Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG-->CmCG-->CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• cytosin metabolismus   MeSH
• DNA rostlinná účinky léků   izolace a purifikace   metabolismus   MeSH
• genetická transkripce MeSH
• genom rostlinný MeSH
• heterochromatin metabolismus   MeSH
• interfáze MeSH
• jedovaté rostliny * MeSH
• kultivované buňky MeSH
• metylace DNA * MeSH
• nukleotidy metabolismus   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• restrikční enzymy MeSH
• satelitní DNA MeSH
• Southernův blotting MeSH
• tabák genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• 9-(2,3-dihydroxypropyl)adenine MeSH Prohlížeč
• adenin MeSH
• cytosin MeSH
• DNA rostlinná MeSH
• heterochromatin MeSH
• nukleotidy MeSH
• restrikční enzymy MeSH
• satelitní DNA 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 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