cr7 was identified as a viable mutant defective in head morphogenesis, somewhat similar in phenotype to
vab-1,-2, and -3 mutants.
cr7 mutants are characterised by having a highly penetrant abnormal dorsal head bulge and a less penetrant tail bulge. The
cr7 allele is unlikely to be null, because animals carrying
cr7 in trans to a deficiency arrest as embryonic lethals. Based on these initial phenotypic observations, the gene identified by
cr7 has been named
vab-17. As a first step toward understanding the role played by
vab-17 in C. elegans morphogenesis, we used positional cloning to identify the gene.
vab-17 was genetically mapped to LGV; its physical location was further refined using PCR to characterise deficiency breakpoints. Cosmids spanning the region were tested for their ability to rescue
vab-17(
cr7) mutants, however, none of these cosmids appeared to rescue. Consequently, fosmid clones that partially cover a cosmid gap in the region were tested and some were shown to rescue
vab-17 (fosmid information kindly provided by Stephanie Chissoe). Further analysis indicated that at least one of these fosmids also rescues the lethality associated with
vab-17/Df. A 10 kb subclone (pPK333) was subsequently identified that also rescued the Vab phenotype; the sequence of this subclone led to the identification of several overlapping Kohara cDNAs. These cDNA clones (kindly provided by Y. Kohara) were found to carry alternatively spliced variants of a single gene. Surprisingly, the sequences of some of these cDNAs extended beyond the 5' end of the rescuing 10 kb subclone. RT-PCR was used to obtain additional cDNAs extending to the 5' end of the
vab-17 transcript;
vab-17 appears not only to have alternatively spliced mRNA variants, but also to have to have multiple 5' ends, each of which is trans-spliced to SL1. Based on our analyses of cDNA sequences, the
vab-17 gene covers a region of 16.5 kb. The largest predicted protein encoded by the
vab-17 locus has a molecular mass of 180 kDa. The sequence of this protein is mostly novel, but it contains a series of potential WD-40 repeats, a motif that has been shown to mediate the assembly of macromolecular complexes. Although pPK333 does not carry the entire
vab-17 coding region, it is capable of rescuing
vab-17(
cr7). Therefore, in order to obtain a preliminary picture of VAB-17 expression, pPK333 was used to create an in-frame GFP fusion. The fusion protein appears to localise to the cytoplasmic face of adherens junctions, but unlike the adherens junction staining displayed by MH27, the VAB-17 fusion is not associated with embryonic hypodermal adherens junctions. Further characterisation of VAB-17 expression is in progress. In the course of genetically mapping
vab-17, several marker genes were found to exhibit synthetic lethality with
vab-17(
cr7). Understanding the basis of this lethality may provide clues to the role
vab-17 plays in morphogenesis. We have taken advantage of this synthetic lethality to identify a series of
vab-17 intragenic revertants, which are weakly Vab, yet suppress the synthetic lethality. We are in the process of identifying the molecular lesions of these
vab-17 mutants.