Worm Breeder's Gazette 14(5): 25 (February 1, 1997)

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.

SuperCosmid Strikes Again

Greg P. Vatcher, Nigel J. O'Neil, Lynnette M. Kuervers, David L. Baillie

Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada, V5A 1S6

In recent articles, WBG 14(2), and this issue, we reported on the use of a cosmid transgenic library to correlate the physical and genetic maps. Using these transgenic strains we have rescued the mutant phenotypes of several lethal genes on LGIII left in the region defined by the overlap of sDf125 and sDf127.

The phenotypes of mel-32, let-721, and let-725, F2 egg lethal, were completely rescued by cosmid C05D11 but not by the flanking cosmids C16A3 or T26A5. C05D11 also completely rescued the lethal phenotypes of let-713 (arrests as sterile adults), and let- 756 (an early larval lethal). C05D11 has been affectionately termed SuperCosmid, based on the fact that it rescued 5! essential genes from our collection. All rescued strains were tested for the presence of cosmid with PCR using a vector specific primer set. Outcrossing to wild type and recovery of the lethal or maternal effect lethal phenotype also confirmed a valid rescue.

C05D11, a 50kb cosmid, has been subcloned into many smaller fragments which have been injected into the syncitial gonad of C. elegans to produce new transgenic strains. In collaboration with Danielle Thierry-Mieg and Regine Roubin, WBG 14 (4), let-756 was determined to be ORF 4, a heparin-binding growth factor gene (a fibroblast growth factor homolog). The rescue of let-721 with overlapping subclones has shown that let-721 corresponds to ORF 12. This gene has a very high homology with a human cDNA of unknown function. mel-32 has been rescued with a subclone containing only ORF 11 which is the only known C. elegans homolog of the ubiquitous serine hydroxymethyltransferase. In collaboration with Heinke and Ralf Schnabel we are further characterizing this gene. Rescue experiments have narrowed the position of let-713 down to two ORFs and let-725 down to an operon containing three ORFs.

Future sequence analysis of these mutant genes will allow the correlation of changes in the DNA sequences (and the subsequent protein sequences) with their lethal phenotypes. Lethal and maternal effect lethal genes are essential for survival; therefore, their assignment to specific ORFs will help define which genes are required for normal growth and development.