The C. elegans sex determination gene,
tra-2, is required for proper development of both somatic and germ-line sexual development. XX animals mutant for loss-of-function (lf) alleles of
tra-2 develop as males (Hodgkin, 1986) and gain-of-function (gf) alleles feminize both XX and XO animals (Doniach, 1986). Previous work has shown that
tra-2 is regulated at the transcriptional as well as the translational level (Okkema and Kimble, 1991: and Goodwin et al., 1993). The (gf) alleles all disrupt a region in the 3' untranslated region (UTR) that contains two 28 nt direct repeat elements (DREs) separated by 4 nts. The DREs confer translational regulation of a reporter transgene and bind a factor (DRF) in worm extracts (Goodwin et al., 1993). Our working model is that binding of DRF to the DREs represses translation, consequently, male development ensues. To isolate trans-acting factors that are involved in
tra-2 translational control, we have designed a mutagenesis screen that specifically targets mutations in genes that disrupt the regulation of a reporter transgene under the control of the
tra-2 3' UTR. The reporter transgene consists of the
ges-1 (gut esterase-1) promoter upstream of a fusion of the green fluorescent protein and LacZ coding sequences (
ges-1:: GFP/LacZ was a kind gift from Steve Gendreau). The wild-type
tra-2 3' UTR has been placed down-stream of the coding sequences.
ges-1 is a gut-specific promoter that is expressed from the 150 cell stage of embryogenesis through adulthood (Kennedy et al., 1993). We chose to focus on the 3' UTR regulation in the gut since this tissue is affected by
tra-2(gf) mutations. In this screen, transgenic hermaphrodites will be mutagenized, and the F1s will be cloned onto individual plates and allowed to self-fertilize. F2 embryos will be screened for inappropriate expression of the transgene. We will present data on optimizing the screen and initial mutations that were obtained. Doniach, T. (1986). Genetics 114, 53-76. Goodwin, E.; Okkema, P.; Evans, T.; and Kimble, J. (1993). Cell 75, 329-339. Hodgkin, J. (1986). Genetics 114, 15-52. Kennedy, B.; Aamont, E.; Allen, F.; Chung, M.; Heschl, M; and McGhee, J. (1993). J. Mol. Biol. 229, 890-908. Okkema, P.; Kimble, J. (1991). EMBO 10, 171-176.