Worm Breeder's Gazette 12(3): 70 (June 15, 1992)

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-1 Proto-Oncogene Homolog Involved In C. elegans Vulval Development

Andy Golden, Paul Sternberg

Division of Biology, California Institute of Technology and Howard Hughes Medical Institute, Pasadena, CA.

Three genes involved in the induction of the hermaphrodite vulva, lin-3 , let-23 ,and let-60 ,show extensive similarity with signal transduction components of Drosophila, yeast, and mammals. From what we know of signal transduction pathways of these other systems, there are a number of other signalling proteins that we anticipate to participate in the induction of the C. elegans vulva. We therefore sought to clone the cytoplasmic raf-1 serine/threonine protein kinase. This gene product associates with some receptor tyrosine kinases and is activated upon ligand stimulation. we anticipated that such a gene would act downstream of the receptor tyrosine kinase, let-23 ,in the vulval induction pathway. Physical mapping of a raf-1 gene may allow us to determine if it corresponds to an already known genetic locus. It should also be possible to generate dominant and dominant negative variants of this gene to determine its role in vulval differentiation and other developmental processes.

Using degenerate oligonucleotides and P CR technology, and then cDNA cloning, we identified a raf-1 cDNA. This cDNA was used to probe the ordered YAC grids to physically map the raf-1 gene. This gene maps to the middle of a 500 kb interval between unc-44 and deb-1 on chromosome IV. The raf-1 gene does not reside on any cosmids from this region. From a YAC in this interval, we cloned a 10 kb genomic fragment that contains the entire raf-1 coding sequence. There are three regions highly conserved among the raf family and these regions are also found in the C. elegans raf-1 homolog. Conserved region 1 is a cysteine-rich region believed to be involved in regulation by an "activating factor". Conserved region two is a short serine/threonine-rich region that presumably contains regulatory autophosphorylation sites. Conserved region 3 is the kinase domain. This domain in the C. elegans raf-1 gene product is the most conserved and has 80% similarity with other members of the raf family. This raf-1 gene has all the landmark features that would place it in the raf family of protein kinases.

Interestingly, the gene lin-45 genetically maps to this same interval on chromosome IV. Min Han (U. of Colorado at Boulder) had previously isolated a single allele of this gene ( sy96 )in a suppression screen of lin-15 Muv animals. Animals homozygous for lin-45 ( sy96 )are E g1 and have decreased vulval differentiation. Min Han has performed germline transformation with the above 10 kb raf-1 genomic fragment and has recently demonstrated that this gene rescues the E g1 phenotype of lin-45 ( sy96 )animals. He has also determined that the molecular defect associated with sy96 is within the raf-1 transcription unit.

The rescue results and the sequencing of the sy96 allele indicate that the lin-45 gene encodes a raf-1 homolog. To examine the temporal and spatial pattern of expression of the lin-45 / raf-1 gene, the 5' regulatory sequences of the raf-1 gene were fused to the lacZ gene of E. coli. Preliminary results suggest that this gene fusion is expressed in the vulva, hypodermis, and other tissues of L4 and adult animals. We have yet to observe staining in the VPCs of L3 hermaphrodites; this could be explained by a number of caveats common to such lacZ fusion constructs.