Worm Breeder's Gazette 11(3): 28
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The X-linked sdc-1 gene is required for the hermaphrodite mode of sex determination and dosage compensation, acting at a point before the two pathways diverge. Temperature-shift experiments further suggest that sdc-1 acts at an early step in the regulatory hierarchy controlling the choice of sexual fate since they demonstrate a requirement for sdc-1 during the first half of embryogenesis ( Villeneuve and Meyer, Genetics 124:91-114, 1990). We have undertaken a molecular approach to examine the function of this gene in the sex determination and dosage compensation processes in C. elegans. Here we describe the cloning and preliminary molecular examination of this locus. The sdc-1 gene was positioned to a two cosmid interval on the clb-1 contig by identifying the breakpoints of two deficiencies that flank the gene, the left breakpoint of mnDf41 (in R09G6) and the right breakpoint of mnDf4 (in R12G12). Restriction mapping of the overlapping R12G12, W06F3 and R09G6 cosmids in conjunction with further molecular characterization of the mnDf41 and mnDf8 breakpoints localized sdc-1 to a 55 kb interval. Micro-injection rescue of sdc-1(y67) with cosmids and cosmid subclones in this interval localized sdc-1 rescuing activity to an 11 kb fragment within R09G6. Rescuing clones completely eliminate the sdc-1 Tra, Egl and Dpy mutant phenotypes in the F1 and in subsequent generations. Concerned that duplications of portions of the X chromosome that do not cover sdc-1 suppress the sdc-1 phenotype, we were relieved that neither adjacent cosmids nor act-4 subclones, which contain a feminizing element defined by McCoubrey et al. (Science 242:1146-51, 1988), are able to rescue sdc-1. [Interestingly, we further noticed that neither of two act-4 clones we utilized will produce stable transmitting lines in co-injections with rol-6(su1006), although F1 roller transformed animals are easily obtainable. ] To analyze sdc-1 transcripts, an overlapping set of fragments spanning the 11 kb rescuing region were subcloned and used to probe Northern blots loaded with 20 g of polyA+ RNA isolated from mixed staged cultures of both N2 and him-8 strains. A 'single' smeary ~3.8 kb transcript derives from the middle 6 kb region of our rescuing fragment. Southern analysis of 14 sdc-1 alleles (13 EMS induced, 1 spontaneous) using these same probes showed no obvious allele-specific polymorphisms in the region. We are in the process of isolating cDNA clones from the region from both him-5 and N2 cDNA libraries, and are determining the genomic sequence of the locus. The molecular analysis of this gene in conjunction with concurrent analysis of the sdc-2, uld expand our understanding of the early steps of both the sex determination and dosage compensation pathways in C. elegans.