THE IN VIVO ANALYSIS OF tRNA-TRYPTOPHAN AMBER SUPPRESSOR EXPRESSION IN Caenorhabditis elegans

The goal of this research was to study expression of the tRNAT, gene family in the nematode Caenorhabditis elegans. Eight of the 12 members, identical in coding sequence, were previously recovered as amber suppressor alleles, and suppression efficiency of amber mutations in different genes was different for individual suptRNAT, genes (Kondo, et al., 1990; 1988). The results suggested that members of the tRNAT, gene family were differentially expressed, possibly in a tissueor development stagespecific manner. Transcription in vitro of individual, cloned suptRNA genes in embryo extracts produces levels of expression roughly paralleling strength of expression in the phenotypic assays (Linning, 1992). An advantage of working with tRNA genes from C. elegans is the availability of individual sup genes; this allows us to study expression of individual family members in vivo, using suppression of an amber mutated reporter gene. An amber site was introduced by unique site elimination site-directed mutagenesis (Deng and Nickoloff, 1992) near the N-terminus of a lac2 gene driven by C. elegans heat shock gene hsp16-48 promoters (Stringham, et al., 1992). The resulting single amber mutation was verified by DNA sequencing and expression testing in E. coli. It was then introduced as a reporter gene into transgenic worms (Fire, 1986; Mello et al., 1991). The presence of transgene arrays was verified by PCR using primers from C. elegans hsp16-48 and E. coli lac2 respectively. Sup7, sup24, sup28 and sup29 strains were chosen because they represented strong, medium and weak levels of transcription in vitro and of suppression in vivo. Suppression of the lac2 amber was studied by in situ histochernical staining techniques (Fire et al., 1990). One strain of sup29/sup29 transformed with an amber lac2 was obtained; other sup worms were too weak to survive the germline microinjection. Heterozygote sup7/+, sup24/+, sup28/+ and sup29/+ worms were generated by appropriate crosses to w.t. worms carrying lacZam. In nervous system, the suppression efficiency order is sup7/+ > sup24/+. No suppression was observed in sup28/+, sup29/+ and sup29/sup29 worms. In pharynx muscle cells, the suppression order is sup7/+ >> sup29/sup29 > sup24/+ > sup28/+ > sup29/+. No suppression was observed in sup29/+ worms. In body muscle cells, the order is sup24/+ 2 sup28/+ 2 sup29/sup29 > sup7/+ 2 sup29/+. In pharynx hypodermis cells, the order is sup7/+ > sup24/+ 2 sup28/+ > sup29/sup29 > sup29/+. In body hypodermis cells, the order is sup24/+ 2 sup28/+ 2 sup29/sup29 > sup7/+ 2 sup29/+. In intestinal cells, the order is sup7/+ > sup29/sup29 2 sup24/+ > sup28/+ > sup29/+. These results suggest that suptRNA genes may be expressed tissue-specifically. Making stable integrated lac2 amber strains will allow me to further confirm these results, and pursue possible molecular mechanisms.