We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.

• MeSH
• alely MeSH
• epigeneze genetická * MeSH
• geneticky modifikované rostliny genetika   metabolismus   MeSH
• kanamycinkinasa genetika   metabolismus   MeSH
• metylace DNA MeSH
• nekódující RNA analýza   MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• reportérové geny MeSH
• tabák genetika   MeSH
• transgeny * MeSH
• umlčování genů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kanamycinkinasa MeSH
• nekódující RNA MeSH

Changes in the distribution of methylcytosine residues along a transgene locus of tobacco (Nicotiana tabacum) in relation to the type of gene silencing were studied in parental plant leaves, calli, and regenerated plants derived thereof. Parental-silenced HeLo1 (hemizygous for locus 1) plants show posttranscriptional silencing of the residing nptII (neomycin phosphotransferase II) transgene and cytosine methylation restricted to the 3' end and center part of the transcribed region. Here, we report that with an increasing number of cell cycles, DNA methylation changes gradually, and methylation is introduced into the promoter during cell culture and more slowly in vegetatively propagated plants. After 24 months of callus in vitro cultivation, an epigenetic variant, designated locus 1E, was obtained in which cytosine methylation of symmetrical (CG and CNG) sites was almost complete within the 5' end of the nptII-transcribed region and the 35S promoter. Further, methylation of nonsymmetrical sites appeared de novo in the promoter, whereas this type of methylation was significantly reduced in the 3' end of the transcribed region when compared with locus 1. The newly established epigenetic patterns were stably transmitted from calli into regenerated plants and their progeny. The protein and steady-state RNA levels remained low in locus 1E, whereas with nuclear run-on assays, no detectable amounts of primary transcripts were found along the nptII gene, indicating that the methylated promoter became inactivated. The results suggest that a switch between posttranscriptional and transcriptional gene silencing could be a mechanism leading to irrevocable shut down of gene expression within a finite number of generations.

• MeSH
• geneticky modifikované rostliny MeSH
• kanamycinkinasa genetika   metabolismus   MeSH
• kultivační techniky MeSH
• metylace DNA * MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u rostlin MeSH
• RNA interference * MeSH
• tabák genetika   metabolismus   MeSH
• transgeny genetika   MeSH
• umlčovací elementy transkripční genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kanamycinkinasa MeSH