Christine R. Preston

Identifying P factors in Drosophila by means of chromosome breakage hotspots

A syndrome of germline abnormalities in Drosophila melanogaster called hybrid dysgenesis is thought to be caused by transposable genetic elements known as P factors. Several lines of evidence presented here show that the chromosomal positions of at least some P factors can be identified as points of frequent chromosome breakage (hotspots). Starting with a strain (pi 2) in which four hotspots had been identified on the X chromosome, we found individual hotspots vanished when their part of the chromosome was replaced by the homologous part from a strain known to lack P factors. All hotspots in the non-substituted parts of the chromosome remained functional, indicating that they can act autonomously. We also observed a new breakage site coinciding with the appearance of an unstable mutation at the singed bristle locus (snW). This mutation was dysgenesis-induced, and previous genetic evidence suggested that it was caused by the insertion of a P factor at that locus. We also present preliminary evidence for rapid scrambling of the positions of hotspots under certain conditions, and we describe a new procedure for efficiently determining the positions of hotspots on a given chromosome.

Age-dependent usage of double-strand-break repair pathways.

A DNA double-strand break (DSB) can be repaired by any of several alternative and competing mechanisms. The repaired sequences often differ from the original depending on which mechanism was used so that the cells choice of repair mechanism can have profound genetic consequences. DSBs can accumulate with age , and human diseases that mimic some of the effects of aging, such as increased susceptibility to cancer, are associated with certain defects in DSB repair . The premeiotic germ cells of Drosophila provide a useful model for exploration of the connection between aging and DNA repair because these cells are subject to mortality and other age-related changes , and their DNA repair process is easily quantified. We used Rr3, a repair reporter system in Drosophila, to show that the relative usage of DSB repair mechanisms can change substantially as an organism ages. Homologous repair increased linearly in the male germline from 14% in young individuals to more than 60% in old ones, whereas two other pathways showed a corresponding decrease. Furthermore, the proportion of longer conversion tracts (>156 bp) also increased nearly 2-fold as the flies aged. These findings are relevant to the more general question of how DNA damage and repair are related to aging.

Spread of P transposable elements in inbred lines of Drosophila melanogaster.

Publisher Summary This chapter describes experiments in which single P elements were observed to spread in M strain genomes. Some of the uncertainties inherent in the previous experimental designs were avoided through the use of in situ hybridization of P-element sequences to polytene chromosomes, as opposed to genomic Southern blots of massextracted DNA. In addition, to follow the chromosomes as closely as possible, the populations were maintained by brother-sister matings, as opposed to mass transfers, during the generations when P-element sites were being monitored. It is considered that control over the population size and structure is a critical feature in this experiment, since the data indicate that these parameters are important factors in determining the fate of invading P elements. The approach discussed in the chapter does not allow detection of heterochromatic P elements. However, it affords much higher resolution, and is less likely to obscure any events that require observation of individual genomes.

Structure and associated mutational effects of the cysteine proteinase (CP1) gene of Drosophila melanogaster

The complete structure of the cysteine proteinase (CP1) gene reveals two large 5 introns as well as a small third intron. Deletion studies have shown that null mutations for the locus are female sterile with partial male sterility as well as wing and pigmentation effects. Null alleles can be produced by either deletions to the left or deletions to the right of a P element insertion in the long second intron of the gene. A nearby phenylalanyl tRNA synthetase gene (Pts) was also identified.