Neutron- and X-ray-induced mutations at the yellow, white, forked and vermilion loci of Drosophila melanogaster; a preliminary analysis

Neutrons and X-rays were used to induce mutations at the yellow, white, vermilion and forked loci of Drosophila melanogaster by irradiation of spermatozoa in males. The mutations were characterized for the presence and location of simultaneously induced rearrangements and recessive lethal mutations. F1 females carrying induced visible mutations were identified, described and tested for fertility. The data are given in this paper. The proportions of mutants at the 4 loci, the ratios of whole-body: mosaic mutations, and the fertility of the mutant-carrying F1 females were similar for both types of radiation. Differences were observed between the frequencies of induced visible mutations and the rates of coincident visible and lethal induction. Although the analysis of the mutant chromosomes has not yet been completed, our data can be interpreted as providing confirmation that there are qualitative differences between the genetic effects of neutrons and X-rays.     

Mutations at six additional loci of Drosophila melanogaster cause alkylation hypermutability

8 mutagen-sensitive strains of Drosophila melanogaster were examined for their effects on alkylation-induced mutagenesis. Using methylnitrosourea as the DNA-damaging agent and the sex-linked recessive lethal test as the monitor of genetic endpoint, 6 of these strains were shown to be hypermutable following exposure to this alkylating agent. Previous studies of 6 other genes have demonstrated that strains exhibiting alkylation hypermutability are completely defective in repair replication following alkylation-induced DNA damage. The present observations suggest that at least 12 loci may be required for excision repair of alkylation DNA damage in this species.     

Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.     

Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters.     

Molecular Analysis of Diepoxybutane-Induced Mutations at the rosy Locus of Drosophila melanogaster

We have analyzed at the molecular level diepoxybutane-induced mutants determined to have lesions affecting expression of the ry locus. Of the 21 mutants analyzed here, genetic analysis suggested that five were putative deficiencies involving ry and adjacent lethal loci. However, molecular analysis confirmed that only two of these five putative deficiencies were in fact deletions detectable by the methods used in the analysis. The remaining 16 mutants were viable as homozygotes, suggesting that their lesions were confined to the ry locus. Seven of these 16 intragenic mutants were determined to be deletions of genetic material as evidenced by altered restriction patterns relative to the wild type patterns. Thus, nine of 21 (43%) diepoxybutane-induced mutants are due to deletions ranging in size from approximately 50 base pairs to more than 8 kilobase pairs. Most of the deletions (seven of nine or 78%) are intragenic and less than 250 base pairs in size; it seems that most, if not all, affect coding rather than regulatory sequences.     

Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster

A genetic analysis of the dominant mutation Glued that perturbs the development of the normal axonal architecture of the fly's visual system was undertaken. Ten new alleles at this locus were identified and characterized. Two complementation groups that were identified failed to complement the original allele, suggesting that it is a double mutant or that it resides at a complex locus. Several of the new alleles display visual-system abnormalities similar to those of the original mutation. Seven of the eight members of one complementation group are embryonic/early larval lethals, like the original mutation. The other allele in this group is temperature sensitive. Homozygous mutant adults exhibit a temperature-sensitive female sterile phenotype. Unsuccessful attempts to recover genetic mosaics carrying clones of cells homozygous for some of these mutations revealed that they are either essential for the viability of individual cells or that they affect some other fundamental cellular function, such as mitosis or the ability to participate in tissue level organization, which prevents them from being recovered in adult mosaics. This also indicates that these mutations do not specifically affect neural cells. A number of X-ray- and EMS-induced partial and complete phenotypic "revertants" of the original allele have also been isolated as material for a comparative analysis of visual system development. All "revertants" that alter the abnormal eye phenotype towards the wild type have similar impact on the organization of the optic lobe.     

Mutations induced by X-rays and some chemical reagents changing Drosophila melanogaster life span

It has been shown that most of Drosophila melanogaster mutant lines obtained as a result of X-rays irradiation (XI) as well as of the combined action of XI and some chemical agents are characterized by decreased indexes of average (7-40 %) and maximal (1-35 %) life span. Insertion-excision processes at the instable genes white and cut are among the reasons of decreased vitality and shortened life span in induced mutants. Collection of neurodegenerative mutants has been obtained under the influence of ENU. Fast dying of flies and decreased vitality correlated with time point of neurodegenerations in brain structure.     

Enzymatic variation at seven loci in nine natural populations of Drosophila melanogaster

Allozyme polymorphisms at seven loci have been studied in nine natural populations of Drosophila melanogaster from the Saône and Rhône valleys sampled in 1973 and 1974. A great deal of polymorphism was observed; an individual was on the average heterozygous at 20.2% of its loci. The populations were genetically very homogeneous throughout the region sampled. The number of ovariolae per female varied from one group of populations to another depending on their geographical separation. Yet the number of ovariolae remained constant from one year to the next. The results show that migration alone cannot explain the homogeneity of the allozyme frequencies. It seems reasonable to conclude that selection plays a major role in maintaining the homogeneity of populations living in proximal biotopes.E.R.A. No. 406: Analyse et mécanismes de maintien du polymorphisme.     

