In-Vitro Recombination

neale at mbcf.stjude.org neale at mbcf.stjude.org
Mon Nov 13 12:13:34 EST 1995


James,

I tried to mail this to you directly, but I got a message saying your host name
was not valid.

As far as I am aware nobody has yet achieved VDJ recombination in vitro.
Several groups are trying. There is a report from Martin Gellert's group (see
below) where they obtained double strand breaks at the recognition signal
sequences using fractionated cell extracts.

If you hear that someone has been successful in vitro, please let me know.

Regards,

Geoff Neale

================================================================================
Dept. of Virology and Molecular Biology
St. Jude Children's Research Hospital
Memphis, TN
Phone: (901) 495-3400
Fax:   (901) 523-2622                        e-mail:geoffrey.neale at stjude.org
================================================================================
                      
van Gent DC  McBlane JF  Ramsden DA  Sadofsky MJ  Hesse JE  Gellert M
Initiation of V(D)J recombination in a cell-free system.

In: Cell (1995 Jun 16) 81(6):925-34

Cells performing V(D)J recombination make specific cuts in DNA at
  recombination signal sequences. Here, we show that nuclear extracts
  of pre-B cell lines carry out this specific cleavage. The products of
  cleavage are the same as found previously in thymocytes: full-length,
  blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin)
  coding ends. A complete signal sequence is required. Recombinant RAG1
  protein greatly increases activity and complements an inactive
  extract from a RAG1 (-/-) pre-B cell line. When the extracts are
  fractionated, cleavage activity correlates with the presence of RAG2
  protein. These results suggest that RAG1 and RAG2 are components of
  the V(D)J recombinase.
                      
Institutional address:
     Laboratory of Molecular Biology
     National Institute of Diabetes and Digestive and Kidney Diseases
     National Institutes of Health
     Bethesda
     Maryland 20892   

In article <48385d$75m at news.umbc.edu>, James Mellott <jmello1 at umbc7.edu> writes:
> 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.  
> 
> Thanks,
> 
> James Mellott 
> jmello1 at umbc.edu 
> 
> 



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