Worm Breeder's Gazette 12(1): 20 (September 1, 1991)

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.

Molecular cloning of the emb-5 gene

Kiyoji Nishiwaki, Tohru Sano, Johji Miwa

NEC Fundamental Research Laboratories

Temperature-sensitive maternal effect mutations in the emb-5 gene (1) show premature second E cell division and abnormal gastrulation at nonpermissive temperatures. Embryos arrest as balls of several hundred differentiated cells around the lima bean stage (2). We previously reported identification of a Bergerac Tc1 closely linked to emb-5 (WBG 11, 1, 39). We obtained several overlapping cosmids (from J. Sulston and A. Coulson) closely linked to this Tc1 containing region so that some of the cosmids possibly contain the emb-5 gene. We injected 4 overlapping cosmids individually into emb-5 ( hc61 )according to the method of C. Mello and V. Ambros (personal communication). Of these, two overlapping cosmids, C56E12 and C17H4 ,were found to have transforming activity, indicating that the activity was within a 20kb overlapping region. To limit the region harboring the transforming activity, we examined transformation after cutting C56E12 with various restriction enzymes, as described by S. Kim et al. (3), and found that region within the 10.3kb fragment. Germline transformants grew slowly at nonpermissive temperature and revealed a wide range of E cell phenotypes from mutant type to completely rescued type similar to wild type. Using an internal 1.7kb SalI fragment (SalI digestion resulted in no rescue) as a probe, we detected a single 5.4kb mRNA in both wild type and hc61 .We obtained several cDNA clones from B. Barstead's cDNA library with the same probe. One of the clones had an insert of about 3.6 kb cDNA and the sequence of this cDNA revealed the 1121 aa C-terminal portion of the putative gene product. No significant homology was found when this amino acid sequence was compared with those for other proteins in the SwissProt and the NBRF databases. Preliminary results showed amino acid change from Ala to Glu at 663rd aa from C-terminus in hc61 .Currently, we are attempting lacZ fusion experiments using the vectors provided by A. Fire (4) to understand the localization of the gene product.