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 genetics   MeSH
• Distamycins MeSH
• DNA Primers genetics   MeSH
• DNA, Plant chemistry   genetics   MeSH
• Plants, Toxic MeSH
• Nucleic Acid Conformation * MeSH
• Potassium Permanganate MeSH
• Chromosome Mapping MeSH
• Molecular Sequence Data MeSH
• Molecular Probes MeSH
• Polymorphism, Genetic MeSH
• Repetitive Sequences, Nucleic Acid * MeSH
• Base Sequence MeSH
• Nicotiana genetics   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Distamycins MeSH
• DNA Primers MeSH
• DNA, Plant MeSH
• Potassium Permanganate MeSH
• Molecular Probes MeSH
• stallimycin MeSH Browser

We have recently shown that hypomethylation of cytosine residues in the HRS60 family of repetitive DNA sequences can be induced with 5-azacytidine (5-azaC) in tobacco tissue cultures. We have also proven that such a DNA methylation status is maintained during the recovery of protoplasts, plant regeneration, and vegetative development. In the present paper we follow meiotic transmission of hypomethylated HRS60 DNA. Plants obtained from seeds treated with 5-azaC were either self pollinated or crossed with a non-treated control in a reciprocal way. Analysis of the methylation status of the HRS60 DNA revealed that these sequences were hypomethylated in the progenies up to the extent found in the parental 5-azaC-treated plant. Since no parent-of-origin effect was observed, we presume that both male and female gametes transmit an artificial methylation imprint to a similar extent. This result is supported by methylcytosine evaluation in the total genomic DNA samples. A temporal analysis of 5-azaC effects on germinating seeds and a phenotypic evaluation of 5-azaC-treated tobacco plants are also presented.

• Publication type
• Journal Article MeSH

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TT-TAGGG)n are many times larger than in other plants, e.g., Arabidopsis thaliana or tomato. They are resolved as multiple fragments 60-160 kb in size (in most cases 90-130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 bp motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157 +/- 5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 bp periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.

• MeSH
• Chromatin genetics   MeSH
• DNA, Plant analysis   genetics   MeSH
• Plants, Toxic * MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Nicotiana genetics   MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA, Plant MeSH

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, Plant analysis   genetics   metabolism   MeSH
• Species Specificity MeSH
• Genome, Plant MeSH
• Plants, Toxic * MeSH
• Cloning, Molecular MeSH
• Methylation MeSH
• Molecular Sequence Data MeSH
• Nucleosomes MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Repetitive Sequences, Nucleic Acid genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA, Plant MeSH
• Nucleosomes MeSH