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.

ced-3 and ced-4 Germline Transformation

Junying Yuan and Bob Horvitz

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.