Responses to activation tagging

Mauricio bustos at UMBC.EDU
Tue Aug 6 15:12:29 EST 1996


Dear colleagues,

	Some time ago I requested information on activation tagging in
Arabidopsis. Many thanks to Stephen Howell and to Eric Van der Graaff for
their insigths, and for sharing unpublished results. With their
permission, I reproduce their responses below.

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Dr. Mauricio M. Bustos
Department of Biological Sciences, UMBC
1000 Hilltop Circle, Baltimore MD 21228-5398
phone: (410) 455-2769; fax: (410)455-3875
e-mail: bustos at umbc.edu
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Date: Sun, 16 Jun 1996 20:27:28 -0500 From: "Stephen H. Howell"
<shh5 at cornell.edu> To: Mauricio <bustos at umbc.edu> Subject: Re: Enhancer
insertion in Arabidopsis

Rick Walden in Jeff Schell's group has developed an activation-tagging
system in plants and has written several interesting articles.  His Email
address is walden at mpiz-koeln.mpg.de
Steve Howell

Stephen H. Howell
Director of Plant Molecular Biology
Boyce Thompson Institute
Tower Road
Cornell University
Ithaca NY 14853

FAX: 607 254-1242 Email: shh5 at cornell.edu

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Date: Mon, 17 Jun 1996 10:00:58 +0100 (MET)
From: GRAAFF at rulsfb.LeidenUniv.nl

       We have used an activator gene tagging T-DNA construct to
generate a pool of 500 independent transgenic arabidopsis
lines [1]. This constructs harbors the CaMV 35S promoter with
doubled enhancer and AMV leader sequence flanking the right T-
DNA border. In theory such a construct can lead upon integra-
tion to both insertional mutagenesis and sense/antisense ex-
pression of flanking plant DNA.
       This population was screened for altered phenotypes as
individual plants and finally 24 transgenic lines were obtai-
ned exhibiting a consistently altered phenotype. For 15 lines
this phenotype was unlinked. For six of the 9 remaining lines
the altered phenotype was shown to linked with (one of) the T-
DNA insert(s).
       The altered phenotypes were in most lines recessive of
nature (7), but in two lines a dominant altered phenotype was
observed. We are currently performing Northern analysis to
identify the gene which might be upregulated in these lines
due to the T-DNA insert. In one line exhibiting a recessive
altered phenotype a gene is located approximately 1.5kb 3'
from the T-DNA insert. Probably this gene is upregulated by
the T-DNA insert, but the altered phenotype is rather caused
by the lack of normal 'wildtype' expression explaining the
recessive nature of this altered phenotype.
       In five lines analyzed the complete binary transformation
vector was transferred to the plant [2], due to start of T-
strand synthesis on the left T-DNA border and read-through and
the right T-DNA border. In these events the binary vector
(9kb) is situated between the right T-DNA border (35S promo-
ter) and the flanking plant DNA. It is likely that in these
lines only knock-out mutations can be expected, but one of
these five lines however, exhibited a dominant altered pheno-
type.
 
[1]    Van der Graaff E, Hooykaas PJJ: Improvements in the
       transformation of Arabidopsis thaliana C24 leaf discs by
       Agrobacterium tumefaciens. Plant Cell Reports (1996) 15-
       :572-577.
[2]    Van der Graaff E, Den Dulk-Ras A, Hooykaas PJJ: Deviating
       T-DNA transfer from Agrobacterium tumefaciens to plants.
       Plant Molecular Biology (1996) in press.

********************************************************
* email: GRAAFF at rulsfb.univleiden.nl                   *
*                                                      *
* Eric van der Graaff                                  *
* Institute of Molecular Plant sciences                *
* Clusius Laboratory, Wassenaarseweg 64                *
* 2333 AL, Leiden                   FAX 31-71-5274999  *
* The Netherlands                   TEL 31-71-5274835  *
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