Worm Breeder's Gazette 11(4): 54
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.
We previously reported on the genetic characterization and progress towards molecular cloning of the let-60/lin-34 gene (WBG 11 (2): 105; WBG 11(3): 24). Our genetic data, and the data from G. Beitel, S. Clark and R. Horvitz (see their report in this issue), suggested that the level of let-60 activity controls the fates of the vulval precursor cells (VPCs) in response to the inductive signal from the anchor cell. Reduction of let-60 activity causes a vulvaless (Vul) phenotype. We also argued that increase of let-60 activity (resulting from lin-34 mutations) causes a multivulva (Muv) phenotype. Moreover, let-60 likely acts after let-23 and lin-15, but before lin-1 and lin- 12, in the genetic pathway of vulval cell type determination. We have obtained cDNAs of let-60 from a library provided by Bob Barstead and Bob Waterston. Sixty-one positive clones were identified from 1.2x10+E6 plaques. Nine clones were characterized and most of them are different from each other in size, suggesting that the let-60 RNA is relatively abundant. We have sequenced both cDNA and genomic DNA in the let-60 region. We found that a 184 amino acid ORF encodes a ras protein after comparison to other genes in Genbank. Among the first 164 amino acids of the protein, 87% of them are identical to the Drosophila ras-1 protein and 84% of them are identical to a Harvey sarcoma ras protein. The extreme similarity of the overall structure between let-60 and other ras proteins suggests: (1) let-60 has similar biochemical functions to other ras proteins known, including GDP/GTP binding activity, GTPase, membrane binding, target site for farnesylation, and interactions with a variety of other proteins in signal transduction pathways; (2) ras oncogenes in other high eukaryotes might have developmental control functions similar to let-60.To confirm the ras gene is indeed let-60, we mapped let-60/lin-34 mutations (point mutations) by chemical modification and cleavage (we used Bob Barstead's protocol with minor modifications). We have mapped all three classes of let-60/lin-34 mutations within the coding region of the cloned gene, indicating the ras protein is encoded by let-60/lin- 34.Microinjection of let-60 clones cause a gain-of-function phenotype ( Muv). Microinjection of plasmids of greater than 20 g/ml causes a lethal phenotype. We now inject let-60 DNA at concentrations between 1 to 10 g/ml, with the concentration of the marker plasmid (pRF4 containing the rol-6(d) gene, provided by J. Kramer) at 50-65 g/ml. This phenomenon suggests that C. elegans may have a strict requirement for a particular level or pattern of let-60 expression; the exogenous DNA array could either cause high cellular level of the gene activity or an abnormal spatial or temporal expression pattern. We have transferred the let-60-rol-6(d) DNA array from our transformants into three other Vul mutants by genetic crosses. Our results show that the exogenous let-60 DNA, which causes a Muv phenotype (24% of animals in wild-type background) suppresses the Vul phenotypes of lin-3, s. These results suggest that an increase in ectopic let-60 functions bypasses or reduces the requirements of these three genes in vulval induction. let-60 acts after let-23, a receptor tyrosine kinase (see Aroian et al. , this issue). Therefore, we propose that inductive signal acts via the let-23 receptor tyrosine kinase, which leads to the activation of the let-60 ras protein.