Worm Breeder's Gazette 14(1): 92 (October 1, 1995)
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.
HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA
Loss-of-function mutations in ced-4 prevent programmed cell death in
C. elegans. The genomic sequences of ced-4 genes from C. briggsae and C.
vulgaris revealed that the 3' end of the third intron of these genes is
conserved with the respective intron in C. elegans. We suspected that
this region might contain an alternatively-used exon, such that a splice
acceptor 72 bp upstream of the previously identified splice acceptor might
be used to generate an in-frame insertion of 24 amino acids in the
translated product. To test this idea, we hybridized a probe derived from
the conserved region to northern blots of wild-type RNA. We observed a
band slightly larger than the previously known ced-4 transcript of 2.2 kb.
In addition, we used RT-PCR to amplify cDNAs containing the alternatively
spliced message from wild-type RNA and confirmed their identities by
sequence determination. The abundance of the alternative transcript
(designated ced-4L, for long) is approximately 10-30 fold less than that
of the more common transcript (designated ced-4S, for short). We have
previously shown that overexpression of ced-4S can kill cells (WBG,
October, 1993). To test the function of ced-4L, we established transgenic
lines containing a cDNA corresponding to this message under the control of
the worm heat-shock promoters. Transgenic animals treated with a heat
shock had many extra cells, suggesting that normal programmed cell deaths
had been inhibited. We also introduced constructs containing ced-4L under
the control of the constitutive dpy-30 promoter into ced-9(lf) animals,
which die because of massive ectopic programmed cell death. Transgenic
animals were rescued from the ced-9(lf)-associated lethality, suggesting
that overexpression of ced-4L can inhibit ectopic as well as normal cell
deaths and supporting the idea that ced-4L normally protects against
programmed cell death.
Genetic evidence supports the notion that ced-4 encodes two
transcripts of opposite functions. Ellis and Horvitz (Cell 44, 817-829,
1986) showed that while egl-1/+ animals are ~60% Egl (because of the
ectopic deaths of the HSN neurons), ced-4(null); egl-1/+ animals are
non-Egl, providing one line of the original evidence that ced-4 encodes a
killing function. However, egl-1/+; ced-4(null)/+ animals are ~85% Egl,
suggesting that loss of ced-4 function can increase cell death. This
finding is consistent with the hypothesis that ced-4 encodes a protective
function as well. Further genetic evidence of a dual function for ced-4
is provided by the ced-4 allele n2273, which contains a mutation in the
conserved G of the ced-4S-specific splice acceptor of intron 3. This
mutation weakly prevents cell death, yet synergizes with a weak ced-9
allele to enhance ced-9-associated lethality (M. Hengartner, personal
communication). This enhancement is ced-3-dependent, suggesting that
n2273 enhances killing by programmed cell death. Thus, this mutation
affects both the protective and killing functions of ced-4. Sequence
analysis of ced-4 transcripts from n2273 animals obtained by RT-PCR
suggests that both ced-4S and ced-4L transcripts are defective, supporting
the notion that the opposing genetic functions encoded by ced-4 correspond
to the alternative transcripts.
Genetic analysis of n2273 has also suggested that the protective
function of ced-4 is negatively regulated by ced-9, just as is the killing
function of ced-4, consistent with the observation of Hengartner and
Horvitz (Nature 369, 318-320, 1994) that ced-9 can act to induce, rather
than prevent, cell death in certain genetic backgrounds. These
observation have suggested to us a model for the function of the
ced-9(n1950) dominant gain-of-function mutation, which prevents cell
death. We propose that ced-9(n1950) is incapable of negatively regulating
the protective (ced-4L) function of ced-4, yet is still capable of
negatively regulating the killing (ced-4S) function of ced-4, thus
resulting in extra cell survival as a result of increased ced-4L activity.
We suggest, therefore, that ced-9(n1950) causes a loss of a specific ced-9
function rather than an activation of ced-9 function in general.