Characterisation of a novel gene (Dstpk61) encoding a serine/threonine protein kinase in Drosophila melanogaster

Sex determination of somatic tissue in Drosophila melanogasier is governed by the gene Sex-lethal (Sxl). Transcription of Sxl is regulated by X-linked activator proteins and autosomally encoded repressor proteins. Thus, in flies with 2 X chromosomes and 2 sets of autosomes (X:A=1), transcription of Sxl occurs and the presence of Sxl protein commits the fly to the female developmental pathway. When only 1 X chromosome is present (X:A=0.5), transcription of Sxl is repressed and the default male developmental pathway is chosen. Sxl is the first gene in the somatic sex determining pathway. At a molecular level, SXL protein regulates the alternative splicing of transcripts from the next gene in the hierarchy, transformer (tra) such that functional TRA protein is present only in females. In turn, TRA protein, in conjunction with Transformer-2 (TRA-2) protein, ensures female-specific splicing of transcripts from the last gene in the hierarchy—doub/esex (dsx)—resulting in the production of a female-specific DSXF protein. In the absence of TRA protein, dsx transcripts are spliced to give a male specific DSXM protein. Traditionally, it was thought that all aspects of somatic sexual differentiation were governed by DSXF and DSXM, the only known targets of which are the yolk protein genes. However, it is now apparent that both TRA and SXL control DSX-independent sex differentiation pathways: TRA acts via dissatisfaction (dsj) to regulate sexual behaviour, and via fruitless (fru) to govern aspects of male courtship behaviour and innervation of the male-specific Muscle of Lawrence (MOL); and SXL acts independently of TRA and DSX to govern body size and colour. Only a few targets of the sex determining genes are known—dsf, fru, and the yps. Such genes would be expected to fit into one of three categories: Genes expressed exclusively in one sex. Genes expressed at a higher level in one sex than the other. Genes that are expressed at similar levels in both sexes, but produce sex-specific transcripts. In an attempt to isolate genes that fit into these categories a non-gonadal differential screen of an adult body genomic DNA library was performed, using sex-specific radio-labelled cDNA. The genomic recombinant fs(l) was chosen for further analysis based on its preferential hybridisation to female non-gonadal cDNA. Screening of cDNA libraries using fs(l) sequence isolated two cDNAs, a 3.0kb cDNA (cDNA A) and a 4.5kb cDNA (cDNA B). Sequence analysis revealed that both these cDNAs encoded a novel opa repeat-containing serine/threonine protein kinase, the difference in the cDNAs being the length of the 5' and 3' untranslated regions (UTRs). The gene, which maps to polytene position 61A, was named Drosophila serine/threonine protein kinase@61 (Dstpk6l). Northern analysis showed there to be at least 4 different Dstpk6l transcripts: a 3.0kb testis-specific transcript (possibly represented by cDNA'); a 3.5kb ovary-specific transcript; a 4.5kb female-specific carcass transcript (possibly represented by cDNA B); and a slightly larger non-sex-specific transcript. The work presented here shows that Dstpk6l is a complex gene that produces multiple transcripts by using alternative promoters, alternative polyadenylation sites, and alternative splicing. It is now known to have significant sequence and functional homology to mammalian Phosphoinositol-dependent kinase 1 (PDK1), which has been implicated in the insulin signal transduction pathway and the regulation of apoptosis. Four mutant P-insertion lines have been identified that disrupt Dstpk6l at different sites. The phenotypes of these mutants, possible Dstpk6l transcripts, and the observed rnRNA expression pattern are discussed in relation to the possible role of DSTPK61 protein in Drosophila development.