in vitro transcription using N. crassa nuclear extracts?

rlab davis rdlab at gandalf.bio.uci.edu
Fri Dec 1 14:51:00 EST 1995


I am attempting to determine if changes in the abundance of a particular N. 
crassa mRNA under two different growth conditions are due changes in the 
cytoplasmic stability of the message.  As a control I would like to determine 
if there are any differences in transcription of the gene in the two growth 
conditions.  (Nuclear run-on assays are messy owing to the normally low level 
of expression of this gene which leads to high signal to noise ratios.) 

Brett Tyler and Norman Giles have published a protocol for making crude RNAPII 
exctracts using conidia.  My concern is that if the stability of my mRNA of 
interest is regulated by a cytoplasmic factor, it might be present in these 
extracts.  Is this a valid concern?  The solution to this problem would seem 
to be to use nuclear extracts for the in vitro assays.  Has anyone ever made a 
nuclear RNAP II transcription extract? (I didn't find one in a search of 
current literature)  Could I use one of the nuclear isolition procedures for 
Neurospora and then modify the Tyler and Giles protocol?  Or, could I obtain 
extracts from isolated nuclei using a mammalian or yeast protocol?  

I would appreciate any feedback/help I can get.

Thanks in advance,

Martin Hoyt (Rowland Davis lab)
mahoyt at uci.edu
Dept. Mol. Biol. and Biochem.
Univ. of Calif., Irvine



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