Plant Molecular Biology 52: 203–215, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Studies on the effects of a ﬂanking repetitive sequence on the expression of
single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N.
, Jarunya Narangajavana
, Johannes Jakowitsch
, Young-Doo Park
, Ales Kovarik
, Blazena Koukalov
, Johannes van der Winden
, Werner Aufsatz
, M. Florian Mette
, Marjori Matzke
and Antonius J.M.
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, 5020 Salzburg, Austria (
for correspondence; e-mail email@example.com);
Seita, Institut du Tabac de Bergerac, 24100 Bergerac,
Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, Czech Republic);
Kleinhuningerstrasse 61, 4057 Basel, Switzerland;
Department of Biotechnology, Mahidol
University, Bangkok 10400, Thailand;
Department of Internal Medicine II, Division of Cardiology, University
of Vienna, 1090 Vienna, Austria;
School of Biotechnology, KyungHee University, Yongin, 449-701, South Korea;
Instituto Multidisciplinario de Biologia Vegetal, Casilla de Correo 495, 5000 Cordoba, Argentina
Received 12 July 2002; accepted in revised form 20 November 2002
Key words: DNA methylation, gene silencing, Nicotiana sylvestris, polyploid, repetitive sequence, retroelement
To test the inﬂuence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in
N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline
synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris
lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became
silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methy-
lation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of
the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had
sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active,
unmethylated NOSpro- NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced,
methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded
to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not
become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not
spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expres-
sion was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in
the context of other possible triggers of silencing.
Abbreviations: dsRNA, double stranded RNA; GUS, β-glucuronidase; HDGS, homology-dependent gene silenc-
ing; IR, inverted DNA repeat; Kan
, kanamycin-resistant; MARs, matrix attachment regions; NOS, nopaline
synthase; NOSpro, nopaline synthase promoter; NPTII, neomycin phosphotransferase II; siRNAs, short interfering
RNAs; ST hybrids, hybrids between Nicotiana sylvestris and N. tomentosiformis; T subgenome, subgenome of
tetraploid tobacco contributed by N. tomentosiformis.