The first developmental choice faced by germ cells in the majority of higher eukaryotes is oogenesis versus spermatogenesis, but very little is known about how this basic event is genetically encoded in any organism. We have chosen to study the developmental genetics of this germline sex determination process in the fruit fly, Drosophila melanogaster. Our long-term goals are to: 1) identify the major germline sex determination regulatory genes; 2) determine how they are related in terms of a developmental hierarchy; and 3) understand in detail how these regulatory gene products function. We are question oriented and take advantage of genetic, molecular, and biochemical techniques as needed. Current molecular studies are focused on OVO, a highly conserved zinc finger domain protein functioning at a key intersection in the hierarchy. We have found that the ovo[+] locus has alternate promotors and 5' exons, only one of which has an in frame AUG. We have sequenced two dominant negative mutations in the locus and find that they add new in frame AUGs downstream of the exon that bears no AUG codon of its own. The potential protein products encoded by these alternative RNAs differ by the presence of an acidic domain at the N-term portion of the protein. Given the activation role of acidic domains in DNA- binding proteins (which OVO is likley to be based on sequence), it seems likely that OVO is both a transcriptional activator and repressor. We are investigating the idea that these alternative forms are important for ovo[+] regulation by transforming flies with ovo genes with new AUGs, and by adding stop codons to selectively disrupt subsets of the potential protein products. We have also developed antibodies to OVO, which we are testing in situ for localization patterns and in western blots to detect different isoforms. We are also looking for biochemical function by assaying for the binding of bacterially expressed OVO to its own promotor. This a likely target for OVO. Genetic data indicates that the ovo[+] locus shows both positive and negative auto-regulation. Specifically, the dominant negative ovo mutations, which mimic the long isoform, down-regulate ovo::lacZ reporters in the fly and duplication of the intact ovo[+] gene up-regulates these same reporter constructs.
| Chen, Zhen-Xia; Sturgill, David; Qu, Jiaxin et al. (2014) Comparative validation of the D. melanogaster modENCODE transcriptome annotation. Genome Res 24:1209-23 |
| Malone, John; Oliver, Brian (2009) The genomic 'inner fish' and a regulatory enigma in the vertebrates. J Biol 8:32 |
| Malone, John H; Oliver, Brian (2008) The sex chromosome that refused to die. Bioessays 30:409-11 |
| Larracuente, Amanda M; Sackton, Timothy B; Greenberg, Anthony J et al. (2008) Evolution of protein-coding genes in Drosophila. Trends Genet 24:114-23 |
| Kalamegham, Rasika; Sturgill, David; Siegfried, Esther et al. (2007) Drosophila mojoless, a retroposed GSK-3, has functionally diverged to acquire an essential role in male fertility. Mol Biol Evol 24:732-42 |
| Sturgill, David; Zhang, Yu; Parisi, Michael et al. (2007) Demasculinization of X chromosomes in the Drosophila genus. Nature 450:238-41 |
| Zhang, Yu; Sturgill, David; Parisi, Michael et al. (2007) Constraint and turnover in sex-biased gene expression in the genus Drosophila. Nature 450:233-7 |
| Zhang, Yu; Oliver, Brian (2007) Dosage compensation goes global. Curr Opin Genet Dev 17:113-20 |
| Oliver, Brian (2006) Tiling DNA microarrays for fly genome cartography. Nat Genet 38:1101-2 |
| Gupta, Vaijayanti; Parisi, Michael; Sturgill, David et al. (2006) Global analysis of X-chromosome dosage compensation. J Biol 5:3 |
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