Worm Breeder's Gazette 9(2): 25
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ced-3 and ced-4 are two genes required for the initiation of programmed cell death during the development of C. elegans (Ellis & Horvitz, Cell 44: 817-829 1986). We are trying to clone ced-3 and ced- 4. We have started by walking from a restriction fragment length polymorphism closely linked to ced-3. A 5.1 kb EcoRI Bristol-specific Tc1 band and a 2.7 kb EcoRI Bergerac-specific Tc1 band had been mapped between ced-3 and unc-26 (which are about 0.2% apart) by Hilary Ellis ( C. elegans Newsletter Vol. 8 No. 3). We have identified two cosmids that contain the 5.1 kb Bristol-specific Tc1 band. Alan Coulson and John Sulston have identified two more cosmids that overlap with our cosmids. We will now use these clones to probe genomic blots of the existing ced-3 mutants, including both the seven EMS-induced alleles and the three putative Tc1-induced alleles (see below). We are also using the mut-2 mutator strain TR679 generated by Phil Anderson's laboratory (C. elegans Newsletter Vol. 9 No. l) to isolate spontaneous ced-3 and ced-4 mutants, in the hope of identifying Tc1-induced mutations. Such mutants could be used for the direct cloning of these genes. We have used the fact that ced-3 and ced-4 are recessive suppressors of egl-1 to do complementation screen experiments to identify new ced-3 and ced-4 alleles. egl-1 males were mated with mut-2 hermaphrodites to construct a mut-2; unc-76 strain. Several putative mut-2; unc-76 strains were identified on the basis of expressing an Egl e as well as being weakly Him and giving rise to spontaneous mutants. We have mated ced-3 egl-1 males with mut-2; unc-76 hermaphrodites and looked for rare nonEgl F1 progeny. So far, we have screened about 16,000 F1's and have found three new alleles of ced-3. All three seem to be linked to a recessive lethal mutation. We do not know yet whether these lethals are in ced-3. We also have mated unc- 79 egl-1 males with mut-2; unc- 76 hermaphrodites and looked for rare non-Egl F1 progeny. We have screened about 30,000 F1's and have found one new allele of ced-4. This allele is homozygous viable. We have identified three spontaneous revertants of this new ced-4 allele. We are now mapping the Tcl's in our new ced-3 and ced-4 strains to see if these new ced-3 and ced-4 mutations were generated by Tcl transposition.