Worm Breeder's Gazette 12(2): 40 (January 1, 1992)

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.

Isolation and Characterization of Vulval-specific cDNA Clones

Paul S. Kayne, Shahla Gharib, Paul W. Sternberg

HHMI, Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125

More than 50 genes involved in vulval development have been identified through a variety of genetic screens. However, it is unlikely that these completely define the vulval developmental pathway. To isolate new genes, we are screening subtracted cDNA libraries. We constructed "plus" libraries from lin-15 ( n309 )animals which have both an anchor cell and 1° and 2° vulval cells. We constructed "minus" libraries from lin-12 ( n676 ); lin-26 ( n156 )animals, which lack both an anchor cell and vulval cells. While lin-12 , lin-15 and lin-26 may affect a few other cells, candidates can be quickly checked against wild type and other vulval mutants to determine their specificity.

All libraries were made from populations that were synchronized in the L3 stage, which encompasses the initial signaling through the beginning of vulval morphogenesis. We constructed libraries using lambda and plasmid vectors to reduce the loss of cDNAs through recombination or vector incompatibility. Two independent libraries were made from each strain, each containing at least 2 x10 +E7independent recombinants. The minus libraries were then subtracted from the plus libraries, thus removing sequences common to both and enriching for vulval specific genes. The resulting library was then hybridized with probe produced from the minus libraries.

From 210,000 recombinants, we selected candidates that did not strongly hybridize to the minus probe (approximately 1-5% of starting colonies, depending on the screen). We screened out undesirable recombinants, rather than selecting, to permit us to identify very rare candidates. We picked 5,000 candidates and probed these with both the plus and minus libraries. 58 of these candidates hybridized only with the plus library. To date, we have checked about half of these by northern hybridization to RNA from L3 plus, minus and N2 animals. One candidate shows at least 20 fold greater expression in lin-15 ( n309 )and N2 animals as compared to lin-12 ( n676 ); lin-26 ( n156 )animals. Seven of the candidates showed no signal on northerns. We are using RNAase protection assays to further characterize these candidates. Of these lower expression candidates, we have found one recombinant that has approximately three-fold greater expression in the plus and N2 strains as compared with the minus strains. We are currently sequencing and placing these candidates on the physical map.