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 function.