Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.
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 methylation 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 expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.
Kunz
Christian
C
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, 5020 Salzburg, Austria.
Narangajavana
Jarunya
J
Jakowitsch
Johannes
J
Park
Young-Doo
YD
Delon
T René
TR
Kovarik
Ales
A
Koukalová
Blazena
B
van der Winden
Johannes
J
Moscone
Eduardo
E
Aufsatz
Werner
W
Mette
M Florian
MF
Matzke
Marjori
M
Matzke
Antonius J M
AJ
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006824:Hybridization, Genetic; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase; D019868:Kanamycin Kinase