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.

Transcription of Pol III Genes in C. elegans Cell-free Extracts

B.M. Honda

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 
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.