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cloning eukaryotic genes

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ng01

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Feb 25, 2003, 1:06:10 AM2/25/03
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Has anyone ever run into a problem such as mine?

I have been attempting to subclone a wild type eukaryotic gene into any
cloning or expression vector. The C-terminal domain of this gene product is
definitely toxic to bacteria. There is a beautiful Shine-Delgarno site
optimal distance 5' from an in-frame AUG a little more than half way down
the gene. Two high-scoring predicted prokaryotic promoter sequences are
present 80 and 400 nucleotides 5' of this AUG. One of these promoter
sequences is basically a textbook polymerase and sigma-70 consensus binding
sequence. I believe that as soon as my gene is subcloned into a vector in
either orientation and stuck into a bacterium, the 3' end of the gene is
being transcribed and translated with no control. Thus, these guys die, and
all I get are the surviving bacteria that have deleted portions of my gene.

Has anyone before heard of such a coincidental residence of promoter and
Shine-Delgarno sequences upstream from an in-frame AUG in a eukaryotic gene?


dudu

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Feb 27, 2003, 6:28:49 AM2/27/03
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"ng01" <add...@page.bottom> wrote in message news:<mJD6a.69541$zF6.4...@bgtnsc04-news.ops.worldnet.att.net>...

I haven't heard of that.And I wonder which gene it is?

dudu

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Feb 27, 2003, 6:32:20 AM2/27/03
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"ng01" <add...@page.bottom> wrote in message news:<mJD6a.69541$zF6.4...@bgtnsc04-news.ops.worldnet.att.net>...

I haven't ever heard of that,and I wonder which gene it is?

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