Dealing w/ Unclonable sequences

[gubbinse at randd.pprd.abbott.com]

In article <3rkevl$bu7 at netnews.upenn.edu>, demuth at biochem.dental.upenn.edu 
(Don Demuth) writes:
> 
>
>
>        I hate when this happens.  We've isolated a Fusobacterium gene by
>immuno logic screening of a lambda EMBL3 library (expression from endogenous
>Fuso promo ter).  However, subcloning of fragments that we suspect contain the
>gene have be en difficult and appear to undergo deletions in E. coli.  Our
>standard protocol involves subcloning into a pUC plasmid.  Tryed lower copy
>number plasmids, no go .  Restriction, rec minus hosts, no go. 
>
>        So I'm curious as to how others deal with situations like these. Also,
>I remember recently seeing a book chapter or published proceedings from a
>meetin g that dealt with strategies for cloning "unclonable" sequences, but I
>can't rec all where or what it was.  Sound familiar to anybody?  Any
>suggestions would be appreciated.  Thanks. 
>
>
> 			Don Demuth 
> _____________________________________________________________________________
> demuth at biochem.dental.upenn.edu
-- 

I think your best strategy is to interrupt the gene.  Try cutting the fragment 
with another or additional enzymes, or random cleavage with DNAse, or 
exonuclease digestion.  That way you can sequence overlapping clones to get 
your information.

If you wish to express the protein (in E. coli) you can subsequently use PCR 
to separate the coding region from the endogenous promoter and use a tightly 
regulated promoter to control expression.

Another strategy, as was used to clone a toxic gene from B. amyloliquifaciens 
(Gene 53: pp.11-19, 1987) was to first introduce a transposon into the gene in 
the original bacteria, then clone the inactivated gene by the transposon marker.

The other approach I have seen was when a group wanted to construct a 
mammalian expression vector (I think the gene was Renin) but the construct was 
toxic due to some read-through expression in E. coli.  They put a 
transcriptional terminator into a unique site within the gene, which 
interrupted it and prevented expression in E. coli.  Once they had the 
construct, they cut the terminator out with the same enzyme, removed the small 
terminator fragment (by gel filtration?) and re-circularized the plasmid under 
dilute conditions (which is very efficient).  They were then able to transfect 
it into mammalian cells.  Something on this order might work for you, 
depending on what you want to do of course.

Good luck with your cloning.

Any opinions are my own, and do not reflect opinions or positions of Abbott.
Dr. Earl J. Gubbins     Abbott Laboratories  Earl.J.Gubbins at Abbott.com