1022-7954/05/4112- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 12, 2005, pp. 1322–1328. From Genetika, Vol. 41, No. 12, 2005, pp. 1601–1607.
Original English Text Copyright © 2005 by Mirza.
In the last two decades, many reports appeared of
the inactivation of foreign DNA sequences introduced
into higher plants [1–3]. The silencing of the transgenes
is often correlated with cytosine methylation of the
gene [4–6]. A variety of experiments suggest a negative
correlation between transgene activity and copy num-
ber of the insert [7, 8]. It has been proposed that
repeated sequences undergo heterochromatinization,
which results in gene silencing [8–10]. However, het-
erochromatinization can also spread from the neighbor-
ing sequences [11, 12]. There are reports suggesting
that insertion of a transgene into hypermethylated or
heterochromatic region can result in inactivation of the
transgene probably by spreading of DNA methylation
into the transgene [12–14]. However, a transgene
inserted into a hypomethylated region can be inacti-
vated and methylated exclusively due to its recognition
as a foreign sequence .
The aim of this work was to study the effect of the
nature of T-DNA integration sequence on the expres-
sion of the transgene present on that T-DNA. For this
purpose, a pale mutant
formed with a construct (pCV002GC) containing the
wild-type copy of the gene
and a selectable drug
). The pale mutant contains a T-DNA tag
locus, 11 bp upstream of the stop codon.
This T-DNA tag contains hygromycin resistance gene,
open reading frame, the pBR322 origin of
replication, and the carbenicillin resistance gene .
The mutant plants are therefore pale, hygromycin-
resistant, and kanamycin-sensitive. Transformation of
plants with pCV002GC usually resulted in
the correction of the phenotype. However, some lines
were identiﬁed which contained an inactive
transgene. Plant DNA ﬂanking the T-DNA containing
transgene was cloned from two inactive and two
active lines as control. This ﬂanking sequence was then
used as probe for Southern blots to study its copy num-
ber and DNA methylation in inactive and active lines.
MATERIALS AND METHODS
Arabidopsis line and plasmid.
allele was initially produced in the Columbia back-
ground . Later, it was backcrossed to Landsberg
and selected for the
in the F
and is referred as
in this study. This
transformed by the plasmid pCV002GC kindly pro-
vided by Koncz. The structure of the T-DNA tag at
locus is shown in Fig. 1a, and the structure of the
pCV002GC T-DNA, in Fig. 1b.
roots was carried out as described by Kilby
gene was used as selectable
maker. The primary transformants were termed the T0
Genomic DNA was extracted from
approximately two-week-old plants employing a proto-
col derived from that of Murray and Thompson .
Southern blots were made using Hybond-N nylon
membrane (Amersham) according to the manufactur-
The plasmid rescue technique was
used to isolate part of the T-DNA and ﬂanking plant
DNA . A sample of 5
g of plant DNA was digested
to completion with
RI. The DNA was ligated using
l (approximately 1000 units) of T4 DNA ligase
Influence of the Nature of the T-DNA Insertion Region
on Transgene Expression in
Department of Biological Sciences, Quaid-I-Azam University, Islamabad, Pakistan; e-mail: firstname.lastname@example.org
Received April 25, 2005
—In the experiment reported here, effect of the nature of T-DNA integration region on the activity of
the transgenes was studied by using a color marker gene in
For this purpose, a pale
mutant was transformed with the wild-type copy of the gene (
) using kanamycin
resistance gene as a selectable marker. Two independent lines were identiﬁed in which
inactive. The T-DNA ﬂanking sequences were recovered from these inactive and two active lines. These ﬂank-
ing sequences were used to examine copy number and DNA methylation of the T-DNA insertion site in active
and inactive lines. Southern blots produced by using
II digested genomic DNA showed signs of meth-
ylation in both inactive lines. Furthermore, in one of the inactive line, the T-DNA ﬂanking sequence probe
hybridized to highly repetitive sequence. The results suggest some correlation between silencing of the trans-
gene and methylation of its insertion region.
* The text was submitted by the author in English.