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.

A raf Proto-Oncogene Homologue in C. elegans

Andy Golden and Paul Sternberg

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 
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.