More In vitro recombination

Moshe Sadofsky sadofsky at helix.nih.gov
Wed Nov 15 15:48:21 EST 1995


Greetings from the Gellert lab. With regard to this thread about in
vitro V(D)J recombination, it is our lab opinion that a plausible model
would be site specific cleavage at the appropriate signal sequences
followed by processing and rejoining of the free DNA ends. Coding ends
must necessarily be processed since they exist first as a hairpinned
intermediate. 
There is nothing about the later steps in this pathway that is beyond
the general capabilities of DNA double strand break repair. In fact
several repair mutant cell lines are inhibited in repair of X-ray
induced breaks and the later steps in V(D)J recombination. This argues
that certain components are at least shared by the two pathways. The
full recombination reaction in all its glory (generating P nucleotides
and N regions on the coding ends) prior to ligation may take several
hours in cells, and poses a formidable biochemical challenge. As steps
along the way, we are interested first in individual reactions. Opening
the hairpins. Processing of the coding ends. Ligation of either the
signal ends or coding ends (And what prevents the erroneous ligation of
one signal end to another coding end). Coordination of (probable)
concerted cleavage at one "12-signal" and one "23-signal". Larger open
questions still include the restriction of recombination to specific
loci. That is, usually B-cells and T-cells don't rearrange genes
appropriate to the other lineage (though not 100%) and in B-cells
usually heavy chain preceedes light chain rearrangement. Again not
100%. There are other fine points.
No we have not achieved recombination in vitro. We are pleased to have
the first step under control. Thanks for the interest.
-Moshe Sadofsky, post-doc in Marty Gellert lab.



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