In vitro recombination

Stacy Ferguson sef at med.unc.edu
Sun Nov 12 11:10:25 EST 1995


In article <483cft$57r at mserv1.dl.ac.uk>,
Andrei Popov  <ANDREI.POPOV at bbsrc.ac.uk> wrote:
>>Does anyone know if in B-cell differentition,  the D-J recombination and
>>the Variable Heavy-DJ recombination reactions have been successfully
>>performed in-vitro.  A friend of mine said that he thought Dr. Stuart
>>Kauffman of the Santa Fe Institute has done this, but I haven't been
>>able to find any references on this.  Any help would be greatly
>>appreciated.    
>
>No. It has not been done.
>M.Sadofsky (spell?) and coauthers published 
>a couple of months ago in Cell a paper on V(D)J recombination
>in vitro but.... it was very far away from total success.
>
>

Perhaps you didn't mean for it to sound that way, but I don't think
it's fair to trivialize this data. It's been a number of years since
the RAG genes were initially described and until this work from the
Gellert lab was published a few months ago, no one has been able to 
show the direct function of RAG-1 and 2 proteins. For one thing, the
solubility problems of these proteins have been a nightmare for many
who have tried to study them (trust me, I've been there) and it's 
not a minor feat to have come up with an assay to which recombinant
RAG proteins can be both added and  functional. Demonstration that
RAG-1 and RAG-2 fractions contain RSS cleavage activities is extremely
important, particularly since aside from a potential topoisomerase-like
site on RAG-1 (which has been mutagenized without any real effect on function
by another group), no obvious DNA binding domains exist. Regardless of
what it takes to set up an in vitro assay for complete recombination
events, I doubt that the ability to purify functional RAG proteins and 
demonstrate their importance in the initial cleavage will have been 
the easiest part and if, in fact, there are no novel DNA binding domains in
either of these proteins, then the system that Gellert's lab described 
should prove useful in identifying those that do. It may only be a start
in establishing a complete in vitro recombinase system, but it's a 
critical one (you can't resolve a structure that hasn't been generated,
after all, and cleavage of RSS sequences has to be a extremely early 
step if not the first.)

Aside from the biochemistry of the reaction, this system is important
in addressing popular models of recombination, including the requirement
(or lack thereof) for the presence of RAG proteins in the nuclear matrix as
it relates to their functions. Given the recent results with the SCID gene
product and DNA repair genes identified as important in V(D)J recombination
by Alt's lab, I suspect that the Gellert paper will be far from trivial
when it comes to developing assays for the functions of the other proteins
involved in the recombination reaction. 


Stacy







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