Worm Breeder's Gazette 9(2): 25

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Progress in the Cloning of ced-3 and ced-4

J. Yuan and B. Horvitz

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.