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 Eg1 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 Eg1 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.