Worm Breeder's Gazette 15(4): 39 (October 1, 1998)
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.
Department of Biochemistry, University of Kentucky College of Medicine. Lexington KY 40536-0298
Our laboratory is investigating the role of lin18 in vulval cell orientation. Worms mutant for lin-18 generate P7.p lineages that are misoriented, forming a pseudovulva posterior to a defective vulva (WBG 11(4):98). We cloned a gene that rescues lin-18 mutants in germline transformation experiments (WBG 15(1):60). The rescuing gene encodes a predicted receptor tyrosine kinase. To confirm that this gene is lin-18, we amplified the gene from lin-18 mutants and sequenced it. We sequenced products of at least two independent PCR reactions for each allele. Regions that contained a sequence change were sequenced on top and bottom strands for each allele. The reference allele, e620, contained a C to T change. This converts a Gln codon to a stop codon at position 84 in the amino acid sequence predicted from cDNA analysis (see below). ga75 contained a 380 bp internal deletion in the predicted extracellular domain. The deletion removes the fourth exon (codons 85-136) and causes a shift in reading frame. This is predicted to generate a truncated gene product ending in a novel sequence of 13 amino acids following codon 84. To confirm the predicted splice pattern reported in ACeDB, we are cloning and sequencing cDNAs. In the first clone analyzed, we found that the splice pattern was not as predicted. The cDNA sequence we obtained is consistent with the e620 sequence analysis, as it places the mutation in an exon; the original splice prediction resulted in the e620 point mutation being in an intron. We are currently examining independent cDNA clones to confirm this result.