Worm Breeder's Gazette 11(4): 37
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.
The genes ced-3 and ced-4 are essential for the 131 programmed cell deaths that occur during normal C. elegans development. Mosaic analysis has shown that the actions of both ced-3 and ced-4 are cell autonomous. We have previously cloned and sequenced ced-4, and found that this gene encodes a protein with two possible calcium-binding domains. We have now initiated molecular studies of ced-3.We identified a Bristol-specific Tc1 polymorphism closely linked to ced-3, cloned this polymorphism and, in collaboration with John Sulston and Alan Coulson, defined a 200 kb contig. By mapping RFLPs between the Bristol and Bergerac strains, we localized ced-3 to a region of about 2-3 cosmids. We microinjected cosmids from this region along with the unc-31(+) cosmid C14G10 into ced-1; ced-3 animals and observed rescue of the ced-3 phenotype using cosmid C48D1. We identified a 7 kb subclone of C48D1 with ced-3(+) activity and showed that a slightly smaller 6 kb subclone lacks this activity. This 7 kb clone detects a 2.8 kb RNA on a Northern blot. This transcript is expressed primarily during embryogenesis, consistent with the observation that most programmed cell deaths are embryonic. This transcript is very abundant, with a level in eggs comparable to that of actin I RNA. The level of this transcript is not altered in eggs derived from ced-4 animals. We have isolated a number of cDNAs that correspond to this transcript, and have sequenced one strand of a 2.5 kb cDNA, pJ87. This cDNA contains a single long open reading frame that can be translated into a protein of 518 amino acids. However, the 5' region of this cDNA consists of a sequence of 24 consecutive T's, which most likely is a cloning artifact. A search of the Genbank protein sequence database (release 63.0) revealed no sequence highly similar to that of ced-3. However, the region near the amino terminus of the putative ced-3 protein is very striking: it has 34 serines within a stretch of 120 amino acids. Serines are common targets for serine and threonine protein kinases. Furthermore, many of these serines are flanked by basic residues, as are a variety of the phosphorylation sites of protein kinase C and cAMP-dependent protein kinases. We have made peptides corresponding to three serine-rich regions of ced-3. Preliminary results indicated that at least one of these peptides can be phosphorylated by cAMP-dependent protein kinase. The high level of expression of the ced-3 transcript suggests that ced-3 expression might not be specific to dying cells. We suggest that ced-3 function might be regulated by phosphorylation, with either phosphorylation or dephosphorylation activating ced-3 and consequently causing cell death.