Expression of EGFP without ATG

David N. Levy levy at removeforspam.uab.edu
Wed Feb 2 17:08:06 EST 2000


EGFP from Clontech has a GTG valine codon immediately following the ATG
start codon.  According to some text books, GTG can be used as a
translation start codon, though with less efficiency than ATG.  I have
tried to find primary articles on this, but I have only been able to
locate experiments showing GTG usage in yeast and bacteria, so I dont
know about higher eukaryotes.  It would be interesting and useful to
find out about this directly.  So I would suggest that you try deleting
the second codon GTG from the gene.  I asked Clontech if the valine can
be deleted and they said that it can without eliminating fluorescence,
though I have not tried it myself. 

Let me know how this comes out.

David N. Levy
University of Alabama at Birmingham
Birmingham, AL 25294



In article <3896C6DD.BEA34344 at hotmail.com>
michaelmoos at hotmail.com (Michael Moos) writes:

> Hi,
> we´ve been trying to determine whether a bacterial intron is splicing
> also in eucaryotes. Therefore we first modified vector pEGFP-N1 by
> deleting the ATG codon and the parts of the Kozak sequence of the EGFP
> gene. We then planned to introduce a new startcodon and a Kozak sequence
> 5´ to the intron and look for expression of the EGFP after transfection.
> However when we tested the modified pEGFP-N1 vector (the one without ATG
> and Kozak seq), we already got fluoresence. Thus apparently the EGFP is
> produced in eucaryotic cells eventhough the ATG is missing.
> Does anyone have an explanation for this? Or has anyone had the problem
> as well??






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