Worm Breeder's Gazette 11(4): 24
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.
In wild-type C. elegans hermaphrodites, 131 cells undergo programmed cell death. Each resulting cell corpse is quickly engulfed and degraded by a neighboring cell. Ed Hedgecock showed that mutations in the genes ced-1 and ced-2 prevent the engulfment of cell corpses. Since then, we have identified five additional genes -- ced- 5, ced-7, required for the engulfment step. In animals with mutations in any of these genes, corpses resulting from programmed cell death can persist for hours. Genetic studies suggest that these genes fall into two groups that might have partially redundant functions in the engulfment process: ced-1, ced-7 and ced-8 constitute one group, while ced-2, te the other. To understand better how the engulfment process works at the molecular level and why there appear to be two classes of genes involved in this process, we have undertaken a molecular analysis of ced-1.The ced-1(n1506) I mutation was isolated in a mut-2 genetic background, in which a number of C. elegans transposons are active. Southern analysis revealed a novel 2.9 kb EcoRI band in DNA from ced-1(n1506) animals, using the transposon Tc3 as a probe. This polymorphic band is within 0.1 map units of ced-1.We cloned this Tc3 polymorphism from a size- fractionated library made with DNA from ced-1(n1506) animals. This clone, skT1, contains 2.0 kb of non-Tc3 sequence in addition to a 0.9 kb EcoRI end fragment of Tc3. We used parts of the non-Tc3 sequence of skT1 to probe a copy of the YAC 'polytene' filters generously provided by John Sulston and Alan Coulson. Three overlapping YACs, Y5D9, Y53C10 and Y47H9, which form part of a previously unpositioned 700 kb contig, were identified. None of the cosmids that overlap all three of these YACs hybridizes to our probe, so we constructed a phage library from the YAC Y5D9 by ligating size-selected SauIIIA partial digests of Y5D9 into BamHI-digested Lambda Dash arms. We obtained approximately 125,000 recombinant phage. We screened 50,000 of these with the probe derived from skT1 and identified several thousand positive plaques. We are currently analyzing these phage, with the goal of obtaining 30 to 40 kb of overlapping phage covering the region of the Tc3 polymorphism. We plan to use these phage in microinjection experiments to attempt to rescue the ced-1 phenotype.