MOLECULAR-CLONING AND CHARACTERIZATION OF THE DPY-20 GENE OF CAENORHABDITIS-ELEGANS

We describe the molecular analysis of the dpy-20 gene in Caenorhabditi s elegans. Isolation of genomic sequences was facilitated by the avail ability of a mutation that resulted from insertion of a Tcl transposab le element into the dpy-20 gene. The Tcl insertion site in the m474::T cl allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tcl element. Genomic dpy-20 clones were isolated from a library of wild-type DNA and were found to lie j ust to the left of the unc-22 locus on the physical map, compatible wi th the position of dpy-20 on the genetic map. Cosmid DNA containing th e dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analys is of the putative dpy-20 homologue in Caenorhabditis briggsae was per formed to confirm identification of the coding regions of the C. elega ns gene and to identify conserved regulatory regions. Sequence analysi s of dpy-20 revealed that it was not similar to other genes encoding k nown cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein t hat may be directly or indirectly involved in cuticle function. Northe rn blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data fro m temperature shift studies using the temperature-sensitive allele e12 82ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and functio n of the DPY-20 protein are closely linked temporally.