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Molecular and Cellular Biology, November 1998, p. 6165-6177, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Position-Dependent Methylation and Transcriptional
Silencing of Transgenes in Inverted T-DNA Repeats: Implications for
Posttranscriptional Silencing of Homologous Host Genes in
Plants
Maike
Stam,
Ada
Viterbo,
Joseph N. M.
Mol, and
Jan M.
Kooter*
Department of Molecular Genetics, Institute
for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije
Universiteit, 1081 HV Amsterdam, The Netherlands
Received 13 January 1998/Returned for modification 17 March
1998/Accepted 7 August 1998
Posttranscriptional silencing of chalcone synthase
(Chs) genes in petunia transformants occurs by introducing
T-DNAs that contain a promoter-driven or promoterless Chs
transgene. With the constructs we used, silencing occurs only by T-DNA
loci which are composed of two or more T-DNA copies that are arranged
as inverted repeats (IRs). Since we are interested in the mechanism by
which these IR loci induce silencing, we have analyzed different IR
loci and nonsilencing single-copy (S) T-DNA loci with respect to the
expression and methylation of the transgenes residing in these loci. We
show that in an IR locus, the transgenes located proximal to the IR
center are much more highly methylated than are the distal genes. A
strong silencing locus composed of three inverted T-DNAs bearing
promoterless Chs transgenes was methylated across the
entire locus. The host Chs genes in untransformed plants were moderately methylated, and no change in methylation was detected when the genes were silenced. Run-on transcription assays showed that
promoter-driven transgenes located proximal to the center of a
particular IR are transcriptionally more repressed than are the distal
genes of the same IR locus. Transcription of the promoterless Chs transgenes could not be detected. In the primary
transformant, some of the IR loci were detected together with an
unlinked S locus. We observed that the methylation and expression
characteristics of the transgenes of these S loci were comparable to
those of the partner IR loci, suggesting that there has been cross talk between the two types of loci. Despite the similar features, S loci are
unable to induce silencing, indicating that the palindromic arrangement
of the Chs transgenes in the IR loci is critical for silencing. Since transcriptionally silenced transgenes in IRs can
trigger posttranscriptional silencing of the host genes, our data are
most consistent with a model of silencing in which the transgenes
physically interact with the homologous host gene(s). The interaction
may alter epigenetic features other than methylation, thereby impairing
the regular production of mRNA.
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Institute for Molecular Biological Sciences,
BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV
Amsterdam, The Netherlands. Phone: 31 20 4447197. Fax: 31 20 4447155. E-mail: kooter{at}bio.vu.nl.

Present address: Department of Plant Sciences, University of
Arizona, Tucson, AZ 85721.
Molecular and Cellular Biology, November 1998, p. 6165-6177, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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