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Abstract:
To study the evolution of developmental processes, we analyse vulva development in P. pacificus and compare it to C. elegans. In P. pacificus, 7 of the 12 ventral epidermal cells undergo apoptosis, whereas the corresponding cells in C. elegans fuse with the surrounding hypodermis. Phylogenetic analysis suggests that cell death of these epidermal cells represents a derived character. To understand the evolutionary changes responsible for these evolutionary alterations in the apoptotic mechanism, we are characterising cell death mutants in P. pacificus. Mutations in the cell death pathway in P. pacificus lead to the survival of P(1-4,9-11).p, which normally die in wild-type. Complementation tests have shown, that these mutants fall into two complementation groups, one of which has been proven to correspond to Ppa-ced-3 (Sommer et al., 1998). The second complementation group has been originally characterized as ipa-2 (inhibitor of P-ectoblast apoptosis) and is a good candidate for the Ppa-ced-4 homolog. To test this hypothesis, we tried to clone ced-4 using different approaches, viz low stringency hybridisation, PCR and yeast two hybrid approach with Ppa-mac-1 as bait. In C. elegans it has been shown that MAC-1, a member of the AAA type ATPase family, interacts with ced-4 at the protein level (Wu et al., 1999). However none of the used approaches were successful. We are presently trying to clone Ppa-ced-4 using positional cloning. Preliminary data suggest a linkage of ipa-2 and Ppa-pal-1. In C. elegans, pal-1 is closely linked to ced-4. Further mapping is in progress to clone P. pacificus ced-4 by chromosomal walking.
