Worm Breeder's Gazette 11(5): 24
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 let-23 gene is involved in at least 5 developmental pathways. It is essential for (1) larval survival, (2) vulval development, (3) male tail development, (4) fertility, and (5) the P11/P12 fate (Aroian and Sternberg, submitted). The let-23 gene has recently been cloned and sequenced and shown to be a member of the epidermal growth factor ( EGF) receptor tyrosine kinase family (Aroian et al., Nature, in press). Assuming that the let-23 gene product is a receptor for a developmental signal, or signals, we are interested in the gene products that act downstream of this receptor tyrosine kinase to transduce such developmental signals. The mammalian EGF receptor has been shown to physically associate with four putative signal transducing molecules: (1) the cellular raf serine/threonine kinase, (2) GAP, the GTPase-activating protein of the ras proto-oncogene product, (3) phospholipase C gamma (PLC-gamma ), and (4) a phosphatidylinositol-3 kinase [for review, Hall, Cell (1990) 61: 921]. These four gene products also serve as substrates for the EGF receptor tyrosine kinase. We have begun studies to identify the raf and GAP homologues in C. elegans in order to elucidate their functions in this conserved signal transduction pathway. We have obtained the following results with the raf gene. Using polymerase chain reaction (PCR) technologies and degenerate oligonucleotide primers designed from conserved regions in the kinase domain of the raf gene product, we have amplified a 350 nucleotide genomic fragment and a 300 nucleotide cDNA fragment. These fragments were sub-cloned and sequenced. The two sequences are identical except for the inclusion of a 48 nucleotide insert in the genomic fragment, which appears to be a classical C. elegans intron. The predicted amino acid sequences of these two DNA fragments share 66% identity (64 of 97 residues) with the corresponding regions of the human cellular raf-1 and Drosophila raf-1 (D-raf-1) gene products. Using the nematode DNAs as probes, a number of candidate cDNAs have been isolated and sub-cloned from the cDNA library kindly provided by Barstead and Waterston. These cDNAs are currently being sequenced. Using our PCR-generated probes, we have also identified 5 overlapping YACs from the YAC grids generously provided by Alan Coulson et al. We have recently narrowed down the physical location of this putative C. elegans raf proto-oncogene within the cosmid C01B10, which has been physically placed to the right of unc-44 on chromosome IV. We will test, by microinjection, the ability of fragments of this cosmid to rescue the phenotypes of animals defective in genes that map in the vicinity of this cosmid. We will also construct dominant versions of this gene to test for effects on vulval development. Strategies similar to the ones described above are also underway in attempts to identify a C. elegans homologue of the GTPase-activating protein of the ras proto-oncogene product.