Worm Breeder's Gazette 13(4): 57 (October 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.
Laboratory of Molecular Biology, Dept of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441, JAPAN
A large set of genes required for embryogenesis has been characterized among temperature sensitive lethal mutants following EMS mutagenesis, using 25260C as non-permissive and 16260C as permissive temperatures (Reviewed by Wood, 1988). To understand molecular basis of such developmental defects, we have used germline transformation technique to rescue the emb30 ( g53 )III,a strict parental effect mutant for which maternal gene function is necessary and sufficient (Cassada et al., 1981; Isnenghi et al., 1983; Denich et al. 1984). We used cosmid clones located in the emb-30 region of chromosome III together with the rol-6 ( su1006 )marker genomic DNA, and screened for emb-30 (F53)roller transformants. From a selection of several cosmid clones in that region only two overlapping cosmids W07H4 and F58A4 produced roller transformants that were viable at the nonpermissive temperature of 25260C. Since both cosmid clones contain the gene encoding a novel member of the gamma tubulin gene, we investigated if an antisense construct from such a gamma tubulin cDNA may produce a phenotype resembling the emb-30 phene. A gamma tubulin cDNA clone (from Chris Martin's library) cml3 g11 which produced excision plasmid pRATII containing a 1.5 kb insert was used to obtain a 0.5 kb fragment of SacI-ScaI fragment of gamma tubulin cDNA, that was ligated in opposite orientation in the hsp16 -2heat shock expression vector pPD49 .78(A. Fire). The resulting construct called pAG0 .5contains the ligated fragment, downstream of the hspl6 -2in the opposite orientation and can produce antisense transcript for the 5' end of the gamma tubulin. Marker DNA from dpy-20 ( D. Baillie and D. Suleman) together with the antisense pAG 0.5 plasmid DNA was microinjected into dpy20 ( e2017 )animals, and non-dpy germline transgenic animals were tested after given a 30260C heat shock treatment for eight hours. There was great reduction in the brood size only for the non-DPY animals compared to the control set in which no heat shock was administered, when screened at the permissive and nonpermissive temperatures of 16 and 25260C, respectively. Reduction in brood size of the heat shock treated transgenic non-dpy animals clearly suggested that antisense construct disrupted the gamma tubulin function resulting in the embryonic arrest phenotype similar to the emb-30 mutants. Finally using gamma tubulin specific flanking primers, PCR amplification, subcloning in pUC118 plasmid vector, we are in the process of sequencing emb-30 ( g53 )mutant DNA to identify the mutation site of this allele. We thank A. Coulson, J. Sulston, R. Cassada, T. Fukushige, A. Fire, J. Kramer, D. Baillie, D. Suleman, J. Miwa, Y. Kohara, R. Durbin, Y. Hotta , R. Holmgren and the Caenorhabditis Genetics Center, for their help in this project.