Worm Breeder's Gazette 10(3): 26
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.
The genes ced-3 and ced-4 are required to initiate the process of programmed cell death during C. elegans development. We have succeeded in rescuing both ced-3 and ced-4 mutants by germline transformation using genomic DNA fragments that were identified by transposon insertion (for ced-4) and chromosome walking (for ced-3) as candidates to contain these two genes. We have coinjected cosmids C14G10, which contains the wild type allele of unc-31 and C10D8, identified by Tc4 transposon insertion as a candidate to contain ced-4, into ced-1; unc-31 animals and looked for non-Unc transformants among the F1 progeny of injected animals. This method of co-rescuing unc-31 to identify transformed animals was developed by Stuart Kim in our laboratory. Non-Unc progeny of F1 nonUnc transformants were checked for the presence of cell deaths, aided by the ced-1 mutation which blocks the engulfment of dying cells. One nonUnc transformant was isolated after injecting 21 parental animals, and its progeny were found to have a wild type pattern of cell deaths. This result shows that C10D8 contains the complete ced-4 gene. We have subcloned a 4.5 kb fragment from C10D8. This fragment contains the complete coding region for a 2. 2 kb transcript previously believed likely to be the ced-4 transcript, as well as 1 kb upstream and few hundred base pairs downstream of the coding region. Coinjection of this 4.5 kb fragment with C14G10 into ced-1; unc-31 animals has produced two lines of non-Unc non-Ced transformants. Thus, this 4.5 kb fragment contains the complete ced-4 gene. We have also coinjected C14G10 and a series of cosmids previously identified by polymorphism mapping as close to ced-3 to look for complementation of the Ced-3 phenotype. Specifically, we have coinjected C14G10 with C43C9, C11B2, W07H6 and C48D1 individually into ced-1; ced-3 animals. Only C48D1 was found to be able to rescue the Ced-3 phenotype. We have obtained two lines of non- Unc non-Ced-3 transformants, one of which is a stable integrant. We are currently subcloning C48D1 to define more precisely the limits of the ced-3 gene.