The mus308 locus of Drosophila melanogaster is implicated in the bypass of ENU-induced O-alkylpyrimidine adducts

The mus308 locus of D. melanogaster was originally characterized by virtue of a mutant phenotype that resulted in specific hypersensitivity to cross-linking agents. However, the gene product has also been implicated in the repair of lesions other than cross-links. The gene was recently sequenced, and it encodes a protein with motifs characteristic of both DNA polymerases and helicases. We present mutability studies, using the recessive lethal (RL) test, which show that N-ethyl-N-nitrosourea (ENU) induces hypermutability in mus308-deficient conditions, although only in early broods. Further studies elucidated the role of MUS308 in repair processes by characterizing the spectrum of molecular mutations induced by in vivo ENU in postmeiotic germ cells, in mus308 conditions. These revealed that, in comparison to repair-proficient conditions, there is an increase in the frequency of GC → AT and AT → GC transitions, and AT → TA transversions. Moreover, frameshift mutations, which have not previously been reported to form part of the ENU spectrum, were also found. These results indicate that MUS308 is needed to process ENU-induced lesions, and support the hypothesis that the mus308 gene plays a role in post-replication bypass of O-alkylpyrimidines, probably mediated by recombination, which serves to increase the time available for error-free repair of these persistent and highly mutagenic lesions. Received: 22 March 1999 / Accepted: 17 November 1999     

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.     

The genetics of a new mutable allele at the white locus in Drosophila melanogaster

Summary The genetics of a third case of high mutation frequency at the white locus in Drosophila melanogaster has been analyzed. The new mutable allele, w ^+u, mutates from a wild-type to a white-eyed phenotype in both males and females. The mutational event is 1) premeiotic, 2) not associated with crossingover, 3) sensitive to genetic modification, and 4) restricted to germinal tissue. The only mutants produced by w ^+u are deletions of the white locus. These deficiencies include subsites 4 and 5 of the white locus, but are cytologically unobservable. The mutable allele itself maps to subsite 4.The mutational properties of w ^+u are unlike those of the other highly mutable white alleles which have been interpreted in terms of phage-like controlling elements. Rather, the properties of w ^+u favor a model based on the premature termination of chromosome replication near the terminus of a replicon which leads to a chromosome deficient for the material between the point of premature termination and the end of the replicon.Supported by NIH predoctoral traineeship GM-150 and by NIH research grant GM-07428 to Dr. W. K. Baker.From a dissertation submitted to the Division of Biological Sciences of The University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Transposition of the bobbed locus in Drosophila melanogaster

Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

The Drosophila melanogaster Ku70 coding sequence is wild-type in mus309 mutants

Drosophila melanogaster mus309 mutations have been identified as being in the Ku70 gene, encoding one subunit of the DNA-dependent protein kinase. However, recent work has suggested that these mutations are in the Bloom's syndrome homologue. Here, we demonstrate that the coding sequence of Ku70 gene is indeed wild-type in two mus309 mutant lines.     

Mutagenesis at the cinnabar locus in Drosophila melanogaster

A study was undertaken to isolate mutations affecting the temporal appearance of kynurenine hydroxylase in Drosophila melanogaster. Such mutations, lacking or having reduced enzyme activity at the larval or pupal stage only, could represent changes in regulatory functions. Mutagenesis was carried out using EMS. Potential mutations were isolated from mass F₁ cultures. The screening of large numbers of individuals was made possible by the use of the mutant red, which allowed visual classification for the presence or absence of the enzyme at both stages. From a series of six mutagenesis experiments 111,561 chromosomes were tested, and 122 phenotypically mutant F₁ individuals were found. From these, 38 inheritable mutations were isolated which, by phenotypic observation, lacked or had reduced enzyme activity at the larval and pupal stages. Assay of enzyme activity levels in several of the mutants confirmed the phenotypic data. All of the 27 mutations that could be tested further are recessive and behave as cinnabar alleles. Complementation tests were performed between these 27 mutant stocks, and no complementation in the production of eye color has been seen between the mutants examined. When extended collection periods were used, a significantly higher percentage of inheritable mutations was isolated from the first 3 days of the screen. Over 80% of the F₁ phenotypic mutants could be classified as mosaics, which indicates that cinnabar can be autonomous under certain conditions. The failure to isolate mutations in possible regulatory function is discussed.     

