Poly(A) addition signals?

Ian A. York iayork at panix.com
Fri Jun 11 09:31:56 EST 1999


I want to express a full-length mRNA from a transient expression vector in
COS.  I suspect (but don't know) that it might be important to express
precisely the authentic mRNA; that includes having the poly(A) tail start
at exactly the right place.  Normally, of course, in an expression vector
there's a poly(A) addition signal downstream of the insert, in the vector,
so that the poly(A) tail starts at a point downstream of the usual place.

What I've done is to make a nearly full-length cDNA in the plasmid.  Then
I PCRed from genomic DNA across the usual stopping point.  I went 190 bp
downstream and then spliced that into my cDNA piece.

I don't know much about poly(A) addition, but my insert now has:
AUUAAA around 20 bases upstream of the nominal stop;
U/G rich region around 50 bp downstream of the nominal stop (GTGGTCTTTT 
TTGGGGGTGG GGAGGGTTT)

As well as this (hopefully) authentic poly(A) signal, the plasmid includes
the SV40 poly(A) addition signal, downstream of my insert.

My question is:  Will the SV40 signal override the 'authentic' signal?  Is
there a simple way of telling if I have the authentic mRNA, or would I
have to clone and sequence?  

I don't even know if the proper termination *is* important, but there's
circumstantial evidence for it.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England



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