Dosage-Dependent Modifiers of Homoeotic Mutations in Drosophila melanogaster

The determination of segment identity in Drosophila melanogaster appears to be controlled by a small number of genes. In order to identity new components in the process, we have systematically screened the autosomal complement for loci that show a dosage-dependent interaction with mutations in previously characterized genes thought to be important in the determination of segment identity. The dominant homoeotic phenotype of mutations at four loci involved in thoracic leg determination (Pc, Pcl, Antp and Scr) were quantitated in flies bearing a series of synthetic duplications covering more than 99% of the autosomal complement. Twelve regions were identified that when present in three wild-type copies strongly enhanced or suppressed the phenotype of mutations at one or more of the four homoeotic loci examined. The effects of five of these regions appear to correspond to previously described homoeotic loci; the effects of the remaining seven appear to identify new loci involved in the determination of segment identity.     

The nature of N-ethyl-N-nitrosourea-induced mutations at the white locus of Drosophila melanogaster

The molecular basis of 36 mutations induced by N-ethyl-N-nitrosourea (ENU) at the white locus of Drosophila melanogaster was analyzed. Blot-hybridization showed that only two of them are rearrangements. One is a 200-bp deletion and the second mutant is an insertion of about 10 kb. The latter might be of spontaneous origin. 34 mutants did not show a detectable alteration of the normal restriction enzyme profile. 21 mutants were also analyzed by Northern blot-hybridization. Normal or nearly normal levels of white mRNA were observed in 8 pigmented and 7 non-pigmented mutants. In 5 other non-pigmented mutants a strong reduction of the amount of mature white mRNA was seen. In one of the pigmented mutants, hybridization occurred with 2 RNAs. When taken together, these results strongly indicate that most of the ENU-induced mutations are caused by base-pair changes or rearrangements smaller than 50-100 bp.     

The nature of radiation-induced mutations at the white locus of Drosophila melanogaster

X-Ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions.     

Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster

Primary cell cultures derived from mutants in seventeen different genes were analyzed for their ability to excise pyrimidine dimers from DNA. Five of these mutagen-sensitive mutants [mus(2)205^A1, mus(3)302^D1, mus(3) 304^D3, mus(3)306^D1, mus(3)308^D2] display a significantly reduced excision capacity relative to control cultures. In addition, two of the five [mus(3)306^D1, mus(3)308^D2] are defective in the accumulation of single-strand breaks normally seen after ultraviolet irradiation. This study, therefore, brings the total number of Drosophila mutants known to be defective in excision repair to seven. The results are discussed relative to other genetic and biochemical properties of these mutants. This work is dedicated to Professor W. Beermann whose own contributions were instrumental in focusing a modern analysis of the eukaryotic genome on the diptera. Those of us who benefitted so much from his personal guidance recognize that we did so as a result of some sacrifice on his part. One of Boyd's contemporaries in Tübingen once remarked: “It's terrifying to think what Professor Beermann could do if he were in the lab full time.”     

Comparative studies of allozyme loci in Drosophila simulans and Drosophila melanogaster. I. Three dipeptidase loci

Genetic variation at three dipeptidase loci (Dip-A, Dip-B, and Dip-C) in Drosophila simulans was analyzed by starch gel electrophoresis. Dip-A was found to be polymorphic in four populations, while Dip-B and Dip-C were found to be polymorphic in one. The numbers of different alleles found at each respective locus were: Dip-A, two; Dip-B, two; and Dip-C, three. Dip-A was genetically mapped at 57.9 on the second chromosome, and Dip-B and Dip-C at 80.9 and 87.9 on the third chromosome, respectively. Neither Dip-B nor Dip-C has been mapped in D. melanogaster because both loci are apparently monomorphic. Their map positions in D. simulans with respect to flanking markers whose homologous genes have been cytogenetically localized in D. melanogaster suggested that they might be mapped cytogenetically by using available deficiencies in D. melanogaster. Accordingly, by the construction of interspecific hybrids which carried deficiencies of melanogaster and an allele of simulans with a mobility different from that of the fixed melanogaster allele, Dip-B and Dip-C were localized between 87F12-14 and 88C1-3 and between 87B5-6 and 87B8-10, respectively, in the salivary gland chromosomes of D. melanogaster. The similarity between these two species is discussed on the basis of these findings.     

[A dual system of superinstability at the yellow and scute loci in Drosophila melanogaster]

We have characterized the phenomenon of super-unstability in yellow and scute loci of Drosophila melanogaster. A few derivatives with different combinations of yellow and scute phenotypes appeared after dysgenic cross between potentially super-unstable stock y2nsscme and P[ry+, (delta 2-3)] (99B). Essentially, the double alterations of yellow and scute phenotypes constitute more than 40% of all derivatives. Most frequently the mutations in both yellow and scute loci change coordinately giving rise to the y+nssc+ allele. Lethal derivatives were not observed. The spectrum and the frequency of the y2nsscme mutagenesis in females differ considerably from the analogous in males. Possibly, the neutral homologous chromosome in females changes contacts between two insertions, and because of that mutagenesis changes too. Thus, all alterations in the double super-unstable system seem to be connected with the recombination between two unstable insertions A genetic exchange may by initiated by a double-strand breaks induced by the transposase of the ends of insertions.