in vitro transcription using N. crassa nuclear extracts?
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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