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 (ovo-a and ovo-b), only one of which has an in frame AUG (ovo-a). OVO-B mRNA is translated from an AUG in exon 2. We found that OVO-B mRNA is produced only in females and is controlled by the sex-karyotype and somatic inductive signals, while OVO-A mRNA is produced in both males and females. To test the function of OVO-A and OVO-B, we constructed multiple transgenes. Analysis of these constructs in an ovo minus genetic background indicate that the female-specific OVO-B isoform is sufficient for zygotic female fertility (female sterility is fully rescued), but is insufficient for full germline viability in the next generation (there is incompletely penetrant maternal effect sterility). This suggests that maternally deposited OVO-A activity is important in the early embryo, while positive OVO-B activity is required for female germline development in the zygote. Flies bearing a ovo-b promoter knockouts or with new in-frame AUG initiation codons in the ovo-B 5'-UTR show dominant negative female sterility suggesting that excessive OVO-A activity is detrimental to the female germline. Interestingly, the ovo-b promoter knockouts also show significant rescuing activity in an ovo minus genetic background. We are investigating whether this simultaneous dominant negative and wild-type allele results in the production of both OVO-A and OVO-B proteins (the later a result of either ectopic ovo-b promoter activity or OVO-A AUG initiation codon suppression). We are also looking for biochemical function by assaying for the binding of bacterially expressed OVO to its own promotor and to the ovarian tumor promoter (we have shown that both are regulated by ovo+ dose). Interestingly, OVO binds at the transcription initiation site of both these TATA-less promoters, in vitro, as well as to upstream sites. We have shown that the upstream sites are functional by using reporter genes. Deletions of the upstream OVO binding sites abolish transcriptional activity and transcription can be reconstituted by adding back any of several slightly different OVO binding sites. The core promoter OVO binding sites also appear to be important. Point mutations and core promoter swapping experiments suggest that only a subset of core promoters can respond to the enhancers at the ovarian tumor locus. For example the ovo-b and ovarian tumor promoters, both of which have strong OVO binding sites at the core promoter, support good transcription using the ovarian tumor enhancers, while the ovo-a promoter, or the ovo-b promoter with a mutated OVO binding site do not.
| 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 |
| 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 |
| 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 |
| 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 |
Showing the most recent 10 out of 36 publications
