BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.

• Keywords
• GFP, Inverted repeat, Promoterless, RNAi, Read-through transcription, T-DNA, Tobacco BY-2 cell line,
• MeSH
• Agrobacterium tumefaciens genetics   MeSH
• Arabidopsis genetics   MeSH
• Cell Line MeSH
• DNA, Bacterial genetics   MeSH
• Transcription, Genetic * MeSH
• Plants, Genetically Modified MeSH
• RNA, Messenger genetics   MeSH
• Inverted Repeat Sequences genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Genes, Reporter MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Bacterial MeSH
• RNA, Messenger MeSH
• T-DNA MeSH Browser

In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.

• Keywords
• DNA methylation, callus, dedifferentiation, histone modification, tobacco, transgene silencing,
• MeSH
• Chromatin genetics   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Histones genetics   metabolism   MeSH
• Bony Callus metabolism   MeSH
• DNA Methylation MeSH
• Gene Expression Regulation, Plant MeSH
• Nicotiana genetics   metabolism   MeSH
• Transgenes MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.

• MeSH
• Alleles MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic MeSH
• Plants, Genetically Modified genetics   MeSH
• Genomic Imprinting MeSH
• RNA, Small Interfering genetics   MeSH
• DNA Methylation * MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Plant * MeSH
• RNA Interference MeSH
• Nicotiana genetics   MeSH
• Transgenes genetics   MeSH
• Silencer Elements, Transcriptional genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Small Interfering MeSH

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome.

• MeSH
• Diploidy MeSH
• DNA, Plant genetics   MeSH
• DNA (Cytosine-5-)-Methyltransferases classification   genetics   metabolism   MeSH
• Epigenesis, Genetic MeSH
• Gene Expression MeSH
• Phylogeny MeSH
• Genome, Plant MeSH
• Cloning, Molecular MeSH
• DNA Methylation genetics   MeSH
• Molecular Sequence Data MeSH
• Multigene Family * MeSH
• Polyploidy MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Genes, Plant * MeSH
• Base Sequence MeSH
• Selection, Genetic MeSH
• Nicotiana enzymology   genetics   MeSH
• Tissue Distribution MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA (Cytosine-5-)-Methyltransferases MeSH

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.

• MeSH
• Alleles * MeSH
• Cell Culture Techniques MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified MeSH
• DNA Methylation MeSH
• Inverted Repeat Sequences genetics   MeSH
• Open Reading Frames genetics   MeSH
• Cellular Reprogramming genetics   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Regeneration genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• RNA, Plant metabolism   MeSH
• Sequence Analysis, DNA MeSH
• Sulfites MeSH
• Blotting, Southern MeSH
• Nicotiana genetics   physiology   MeSH
• Transgenes * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• hydrogen sulfite MeSH Browser
• RNA, Plant MeSH
• Sulfites MeSH

Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE), followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

• Keywords
• DNA hybridization, PCR, electrochemical detection, enzyme-linked assay, gene expression, primer extension,
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In plants, 5S rRNA genes (5S rDNA) encoding 120-nt structural RNA molecules of ribosomes are organized in tandem arrays comprising thousands of units. Failure to correctly terminate transcription would generate longer inaccurately processed transcripts interfering with ribosome biogenesis. Hence multiple termination signals occur immediately after the 5S rRNA coding sequence. To obtain information about the efficiency of termination of 5S rDNA transcription in plants we analyzed 5S rRNA pools in three Nicotiana species, N. sylvestris, N. tomentosiformis and N. tabacum. In addition to highly abundant 120-nt 5S rRNA transcripts, we also detected RNA species composed of a genic region and variable lengths of intergenic sequences. These genic-intergenic RNA molecules occur at a frequency severalfold lower than the mature 120-nt transcripts, and are posttranscriptionally modified by polyadenylation at their 3' end in contrast to 120-nt transcripts. An absence of 5S small RNAs (smRNA) argue against a dominant role for the smRNA biosynthesis pathway in the degradation of aberrant 5S rRNA in Nicotiana. This work is the first description of polyadenylated 5S rRNA species in higher eukaryotes originating from a read-through transcription into the intergenic spacer. We propose that polyadenylation may function in a "quality control" pathway ensuring that only correctly processed molecules enter the ribosome biogenesis.

• MeSH
• Arabidopsis genetics   MeSH
• Transcription, Genetic * MeSH
• DNA, Intergenic * MeSH
• RNA, Small Interfering metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Models, Genetic MeSH
• Molecular Sequence Data MeSH
• Polyadenylation * MeSH
• Gene Expression Regulation, Plant * MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• Base Sequence MeSH
• Sequence Homology, Nucleic Acid MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Intergenic * MeSH
• RNA, Small Interfering MeSH
• RNA, Messenger MeSH
• RNA, Ribosomal, 5S MeSH