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
• diploidie MeSH
• DNA rostlinná genetika   MeSH
• DNA-(cytosin-5-)methyltransferasa klasifikace   genetika   metabolismus   MeSH
• epigeneze genetická MeSH
• exprese genu MeSH
• fylogeneze MeSH
• genom rostlinný MeSH
• klonování DNA MeSH
• metylace DNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina * MeSH
• polyploidie MeSH
• repetitivní sekvence nukleových kyselin MeSH
• rostlinné geny * MeSH
• sekvence nukleotidů MeSH
• selekce (genetika) MeSH
• tabák enzymologie   genetika   MeSH
• tkáňová distribuce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH

A single-strand-specific chemical probe, potassium permanganate (KMnO4), was used to study the sequence-dependent conformation periodicity of tandem multicopy repetitive DNA sequences HRS60 and GRS (Nicotiana Species) at the level of single base pair and dinucleotide step. Local DNA structures, sensitive to KMnO4, revealed periodicity of 182 +/- 2 bp, equal to the length of repeat units. Permanganate-sensitive local structures were mapped to both DNA strands of genomic HRS60 sequences and were found to be linked to d(A)n tracts. These adenine tracts are located in the proximity of the intrinsically curved domains. Distamycin A increased reactivity of the DNA but decreased the specificity of DNA cleavage. Similar conformation periodicity has been detected also in the 'canrep' family of repeats (Brassica species). All studied repetitive sequences are predominantly located in the constitutive heterochromatin. We discuss the role of conformation periodicities in relation to a structural code for nucleosome phasing at tandem arrays of DNA repeats.

• MeSH
• Brassica genetika   MeSH
• distamyciny MeSH
• DNA primery genetika   MeSH
• DNA rostlinná chemie   genetika   MeSH
• jedovaté rostliny MeSH
• konformace nukleové kyseliny * MeSH
• manganistan draselný MeSH
• mapování chromozomů MeSH
• molekulární sekvence - údaje MeSH
• molekulární sondy MeSH
• polymorfismus genetický MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekvence nukleotidů MeSH
• tabák genetika   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• distamyciny MeSH
• DNA primery MeSH
• DNA rostlinná MeSH
• manganistan draselný MeSH
• molekulární sondy MeSH
• stallimycin MeSH Prohlížeč

Members of a new family of highly repetitive DNA sequences called GRS were isolated from Nicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180-182 bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (10(4) copies per haploid genome) and could be derived from either Nicotiana tomentosiformis or Nicotiana otophora, two possible ancestors of the T genome of the amphidiploid N. tabacum. Sequence homology between the HRS60 (Koukalová et al. 1989) and the GRS family has been estimated to be 57%. In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploid N. tabacum. GRS sequences, like HRS60 (Fajkus et al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conserved Mbol site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.

• MeSH
• chromatin MeSH
• DNA rostlinná analýza   genetika   metabolismus   MeSH
• druhová specificita MeSH
• genom rostlinný MeSH
• jedovaté rostliny * MeSH
• klonování DNA MeSH
• metylace MeSH
• molekulární sekvence - údaje MeSH
• nukleozomy MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• tabák genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• DNA rostlinná MeSH
• nukleozomy MeSH

The methylation status and 5-azacytidine-induced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leaf-derived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine.

• Publikační typ
• časopisecké články MeSH

Two DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.

• Publikační typ
• časopisecké články MeSH