Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.

• MeSH
• Alternative Splicing MeSH
• Exons MeSH
• Transcription, Genetic MeSH
• Genetic Loci MeSH
• Genome, Plant MeSH
• Gene Rearrangement MeSH
• Introns MeSH
• RNA Isoforms MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Gene Order MeSH
• Pseudogenes MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Nicotiana genetics   MeSH
• Telomerase genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Isoforms MeSH
• Telomerase MeSH

Satellite sequences of the VicTR-B family are specific for the genus Vicia (Leguminosae), but their abundance varies among the species, being the highest in Vicia sativa and Vicia grandiflora. In this study, we have sequenced multiple randomly cloned VicTR-B fragments from these two species and analyzed their sequence variability, periodicity, and chromosomal localization. We have found that V. sativa VicTR-B sequences are homogeneous with respect to their nucleotide sequences and periodicity (monomers of 38 bp), whereas V. grandiflora repeats are considerably more variable, occurring in at least four distinct sequence subfamilies. Although the periodicity of 38 bp was conserved in most of the V. grandiflora sequences, one of the subfamilies was composed of higher-order repeats of 186 bp, which originated from a pentamer of the basic repeated unit. Individual VicTR-B subfamilies were preferentially located in either intercalary or subtelomeric regions of chromosomes. Interestingly, two V. grandiflora subfamilies with the highest similarity to V. sativa VicTR-B sequences were located in intercalary heterochromatic bands, showing similar chromosomal distribution as the majority of VicTR-B repeats in V. sativa. The other two V. grandiflora subfamilies showing a considerable divergence from V. sativa sequences were found to be accumulated at subtelomeric regions of V. grandiflora chromosomes.

• MeSH
• Chromosomes, Plant chemistry   MeSH
• DNA, Plant analysis   MeSH
• Genetic Variation MeSH
• In Situ Hybridization, Fluorescence MeSH
• Conserved Sequence * MeSH
• Chromosome Mapping * MeSH
• Molecular Sequence Data MeSH
• DNA, Satellite analysis   MeSH
• Base Sequence MeSH
• Vicia genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Satellite MeSH

This paper examines telomeres from an evolutionary perspective. In the monocot plant order Asparagales two evolutionary switch-points in telomere sequence are known. The first occurred when the Arabidopsis-type telomere was replaced by a telomere based on a repeat motif more typical of vertebrates. The replacement is associated with telomerase activity, but the telomerase has low fidelity and this may have implications for the binding of telomeric proteins. At the second evolutionary switch-point, the telomere and its mode of synthesis are replaced by an unknown mechanism. Elsewhere in plants (Sessia, Vestia, Cestrum) and in arthropods, the telomere "typical" of the group is lost. Probably many other groups with "unusual" telomeres will be found. We question whether telomerase is indeed the original end-maintenance system and point to other candidate processes involving t-loops, t-circles, rolling circle replication and recombination. Possible evolutionary outcomes arising from the loss of telomerase activity in alternative lengthening of telomere (ALT) systems are discussed. We propose that elongation of minisatellite repeats using recombination/replication processes initially substitutes for the loss of telomerase function. Then in more established ALT groups, subtelomeric satellite repeats may replace the telomeric minisatellite repeat whilst maintaining the recombination/replication mechanisms for telomere elongation. Thereafter a retrotransposition-based end-maintenance system may become established. The influence of changing sequence motifs on the properties of the telomere cap is discussed. The DNA and protein components of telomeres should be regarded--as with any other chromosome elements--as evolving and co-evolving over time and responding to changes in the genome and to environmental stresses. We describe how telomere dysfunction, resulting in end-to-end chromosome fusions, can have a profound effect on chromosome evolution and perhaps even speciation.

• MeSH
• Chromosomes, Plant genetics   metabolism   MeSH
• Phylogeny MeSH
• Genome, Plant * MeSH
• Minisatellite Repeats MeSH
• Evolution, Molecular * MeSH
• Telomere-Binding Proteins physiology   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Retroelements MeSH
• Telomere genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Comparative Study MeSH
• Names of Substances
• Telomere-Binding Proteins MeSH
• Retroelements MeSH

An approximately 135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S(3) generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

• MeSH
• DNA Primers MeSH
• Species Specificity MeSH
• Phylogeny MeSH
• In Situ Hybridization, Fluorescence MeSH
• DNA, Intergenic genetics   MeSH
• DNA, Complementary genetics   MeSH
• Evolution, Molecular * MeSH
• Molecular Sequence Data MeSH
• Ploidies MeSH
• Restriction Mapping MeSH
• DNA, Ribosomal genetics   MeSH
• DNA, Satellite genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Cluster Analysis MeSH
• Blotting, Southern MeSH
• Nicotiana genetics   MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• DNA Primers MeSH
• DNA, Intergenic MeSH
• DNA, Complementary MeSH
• DNA, Ribosomal MeSH
• DNA, Satellite MeSH

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
• Adenine analogs & derivatives   pharmacology   MeSH
• Cytosine metabolism   MeSH
• DNA, Plant drug effects   isolation & purification   metabolism   MeSH
• Transcription, Genetic MeSH
• Genome, Plant MeSH
• Heterochromatin metabolism   MeSH
• Interphase MeSH
• Plants, Toxic * MeSH
• Cells, Cultured MeSH
• DNA Methylation * MeSH
• Nucleotides metabolism   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• DNA Restriction Enzymes MeSH
• DNA, Satellite MeSH
• Blotting, Southern MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• 9-(2,3-dihydroxypropyl)adenine MeSH Browser
• Adenine MeSH
• Cytosine MeSH
• DNA, Plant MeSH
• Heterochromatin MeSH
• Nucleotides MeSH
• DNA Restriction Enzymes MeSH
• DNA, Satellite 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 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

In order to investigate possible interactions between parental genomes in the composite genome of Nicotiana tabacum we have analyzed the organization of telomeric (TTTAGGG)n and ribosomal gene (rDNA) repeats in the progenitor genomes Nicotiana sylvestris and Nicotiana tomentosiformis or Nicotiana otophora. Telomeric arrays in the Nicotiana species tested are heterogeneous in length ranging from 20 to 200 kb in N. sylvestris, from 20 to 50 kb in N. tomentosiformis, from 15 to 100kb in N. otophora, and from 40 to 160kb in N. tabacum. The patterns of rDNA repeats (18S, 5.8S, 25S RNA) appeared to be highly homogeneous and speciesspecific; no parental rDNA units corresponding to N. sylvestris, N. tomentosiformis or N. otophora were found in the genome of N. tabacum by Southern hybridization. The results provide evidence for a species-specific evolution of telomeric and ribosomal repeats in the tobacco composite genome.

• Publication type
• Journal Article MeSH