Worm Breeder's Gazette 13(2): 89 (February 1, 1994)

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.

Cloning and characterization of a C. elegans homologue of 14-3-3, a highly conserved gene thought to be involved in intracellular signalling.

Wenfu Wang, Diane Shakes

Dept. of Biology, University of Houston, Houston, TX, 77204-5513.

In the course of looking for the par-5 gene, we discovered (with help from Sherry Dickens of the Kemphues' lab) a 3.8 kb E coR1 fragment of the cosmid C38H7 which appeared to encode transcripts which were considerably more abundant in wildtype hermaphrodites than in glp-4 (germline-less) hermaphrodites. Finding this to be a promising characteristic, we used this genomic fragment to isolate seven cDNAs from both the early embryonic Schauer/Wood library and the Okkema/Fire mixed stage library. One of these clones has now been sequenced. The sequence results demonstrated that this cDNA clone contains an ORF with 248 amino acids, and deduced the amino acid sequence shares 77.6%, 77.6%, 77.1%, 76.3%, 76.9%, 61.4% and 62.9% identity with bovine, Drosophila, human, sheep, Xenopus, plant(Oenothera) and Saccharomyces cerevisiae forms of 14-3-3 protein respectively. Like these other genes, the C. elegans version includes a putative pseudosubstrate domain for protein kinase C (GARR), an annexin-like domain which is thought to bind to protein kinase C, and a highly acidic C-terminus.

14-3-3 proteins are highly conserved, soluble acidic proteins which appear to be involved primarily in intracellular signalling (Aitken et al., 1992, TIBS 17: 498-501). However, their function remains controversial since different groups have implicated them in the following diverse functions: 1) an activator of tyrosine and tryptophan hydroxylases, 2) an activator, inhibitor, or translocator of protein kinase C, 3) an activator of ADP-ribosyltransferase, and 4) a component of G-box binding transcription factors in plants.

When we used our cDNA clone C e14 -3-3to probe a blot containing the total cellular RNA from both N2 and glp-4 adult hermaphrodites, three bands (transcripts) with the sizes of 1.5, 1.35 and 0.9 kb respectively were seen in both samples. However, all three transcripts were more abundant in wild type worms. This result suggested that A). C e14 -3-3may have isoforms or closely related members as has been observed for other species and B). C e14 -3-3'stranscription is germ-line enhanced.

When C e143 -3was used to probe genomic Southern blots it hybridized strictly to the predicted 3.8 kb E coR1 band under hybridization conditions similar to the Northern blot. This result suggests that the 3 different C e14 -3-3transcripts are products of differential RNA processing rather than different genes. Work is currently in progress to determine if the transcripts differ in their protein coding sequences.