Worm Breeder's Gazette 8(3): 67
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.
We've recently prepared crude C. e extracts which support the transcription of pol III genes. S100 fractions were initially prepared from ncl-1(e1865) (generous gift of Ed Hedgecock) as it was possibly RNase deficient, but more recently N2 extracts have also shown transcription activity. There are still some problems with variability from preparation to preparation; general assay conditions ( optimal salt, temp., etc.) are similar to those described for yeast systems. When a single C. elegans 5S DNA repeat in pBR322 is used as a template, a 120N transcript is produced. The product hasn't yet been fingerprinted, but transcription of 5S DNA minus termination signal ( run of T's) yields the expected (185N) transcript (terminated at a run of T's in psR322), suggesting initiation of 5S transcription at the correct place upstream. Transcription of a Xenopus tRNAmet gene shows products with sizes characteristic of primary transcript and processed product(s)--processing may be aberrant, as product sizes look different from those obtained in a Xenopus oocyte extract. It's interesting that C. elegans extracts will not efficiently transcribe Xenopus 5S DNA; similarly, Xenopus extracts are very inefficient in transcribing heterologous worm 5S DNA. This is perhaps not surprising given the fairly large sequence differences between the internal promoter regions of the two genes. However, the addition of C. elegans protein fractions 'complements' Xenopus extracts and allows efficient transcription of worm 5S DNA. This might provide a good assay to purify a worm 5S specific protein without the problems associated with the instability of fractionated components (this would be analogous to the first scheme used to purify TFIIIA from Xenopus oocytes). We're also looking indirectly for TFIIIA-like or tRNA- specific fractions, and a pol II system to look at more 'interesting' ( ?) genes.