somatic mutation

Dan Jacobson danj at welchgate.welch.jhu.edu
Sat Feb 20 12:37:23 EST 1993


In article <1993Feb19.185444.10607 at ncsu.edu> kepler at stat.ncsu.edu (Thomas B. Kepler) writes:
>
>I would appreciate any information anyone has on the current state of research
>into the molecular mechanisms of B cell somatic mutation: references to recent articles,
>or simply anecdotal comments.  Thanks.
>
>Tom Kepler
>

You can check out all the labs that are funded by the NIH to work
on somatic mutation of Ig by doing the following:

If you have a gopher client point it at merlot.welch.jhu.edu and select
this directory (if you don't know about gopher see the end of this message):

-->  14. Searching For Biologists/

then chose this search:

    -->  2.  Search for All Researchers funded by NIH <?>

Now search for:

ig and mutation and somatic

And you'll see quite a few labs (38) working on this topic. Read and save
as many as you'd like.

Now to get the entries wich contain a slightly different wording
search for:

immunoglobulin and mutation and somatic not ig

And you'll get the other labs (22) working on this subject.

Design your own searches to narrow or broaden the range of results....


A typical entry is as follows:


-----

GEARHART, PATRICIA J
JOHNS HOPKINS UNIVERSITY
615 NORTH WOLFE STREET
BALTIMORE, MD  21205
PERFORMING ORGANIZATION: JOHNS HOPKINS UNIVERSITY
TITLE   Generation of antibody diversity

ABSTRACT:

 Somatic hypermutation in murine immunoglobulin genes, which occurs at a
 rate of 10-3 per bp per generation, is localized to a 2-kb region of DNA
 surrounding and including rearrange variable (V), diversity (D), and
 joining (J) gene segments encoding heavy and light chain variable regions.
 It is proposed that the unique sequence and structure of the VDJ gene and
 flanking regions encodes the information to cause mutation by error-prone
 repair.  The tow major objectives are to identify cis DNA sequences that
 target mutation to the VDJ gene, and to identify enzymes in B cells that
 are involved in error-prone repair.  Since mutation is activated in nearly
 every B lymphocyte that is stimulated with antigen, it is of paramount
 importance to understand this mechanism.  The first aim is to develop
 assays and systems that generate and detect mutation in plasmid vectors
 containing VDJ genes.  Two assays will be developed to rapidly scan for
 mutation.  The fist is a genetic assay that scores for mutation in a tRNA
 reporter gene placed next to the VDJ gene.  The integrity of the tRNA
 molecule is monitored by color of bacterial colonies.  The second is a
 chemical assay which detects mutation by cleavage of mismatched
 heteroduplexes.  Mutated templates are amplified by PCR, and mismatched
 nucleotides are chemically-modified by osmium tetroxide and hydroxylamine.
 For both assays, mutations will be confirmed by sequencing.  Three systems
 will be tried to generate mutation on the plasmid vectors: (i) SP2/0
 hybridoma cells containing plasmids will be fused to antigen-activated B
 cells, and mutation will occur in vitro; (ii) plasmids will be used to make
 transgenic mice which will be immunized, and (ii) plasmids will be
 recombined into the homologous immunoglobulin site in pluri-potent stem
 cells, which will then be developed into chimeric mice and immunized.  The
 second aim is to identify cis sequences around the VDJ gene that are
 involved in generating mutation.  Using the assay and system which gives
 the highest frequency of mutation, plasmids will be constructed to  contain
 deletions of the 2-kb region surrounding the VDJ gene, and the plasmids
 will be tested for mutation.  When a critical sequence is deleted, mutation
 will be abolished.  The third aim is to examine the mechanism of somatic
 mutation by identifying enzymatic activities in B-cell nuclear extracts.
 Nuclear extracts will be prepared from B cells in various stages of
 differentiation and assayed for endonuclease and exonuclease activities.
 Specificity will be shown at one of two levels: (i) cell specificity, where
 B-cell extracts contain the enzyme, but not other cells, or (ii) substrate
 specificity, where VDJ genes mutate, but not other genes.  Exonuclease
 activity will assayed by primer extension to map the sites where nicks have
 occurred.  Exonuclease activity will be assayed by degradation of
 radiolabeled DNA.  Proteins will be purified and tested in vitro to study
 the mechanism of mutation.



-----


If you don't have a gopher client don't worry - they are free and on the 
net - I've included some information below to help get you started.


Best of luck,

Dan Jacobson

danj at welchgate.welch.jhu.edu


------------------------------------------------------------------------

                          Gopher Info


This is a heavily edited version of the Gopher FAQ intended to
give people just starting with gopher enough information to get a
client and jump into Gopher-space - a complete version can be obtained
as described below.

Dan Jacobson

-----

Common Questions and Answers about the Internet Gopher, a
client/server protocol for making a world wide information service,
with many implementations.  Posted to comp.infosystems.gopher, 
comp.answers, and news.answers every two weeks.

The most recent version of this FAQ can be gotten through gopher, or
via anonymous ftp:

pit-manager.mit.edu:/pub/usenet/news.answers/gopher-faq

Those without FTP access should send e-mail to mail-server at rtfm.mit.edu
with "send usenet/news.answers/finding-sources" in the body to find out
how to do FTP by e-mail.

------------------------------------------------------------------- 
List of questions in the Gopher FAQ:

Q0:  What is Gopher?
Q1:  Where can I get Gopher software?
Q2:  What do I need to access Gopher?
Q3:  Where are there publicly available logins for Gopher?
Q4:  Who Develops Gopher Software?
Q5: What is the relationship between Gopher and (WAIS, WWW, ftp)?
Q6: Are papers or articles describing Gopher available?
Q7: What is veronica?

-------------------------------------------------------------------
Q0:  What is Gopher?

A0:  The Internet Gopher client/server provides a distributed
     information delivery system around which a world/campus-wide
     information system (CWIS) can readily be constructed.   While
     providing a delivery vehicle for local information,  Gopher
     facilitates access to other Gopher and information servers
     throughout the world. 

-------------------------------------------------------------------
Q1:  Where can I get Gopher software?

A1:  via anonymous ftp to boombox.micro.umn.edu.  Look in the directory
     /pub/gopher

--------------------------------------------------------------------
Q2:  What do I need to access Gopher?

A2:  You will need a gopher "client" program that runs on your local PC
     or workstation

     There are clients for the following systems.  The directory
     following the name is the location of the client on the anonymous
     ftp site boombox.micro.umn.edu (134.84.132.2) in the directory
     /pub/gopher.

      Unix Curses & Emacs   :  /pub/gopher/Unix/gopher1.12.tar.Z
      Xwindows (athena)     :  /pub/gopher/Unix/xgopher1.2.tar.Z
      Xwindows (Motif)      :  /pub/gopher/Unix/moog
      Macintosh Hypercard   :  /pub/gopher/Macintosh-TurboGopher/old-versions *
      Macintosh Application :  /pub/gopher/Macintosh-TurboGopher *
      DOS w/Clarkson Driver :  /pub/gopher/PC_client/
      NeXTstep              :  /pub/gopher/NeXT/
      VM/CMS                :  /pub/gopher/Rice_CMS/ or /pub/gopher/VieGOPHER/
      VMS                   :  /pub/gopher/VMS/
      OS/2 2.0	            :  /pub/gopher/os2/
      MVS/XA                :  /pub/gopher/mvs/

     Many other clients and servers have been developed by others, the
     follow


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