Worm Breeder's Gazette 11(3): 50
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.
In previous gazette abstracts we have reported the use of a run-on transcription reaction to quantitate and characterize transcription in pregastrulation C. elegans embryos and to identify clones of genes that are preferentially expressed during this time. We report here on further experiments addressing the validity of the in vitro assay and characterizing the early embryonic genes identified so far. The presence of 0.5% sarkosyl or 1 mg/ml heparin, inhibitors of polymerase II initiation, in the embryonic extracts and run-on transcription reactions enhances the activity by about 50-100%. The transcripts produced in the presence and absence of these initiation inhibitors have about the same size range and proportion of sequences homologous to the 3' and 5' ends of several specific genes. Thus the enhancement of activity represents a slight increase in elongation rate (as opposed to inhibition of reinitiation accompanied by a large increase in elongation rate). We conclude that little or no reinitiation occurs in the in vitro reaction. Since initiation therefore occurs in vivo, the in vitro reaction is, at that level, regulated in vivo. We have previously demonstrated correct regulation in the in vitro reaction for several temporally regulated genes (C. elegans meeting abstracts, 1989) and now also for the sex-specific gene, her-1. Radiolabeled run-on transcripts produced in extracts of him-8 embryos hybridize strongly to cloned her-1 DNA while those produced in extracts of N2 embryos do not. This result strongly suggests that the sex-specificity of her-1 transcript accumulation (C. Trent, WBG 10,3) is controlled at the level of transcription. These observations also strengthen the argument that the patterns of transcription observed in vitro do reflect in vivo regulation. We have also continued to screen for early embryonic genes using run- on transcripts to probe an early embryonic cDNA library. Several new clones, including new isolates of two of the genes found earlier, have brought the total number of identified early embryonic genes to fourteen. Hybridization to YAC grids has placed several of the new genes on the physical map. Unfortunately, and against all odds, all of them so far fall into genetically unmapped contigs. Of the fourteen total genes identified three are genetically mapped, two to LGX and one to LGIV, eight fall into genetically unmapped contigs, and three have not yet been mapped. Sequence analysis of cDNA clones of five genes has yielded open reading frames but no significant homologies. We have also extended our RNA gel blot analysis of the early embryonic genes to include RNA preparations from glp-1(q231ts) and fem-1(hc17ts) adults, both grown at the nonpermissive temperature, as well as the early embryonic, late embryonic, and L1 RNAs used previously. None of the early embryonic transcripts are present in the glp-1 (soma only) RNA, but all appear to be present at very low levels in the fem-1 (soma and germline) RNA. Levels of these transcripts increase about 20- to 50-fold in early embryonic RNA. In contrast, levels of several predominantly maternal transcripts increase only about 5- to 10-fold, a consequence of packaging of transcripts into oocytes and of a low level of continuing embryonic expression. The RNA gel blot and run-on transcription results together indicate that there is a small number of genes whose expression is limited to a period of time beginning just prior to fertilization and ending sometime early in embryogenesis. We are continuing to pursue various approaches to determining their functions in the early embryo.