Daniel R. Schoenberg
dscho001 at postbox.acs.ohio-state.edu
Wed Sep 27 09:05:30 EST 1995
In article <446e93$g8e at rs3.rz.uni-hohenheim.de> mmzsm at mmn2.med.nottingham.ac.uk (steven morse) writes:
>From: mmzsm at mmn2.med.nottingham.ac.uk (steven morse)
>Subject: polyadenylation positions
>Date: Mon, 25 Sep 1995 14:27:47 GMT
>Organization: Rechenzentrum Universitaet Hohenheim
>Message-ID: <446e93$g8e at rs3.rz.uni-hohenheim.de>
>X-Newsreader: Forte Free Agent 1.0.82
>Hope someone out there will be able to give me some advice with
>regards polyadenylation sequences.
>1) I'm attempting to modify a vector and in the process have to
>remove the polyadenylation sequence - ideally I'd like to put a
>multiple cloning site before the polyadenylation sequence but will
>this affect gene expression ?
>2) One of the genes I'm interested in is CAT - does CAT require a
>polyadenylation signal or is it stable enough without one ?
>3) Is there a minimum length required between the polyA sequence and
>the gene for the polyA to be effective ?
>4) Is there a good review on polyA sequence structure and function ?
>I did a lit. search but found very little info.
>Thanks for all your help.
>CRC Department of Clinical Oncology
>University of Nottingham
>e-mail: stewart.martin at nott.ac.uk
1. We have some experience in this area. It is fine to put a multiple
cloning site before the polyadenylation sequence. We've done this with no
problem at all. Be certain that you don't do away with the sequences
downstream of AAUAAA. These GU and U-rich sequences are very important. If
you wish to make a synthetic poly(A) element that is highly functional, see
Leavitt et al., Genes and Development (1989) 3: 1019-1025. You can synthesize
this and just insert it into your plasmid.
2. I would not try to express any gene in eukaryotic cells without poly(A).
3. I'm not certain I understand what you are looking for here. No, there is
no minimum length, just be certain you have poly(A).
4. For some recent reviews see Sachs and Wahle, JBC (1993) 268: 22955-58,
Manley, Current Opinion in Genetics and Development (1995) 5: 222-228.
Department of Pharmacology
Ohio State University
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