Worm Breeder's Gazette 13(3): 46 (June 1, 1994)

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.

DAD-1 ,an evolutionarily conserved suppressor of programmed cell death in vertebrates and Caenorhabditis elegans

Asako Sugimoto, Rebecca R. Hozak[1], Torahiko Nakashima, Takeharu Nishimoto[2], Joel H. Rothman[1]

[1]Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
[2]Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812, JAPAN.

The dad-1 (Defender against Apoptotic Death) gene was originally identified in a mutant hamster cell line (t sBN7 )(Nakashima, et. al, M.C.B., 13, 6367-6374). These cells, which have a point mutation in dad-1 ,undergo apoptosis (programmed cell death) after a shift to a restrictive temperature. This suggests that dad-1 is essential to suppress cell death in these cells. The predicted DAD1 protein is highly hydrophobic. Its sequence is not similar to any other gene products in the database, but it is highly conserved among vertebrates: human and hamster DAD1 are 100% identical, and they are 91% identical to the predicted Xenopus protein. To understand how DAD1 participates in programmed cell death, we are characterizing its function in C. elegans.

First, we tested the ability of human DAD1 to prevent cell death in C. elegans. Human DAD1 was expressed from the hsp16 -2promoter. Heat-shocked transgenic worms showed significantly fewer cell corpses than control worms (at comma stage, heat-shocked transgenic: 8.7; non-heat-shocked transgenic: 14.8; heat-shocked control: 15.8; non-heat-shocked control: 15.1; Both strains carried a ced-1 mutation to facilitate scoring of corpses). These results suggest that human DAD1 is sufficient to prevent cell death in C. elegans.

To initiate genetic analysis of this gene, we isolated a C. elegans dad-1 homologue (Ce- dad-1 ) from B. Barstead's cDNA library. Ce- dad-1 ,like the vertebrate genes, encodes a protein of 113 amino acids. This sequence is highly similar to that of vertebrate DAD1 (63% identical, 83% similar to human/hamster DAD1 ).In addition, the position of the single intron in the coding region is conserved. The Ce- dad-1 cDNA detects a single 550 base transcript on a Northern blot. Expression of Ce- dad-1 from the heat-shock promoter reduces the number of cell corpses, albeit with lower efficiency than did the human gene. However, the C. elegans gene rescues the mutant hamster cell line as well as the human or frog genes.

These findings suggest that dad-1 appears to be both necessary and sufficient to suppress programmed cell death. In order to understand the function of endogenous Ce- dad-1 ,we are currently examining the expression pattern of the gene and attempting to isolate loss-of-function mutants. We have screened for a mutator-induced Tc1 insertion (Rushforth, Saari and Anderson, M. C. B., 13, 902-910; thanks to Ed Maryon from Phil Anderson's lab for help). Although a positive candidate was isolated in our initial screen, the strain proved not to contain a simple Tc1 insertion. The PCR product which hybridized to a dad-1 probe was not the result of amplification with the Tc1 and dad-1 primers, but instead from the dad-1 primer alone. This suggested that there may be a duplication and inversion around the dad-1 locus in this strain. Genomic Southern hybridization confirmed that there is a rearrangement of several hundred bases. This rearrangement is likely to be caused by Tc1 insertion/excision, but we do not understand how it arose. We are examining whether this rearrangement gives any phenotype, and are performing additional screens for insertions.