bernard at elsie.nci.nih.gov
Fri Nov 17 15:33:27 EST 1995
This may belong in bionet.viruses (?exists) but I feel more at home in
this newsgroup so....
BOTTOM LINE: For the usual (eg. 369 bp) SV40 polyadenlation sequence
hom much of the actual sequence appears in the mature mRNA?
I am trying to differentiate the expression of an endogenous mouse
gene from a mouse transgene in the same animal. The construct has
a heterologous promoter and the SV40 polyadenylation sequence
(the usual HincII fragment used in commercial expression vectors).
A sure way would be to use RNase protection analysis with a
probe generated from the 5' end of the transgene construct as this
would protect from the 3' end of the promoter into the cDNA and
would thus be larger than the protected fragment from the endogenous
mRNA (which would comprise just the cDNA portion). I realise that
this could also be achieved by primer extension analysis.
However, since the transgene construct is already in a
vector with a viral promoter at the 3' end I was wondering if I
could save some time and just run a fragment at the 3' end of the
construct. The key question is thus how much of the foreign
polyadenylation sequence actually appears in the mature mRNA. Will
this piece of sequence be long enough to make the whole fragment run
slower than the endogenous fragment on the gel?
I have delved into SV40 and polyadenylation biology and have
found no hard answers in the literature. There are two AAUAAA
sequences in the SV40 signal but I don't know which of these (or both)
Any insight gratefully received. I can e-mail anyone the actual
sequence of the polyadenylation signal if they need it. I confess to being
a complete amateur at understanding such sequences. Those working on them
have my admiration.
Bernard (not shut down)
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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