Worm Breeder's Gazette 10(1): 31

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.

Molecular Cloning of ced-4

J. Yuan and B. Horvitz

Mutations in ced-4 eliminate almost all of the programmed cell 
deaths that normally occur during C.  elegans development.  Mosaic 
analysis has shown the action of ced-4 to be cell autonomous.  To 
study at the molecular level how ced-4 acts within cells to cause 
their deaths, we decided to clone this gene.  We isolated a ced-4 
allele, n1416, in a TR679 mutator background by a complementation 
screen (WBG, 9(2), p.  25).  Southern blot analyses revealed that 
n1416 is not caused by the insertion of Tc1 into ced-4.  However, when 
we used the newly isolated Tc4 (see abstract by Junying Yuan, Mike 
Finney and Bob Horvitz in this WGB) to probe appropriate recombinants, 
we found a novel 2.0 kb HindIII-BamHI Tc4-containing band that 
cosegregates with the Ced-4 phenotype.  Five dpy-17 
79 recombinant chromosomes and five unc-79 
17 recombinant chromosomes had the band, 
while seven dpy-17 non-unc-79 non-ced-4 and two unc-79 non-dpy-17 non-
ced-4 did not, which suggests that the novel 2.0 kb Tc4 band maps 
between 0.02 mu to the left of ced-4 and 0.01 mu to the right of ced-4.
Furthermore, three independent non-Ced revertants of n1416 did not 
have the band, which suggests that Tc4 had inserted into the ced-4 
gene to cause the n1416 mutation.  We cloned a 5.5 kb HindIII fragment 
that contains this insertion.  Probing the Southern blot containing 
our recombinant chromosomes with the flanking region (non-Tc4) of this 
Tc4-containing band (a 3.5 kb HindIII-BamHI Band), we have confirmed 
that we have cloned a piece of DNA that contains at least part of the 
ced-4 gene.
Northern blot analysis using this 3.5 kb fragment as a probe showed 
two hybridizing transcripts from N2 animals: one about 2.2 kb and one 
about 0.9 kb.  In n1416 animals there are three hybridizing 
transcripts: one about 4.0 kb and containing Tc4, another a little 
bigger than the 2.2 kb transcript in N2 and a third a little smaller 
than the 0.9 kb transcript in N2.  In two revertants of n1416, there 
are two hybridizing transcripts with close to wild-type mobility: one 
0.9 kb and the other about 2.2 kb (it is a little bigger than 2.2 kb 
in one revertant).  It is most likely that the 4.0 kb band was caused 
by the insertion of Tc4 (1.8 kb) sequences into the 2.2 kb transcript 
in N2.  It is possible that in n1416 animals Tc4 sometimes is removed 
imprecisely by splicing to produce a band that is a little bigger than 
the 2.2 kb band in N2.  The origin of the 0.9 kb band is unclear.  It 
may share some sequence homology with the 2.2 kb RNA, since we have 
isolated cDNA's that hybridize strongly to the 0.9 kb band but weakly 
to the 2.2 kb band.
Preliminary developmental Northern analyses have shown that the 
expression of the 2.2 kb band is predominantly in eggs and the 
expression of the 0.9 kb band is mostly in eggs and young adults.
We are trying to isolate a larger piece of genomic DNA from the ced-
4 region, since it is possible that we have isolated only a part of 
the ced-4 gene.  We are also trying to isolate complete cDNA clones 
for both of the transcripts.  Once we get complete cDNA clones, we 
will start sequencing and microinjecting to study the relationship 
between the 0.9 kb and the 2.2 kb transcript and to ascertain whether 
one or both of these transcripts are responsible for ced-4 gene