Path: utzoo!mnetor!uunet!husc6!uwvax!speedy!noordewi
From: noordewi@speedy.cs.wisc.edu (Mick Noordewier)
Newsgroups: sci.bio
Subject: Re: Antisense-RNA-derived mutants
Message-ID: <5703@spool.cs.wisc.edu>
Date: 6 May 88 17:26:50 GMT
References: <1784@aecom.YU.EDU>
Sender: news@spool.cs.wisc.edu
Lines: 44
Summary: dubious technique

In article <1784@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes:
> I am interested in generating mutant strains of E. coli in two genes
> I've been working with.  I don't want to mutagenize and hunt for mutants
> (yuck, what a mess!).  The method I use should be simple, effective, and
> specific to the genes in question.  What if I put an antisense code
> for the gene(s) on a plasmid and transform.  The antisense message would
> then prevent translation of the sense message, right?  I'd appreciate some
> advice, although, as always, Dan Davison may not respond.
> -- 
>       dn/dx      Dept Molecular Biology   diaz@aecom.yu.edu
>      Dan Diaz      Albert Grubelstein College of Osteopathy 

If you are looking for a simple, effective method, then anti-sense
is not the answer.  This issue received considerable attention several
years ago, when anti-sense RNA was proposed as a mechanism for natural
gene control.  Many workers made efforts, but little seems to have
come of it.

I actually tried it, in a lambda system, to attempt to regulate
expression of a lac fusion.  I cloned pBR322 p1 promoter to create
anti-beta-galactosidase on a tetracycline-resistant plasmid, then
transformed a lambda lysogen which was producing a lac fusion 
protein.  I confirmed the presence of anti-sense transcript by 
northern blotting, and did dot-blots to  ascertain that it was present
in excess of the sense RNA (I could distinguish between the sense
and anti-sense message because my anti-sense transcript was full-length
lacZ, whereas the lac fusion contained only the first few  amino acids,
therefore I could make a DNA probe that only recognized my anti-sense).

I am not suggesting that because I failed the entire concept is faulty,
but other's experiences seem to be less than unqualified successes
as well.  Even the reported successes have variable quality.  Issues
seem to be stability of the anti-sense transcript, as well as its
ability to interrupt a closely coupled transcription-translation
mechanism.

If you want, I can probably come with some literature citations of
both pros and cons.

If you try it, good luck.  I would be interested in hearing of your
results.

				Mick Noordewier
				noordewi@cs.wisc.edu