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Arabidopsis transformation-competent cosmid library

LAZO,GERARD glazo at aardvark.ucs.uoknor.edu
Mon Nov 9 12:36:00 EST 1992


An Arabidopsis cosmid library has been constructed in Agrobacterium
as referenced in Bio/Technology 1991 9:963-967.  The library is of
A. thaliana GH50 (ALS mutant, ecotype Columbia) and consists of
about 20 kb fragments contained between T-DNA borders and is
harbored within an attenuated recA strain of Agrobacterium. 
Bacterial selectable markers are for tetracycline and gentamicin. 
Plant selectable markers are for kanamycin and streptomycin. 
21,600 clones were plated into 225 microtiter dishes and arrayed in
a 15 by 15 matrix.  Each row and column were pooled into 30
microtiter dishes to assist clone identification by PCR and filter
hybridization.  Ideally, if a clone can be identified using probes,
the corresponding clone can be used directly for plant
complementation.  If plant transformation methodologies were truly
straightforward it may be possible to attempt the screening of all
21,600+ clones.

I believe some efforts are currently in progress by the R.A. Ludwig
laboratory [UC Santa Cruz; rludwig at cats.ucsc.edu] to order the
clones contained within the 225 microtiter dish library.  A pooled
collection of 30 microtiter dishes have been forwarded to the Ohio
State University Arabidopsis Stock Center and may become available
shortly depending on replication efforts, etc... I recommend
contacting K.R. Davis [Ohio State U.; krdavis at magnus.acs.ohio-
state.edu] on its availability.  It may also be possible to get
microtiter dish copies from other labs using the library [e.g.
chory at salk-sc2.sdsc.edu, coupland at jii.afrc.ac.uk, there may be others]. 
If not available, arrangements may be made through R.A. Ludwig 
or myself [S.R. Noble Foundation, Ardmore; glazo at aardvark.ucs.uoknor.edu].
There is also a T-DNA based cosmid library of Arabidopsis which was
maintained in E. coli; this library was constructed by N.E. Olszewski 
[neil at molbio.cbs.umn.edu] et al (Nucl. Acids Res. 1988 16:10765-10782) 
and maintained in E. coli, but I don't know the availability of this 
library.

In general, the Arabidopsis library in Agrobacterium can be useful
ultimately for assisting mapping efforts by providing a
transformation competent clone.  Once mapping efforts with YAC, P1,
cosmid, or phage clones have led you to a locus these clones may be
used as probes to pull out the corresponding clones for
complementation and verification of the phenotype by transformation
in the appropriate plant host.  The Agrobacterium-based library
would probably be most useful in the latter stages of the general
mapping strategies with E. coli- and yeast-based libraries.

I hope this is useful.

Gerard R. Lazo               /\     //====== glazo at aardvark.ucs.uoknor.edu
Plant Biology Division      // \   //            LAB: 405-221-7305
The S.R. Noble Foundation, Inc. \ //======           FAX: 405-221-7380
Ardmore, Oklahoma 73402   //     \/                      MSG: 405-223-5810



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