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From: pell@boulder.Colorado.EDU (Anthony Pelletier)
Newsgroups: sci.bio
Subject: Re: Cloning/Expression vector copy numbers
Message-ID: <1485@sigi.Colorado.EDU>
Date: Tue, 30-Jun-87 14:01:37 EDT
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Posted: Tue Jun 30 14:01:37 1987
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Reply-To: pell@boulder.Colorado.EDU (Anthony Pelletier)
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(Dan Davison) writes:
>> 
>> Have you considered and rejected chloramphenicol amplification?  I've heard
>> it can get 1000s (!) per cell.  The plasmid must be descended from ColE1,
>> though.
>> 
>> dr. dan davison|Los Alamos National Laboratory|Theoretical Biology|T-10,


Craig writes:
>	Only if it retains the _rom_ gene, which is what is repressed
>and leads to amplification.  Many laboratory derivatives of pBR322,
>such as all the pUC derivatives, have had this gene deleted. As a result,
>they cannot be chloramphenicol-amplified.  However, their basal copy
>number does increase from the 20 or so of pBR322 to about 150-250, which
>makes amplification a bit gratuitous.
>
>
ok, i should start with a disclaimer.  I have not read a thing about this
in a while; the last was a pre-print from Tomizawa's lab that I think was due
out in "Cell" in the summer of '84.  i got it in Feb. of that year.
The product of the rom gene (or is rop the gene and rom the protein?)
is involved in mediating the interaction of RNA1 and RNA2.  One of these
serves as the primer for DNA synthesis at the ColE1 ori and the other is made
from a promoter on the opposite strand such that the two overlap.  This second
RNA inhibits initiation by binding to the primer RNA.  This is part of the 
copy-number control of the plasmid.  tomizawa was pushing the RNA as the
"catalyst" (it is nothing of a sort) and claiming that the protein was helpful,
but not a strict reguirement.  At the time it was (and still is) quite in
vogue to claim that "your favorite RNA" had catalytic fuction.  He tried to
fit the kinetics of the reaction to Michaelis-Menton and failed for the
obvious reason that the steady-state assumtions cannot be made.  The fact
that the reaction had a first order rate constant was pointed out to him.

now, i suppose i should attempt a point here.  I thought that knocking out
the rom product was only part of the story where amplification was concerned.
If rop and the inhibitory RNA are made, the plasmid will not continue to
replicate beyond about 40/cell, to be sure.
But, the real gift of amplification is that the cell number
remains constant and the plasmid continues to replicate; you get about the same
number of plasmid molecules as in a stationary culture of the same volume
but have orders-of-magnitude fewer cells.  This makes lysis and extraction
a great deal easier.
The basis for this is that the dnaA gene product, which is essential for
initiation of chromosomal replication, must be made new for each round of
replication.  ColE1 and others replicate in a dnaA-independant fashion and
so continue to replicate.
I have never done careful studies on the amplification of pGems and the like.
However, I do get better yields from amplified than stationary cultures.
This could be largely due to better lysis.
I'd be willing to bet that there is more to it than we know yet.
But, for me, if it works, don't fix it.



>	   Craig "Baby Doc" Werner   (future MD/PhD, 3 years down, 4 to go)
                 
               ^(I like this one--not that i think it is for my approval)^    

tony pelletier (planning to make my fortune selling yeast alpha factor
                to the singles crowd as "mating phermone" produced through
                the miracle of gene splicing)