Sv40Ori acts as a promoter?

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Fri Oct 24 10:14:31 EST 1997


On Fri, 24 Oct 1997 02:51:30 -0600,
oikari at hytti.uku.fi (oikari at hytti.uku.fi) wrote:

>I do have strange pUC119 -based plasmid with Sv40Ori and Human elongation
>factor 1 alpha -promoter in sequence behind each other (Sv40Ori+Ef1a-GFP)
>
>When I cloned Ef1a as a promoter into the pcDNA3 (removing CMV) expression
>of GFP dropped dramatically compared to pUC119 -based vector.
>
>Both plasmids are roughly 10kb, and were transfected into Cos-7 cells
>using standard CaCl precipitation method.
>
>So the question is that if Sv40Ori can enhance expression as a "second"
>promoter (or is there something on pcDNA3 that blocks expression)?
>Any test, results or thoughts available?

The SV40 origin lies between the SV40 early and late promoters, and 
is very close to the early promoter.  

Several plasmids that I have that contain the SV40 origin, contain a 
HindIII to KpnI fragment from the SV40 genome.  I believe that this 
fragment contains:
a) the early promoter
b) the three T-Ag binding sites, and
c) Sp1 binding sites

So it is entirely possible that the SV40 origin cloned in your plasmids 
functions as an "enhancer" of the Ef1a promoter.  

But you also state that you transfect your plasmids into COS-7 cells.
Other COS cells that I have worked with express SV40 large T-antigen,
which will catalyze rapid amplification of plasmids which contain the
SV40 origin.  If COS-7 cells also express large T-antigen, it is equally 
likely that plasmids your SV40 origin-containing plasmids get amplified, 
and thus you detect more GFP expressed from them.

later,
Ashok
-- 
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at ebv.oncology.wisc.edu



-- 
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at ebv.oncology.wisc.edu



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