|Functional Genomics Section
DOE Human Genome Program Contractor-Grantee Workshop
142. Isolation of Drosophila DNA Repair Genes
R. Scott Hawley, Kenneth C. Burtis,
and Gerald M. Rubin1
The ultimate goal of this project is to complete the identification and mapping of all of the genes involved in repair of genome damage in Drosophila melanogaster and to initiate their functional characterization. The substantial quantity of Drosophila genomic and cDNA sequences obtained to date, in combination with the genetic information available for this organism, provide a powerful base from which to begin a comprehensive description of the DNA repair genes operating in Drosophila. We have now initiated a multi-faceted approach to complete this process, using genetic, molecular and bioinformatic approaches.
We have initiated genetic screens to extend previous, non-saturating screens for mutagen-sensitive mutations. These genetic screens have only just begun, and no new mutagen-sensitive loci have yet been isolated. However, using a combined molecular and genetic approach, we have made some progress in identifying two repair genes previously uncharacterized in Drosophila; the Drosophila homolog of the yeast RAD10 gene (now designated mei-10), and the Drosophila homolog of XPG. We have shown that the fly MEI-10 protein physically interacts with the MEI-9 (dm Rad1) protein by yeast two-hybrid studies. We are now in the process of creating mutants in the mei-10 gene. Preliminary mapping suggests that the MEI-10 gene may correspond to the previously identified mus210 locus. We have also obtained strong evidence that the Drosophila XPG homolog corresponds to the mus201 locus. The two extant mus201 alleles display phenotypes expected for an NER defect, but no discernable meiotic defect.
We are also continuing genetic studies of several known repair-deficient loci. Most notably we have identified a null allele of the repair/checkpoint gene mei-41 and demonstrated intermediate levels of repair competency and checkpoint function in heterozygotes for this mutation. We have used this and other such mutants as substrates in screens for dominant enhancer or suppressor mutations. These screens are allowing us to identify mutations that might be lethal or sterile when homozygous and thus be missed in more convential screens for mutagen-sensitive mutations.
A second approach to identifying genes involved in the Drosophila response to genome damage will involve the use of DNA microarrays. We have recently completed construction of an arraying robot using the design developed by Pat Brown's lab at Stanford, and have initiated the production of arrayed collections of EST-characterized Drosophila cDNAs produced by the Berkeley Drosophila Genome Project. These arrays will be used to identify transcription units whose expression is regulated in response to DNA damage. The genes thus identified will be further characterized molecularly and genetically, and correlated to the extent possible with repair genes identified by other means.
Finally, we will present an up-to-date summary of the Drosophila DNA repair genes identified through genomic and EST sequences obtained to date by the Berkeley Drosophila Genome Project.
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