Localization of a gene for a minor chorion protein in Drosophila melanogaster: a new chorion structural locus

A minor chorion protein (called s70) with an approximate molecular weight of 70,000 D has been characterized in Drosophila melanogaster. The Staket geographic strain was found to carry an electrophoretic variant of this eggshell component and was used to determine the chromosomal location of the s70 gene. Our results establish a new locus for a chorion gene near yellow on the X chromosome and represent the first mapping of a quantitatively minor eggshell protein.     

Dipeptidase-A: A polymorphic locus in Drosophila melanogaster

Dipeptidase A (Dip-A), a new peptidase locus in Drosophila melanogaster, is located on the second chromosome at map position 55.2 and in the 41A-B; 42A2-3 interval in the salivary gland chromosomes. Three alleles are reported. In the Carpenter, North Carolina population the allele frequencies are: Dip-A ⁶ (fastest)=0.064; Dip-A ⁴ (intermediate)=0.920; and Dip-A ² (slowest)=0.015.     

Nucleotide variation in the Egfr locus of Drosophila melanogaster

The Epidermal growth factor receptor is an essential gene with diverse pleiotropic roles in development throughout the animal kingdom. Analysis of sequence diversity in 10.9 kb covering the complete coding region and 6.4 kb of potential regulatory regions in a sample of 250 alleles from three populations of Drosophila melanogaster suggests that the intensity of different population genetic forces varies along the locus. A total of 238 independent common SNPs and 20 indel polymorphisms were detected, with just six common replacements affecting >1475 amino acids, four of which are in the short alternate first exon. Sequence diversity is lowest in a 2-kb portion of intron 2, which is also highly conserved in comparison with D. simulans and D. pseudoobscura. Linkage disequilibrium decays to background levels within 500 bp of most sites, so haplotypes are generally restricted to up to 5 polymorphisms. The two North American samples from North Carolina and California have diverged in allele frequency at a handful of individual SNPs, but a Kenyan sample is both more divergent and more polymorphic. The effect of sample size on inference of the roles of population structure, uneven recombination, and weak selection in patterning nucleotide variation in the locus is discussed.     

Transvection at the vestigial locus of Drosophila melanogaster

Transvection is a phenomenon wherein gene expression is effected by the interaction of alleles in trans and often results in partial complementation between mutant alleles. Transvection is dependent upon somatic pairing between homologous chromosome regions and is a form of interallelic complementation that does not occur at the polypeptide level. In this study we demonstrated that transvection could occur at the vestigial (vg) locus by revealing that partial complementation between two vg mutant alleles could be disrupted by changing the genomic location of the alleles through chromosome rearrangement. If chromosome rearrangements affect transvection by disrupting somatic pairing, then combining chromosome rearrangements that restore somatic pairing should restore transvection. We were able to restore partial complementation in numerous rearrangement trans-heterozygotes, thus providing substantial evidence that the observed complementation at vg results from a transvection effect. Cytological analyses revealed this transvection effect to have a large proximal critical region, a feature common to other transvection effects. In the Drosophila interphase nucleus, paired chromosome arms are separated into distinct, nonoverlapping domains. We propose that if the relative position of each arm in the nucleus is determined by the centromere as a relic of chromosome positions after the last mitotic division, then a locus will be displaced to a different territory of the interphase nucleus relative to its nonrearranged homolog by any rearrangement that links that locus to a different centromere. This physical displacement in the nucleus hinders transvection by disrupting the somatic pairing of homologous chromosomes and gives rise to proximal critical regions.