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.