILA MELANOGASTER: The Origin of the Mutation and Cytogenetic Evidence for Site Specificity

During a study of delayed mutations, an unstable X chromosome (Uc) was detected. Spontaneous X-linked recessive lethal mutations were detected in 34 of 993 sperm sampled from 50 males carrying this chromosome. All but three of the 34 lethals originated as clusters in three of the 50 males Cytogenetic and complementation analyses revealed 14 intrachromosomal rearrangements: ten inversions, two reverse repeats, one deficiency and one transposition. Eight of the 14 rearrangements have one break in the 6F1-2 doublet and two rearrangements have a break in 6F1-5 of the X chromosome. The remaining four rearrangements have in addition to the aberrations a lethal point mutation between 6F1 and 6F5. Though each of the lethal lines was established from a single lethal-bearing female, chromosome polymorphism is evident in 17 of the 18 lines having rearrangements, with certain aberrations recurring in several lines. The lethal mutations revert frequently to the nonlethal state, and cytological evidence indicates that more than one mutational event may occur at the unstable locus of the chromosome during one generation. Two lethal lines had more than one type of chromosome rearrangement sharing a common breakpoint. These observations are consistent with the view that the instability of the Uc lines is caused by a transposable element capable of site-specific chromosome breaks and perpetual generation of mutations. The mutagenic and genetic properties of transposable elements can be related to the two-mutation theory of KNUDSON (1971) for cancer initiation.     

Genetic analysis of Drosophila melanogaster polytene chromosome region 44D-45F: loci required for viability and fertility

A genetic screen to identify mutations in genes in the 45A region on the right arm of chromosome 2 that are involved in oogenesis in Drosophila was undertaken. Several lethal but no female sterile mutations in the region had previously been identified in screens for P-element insertion or utilizing X rays or EMS as a mutagen. Here we report the identification of EMS-induced mutations in 21 essential loci in the 45D-45F region, including 13 previously unidentified loci. In addition, we isolated three mutant alleles of a newly identified locus required for fertility, sine prole. Mutations in sine prole disrupt spermatogenesis at or before individualization of spermatozoa and cause multiple defects in oogenesis, including inappropriate division of the germline cyst and arrest of oogenesis at stage 4.     

Comparison of the molecular and genetic map of the 2B6-2B7-8 of Drosophila melanogaster X-chromosome]

Molecular and genetic data were compared for the 2B6-2B7-8 region of the Drosophila melanogaster X chromosome. This region contains the dor (deep orange) and swi (single wing) genes influencing ecdysterone-dependent gene expression. Genes which had not been identified previously by genetic methods were shown to be present in this region. Two novel loci, designated a6 and b6, were characterized in detail. Both genes are expressed throughout Drosophila embryogenesis. The product of b6 has a homology with mammalian pentraxins. This is the first Drosophila gene found to contain the pentraxin motif.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

Mutagenicity of 7,12-dimethyl[a]anthracene and some other aromatic mutagens in Drosophila melanogaster

The optimal conditions for mutagenesis studies with DMBA and some other aromatic carcinogens in Drosophila were investigated in detail. The results presented in this paper indicate the following.The mutagenic effectiveness of DMBA is dependent on the route of administration, injection being far more effective when compared with feeding.The choice of the solvent is a crucial experimental condition. DMBA, when dissolved in oil/DMF, is ineffective whereas a special fat emulsion of DMBA gives high mutation frequencies.There appears to be an extreme strain dependence in the mutagenicity of DMBA. Mutagenic effectiveness in strain Berlin-K was rather low, whereas Oregon-K and Karsnäs-60 proved to be very susceptible to DMBA.Under the conditions of test, DMBA did not induce loss of a ring-X chromosome and did not produce recessive lethal mutations in such a chromosome.DMBA did not produce 2–3 translocations to any significant extent.An increase in DMBA-induced recessive lethal mutations was found upon storage of treated sperm with an optimal storage time of 4–10 days.DMBA is efficient in the production of delayed recessive lethal mutations in strain Berlin-K. Twice as many lethals were recovered with the F3 generation as compared with those in F2. In strain Oregon-K, where the frequency of F2 lethals was much higher than in strain Berlin-K, the ratio of F3/F2 lethals was clearly lower.Enzyme induction with phenobarbital reduces the mutagenic effectiveness of DMBAWith TMBA, similar strain differences in sensitivity were observed as those found for DMBA. Whereas TMBA was not mutagenic in Berlin-K, considerable mutagenicity was observed in Oregon-K and Karsnäs-60.Injection of carcinogenic polycyclic aromatic hydrocarbons and aromatic amines, when dissolved in special fat emulsions, enhances the mutagenic effectiveness of some compounds (DMBA, TMBA, DA and AcO-AAF), but this procedure does not always solve the problems-pertinent to these classes of promutagens in Drosophila.     

Mutations Affecting Functions of the Drosophila Gene Glued

Glued mutations in Drosophila comprise an essential complementation group with complex developmental effects. The original Glued mutation (Gl) has dominant nonlethal effects in heterozygous flies, principally on the morphogenesis of the visual system. Gl also has a recessive lethal effect early in development. Mutations that reverse the dominant visual effects of Gl (GlR mutations) were induced by gamma-radiation or by insertions of the transposable P element. The GlR(G) mutations induced by gamma-radiation do not reverse the lethal effect of Gl; these appear to be null mutations, some of which (and possibly all) delete segments of the Glued region. The GlR(P) mutations induced by insertion of the P element also reverse concomitantly a recessive lethal effect of Gl, suggesting that both the recessive and dominant effects are controlled by the same gene. The reversal of a lethal effect of Gl by the P element is remarkable, since it indicates that an essential gene function can be restored by insertion of unrelated DNA. Another class of lethal Glued mutations was induced in the normal Gl+ strain by ethyl methanesulfonate (EMS). The EMS mutations belong to the same essential complementation group as Gl, but do not have the strong dominant effects of Gl on the visual system. The GlR(P) mutations provide a molecular marker for the Glued gene, which was used to map the gene to the 70C2 band of chromosome 3L by in situ hybridization of a P element probe to polytene chromosomes from the GlR(P) strains and also to isolate clones of Glued genomic DNA for molecular studies of the normal gene and the various Glued mutations.