Ales Kovarik

Abstract: Tragopogon mirus Ownbey and Tragopogon miscellus Ownbey are recent allotetraploids that have formed recurrently within the last 80 years, following the introduction of the diploids T dubius Scop., T pratensis L. and T porrifolius L. from Europe to North America. In some areas, the progenitor diploids still occur along with expanding populations of the allotetraploids, and the polyploids at those locations likely represent descendents of the nearby diploids. In most natural populations of T mirus and T miscellus, there are far fewer rDNA units of the common parent (T dubius) than there are of the other diploid parent and in some rarely occurring individuals, one parental locus was >90% deleted. Nevertheless, in contrast to several ancient Old World allotetraploids, gene copies from both parents are readily detected by molecular methods in both T mirus and T miscellus. In one population of T minis and herbarium specimens collected at the time of species origin the gene ratios were balanced. Several lineages of T mints and T miscellus were recently successfully resynthesized from diploid species. Among 181 synthetic individuals analyzed, we observed frequent deviations from copy-number additivity; that is in most cases, the T dubius homeologs were reduced in copy number. At the epigenetic level, the genes of T. dubius origin dominate expression in most natural and synthetic allopolyploids. The fact that some rDNA genotypes seen in 80-year-old allopolyploids are already evident in the first generation of synthetic lines supports the hypothesis that the extent and tempo of rDNA homogenization in older allopolyploids is largely influenced by genetic and epigenetic changes in the early generations. Thus, T mints and T miscellus that formed repeatedly in the wild within the last century represent a unique system to study the early stages of genome evolution following interspecific hybridization and genome duplication. This review summarizes recent works on the rDNA chromosomal organization, repeat and loci inheritance and gene expression.

Abstract: � Allopolyploidy, a driving force in plant evolution, can induce rapid structural changes in parental subgenomes. Here, we examined the fate of homologous subtelomeric satellites in intrasection allotetraploid Nicotiana arentsii formed from N. undulata and N. wigandioides progenitors <�200�000�yr ago. � We cloned and sequenced a number of monomers from progenitors and the allotetraploid. Structural features of both cloned and genomic monomers were studied using double-strand conformation polymorphism analysis. � Two homologous satellites were isolated from N. undulata (called NUNSSP) and N. wigandioides (NWISSP). While the NUNSSP monomers were highly homogeneous in nucleotide sequences, the NWISSP monomers formed two separate clades. Likewise, the genomic NUNSSP monomers showed less DNA conformation heterogeneity than NWISSP monomers, with distinct conformations. While both satellites predominantly occupy subtelomeric positions, a fraction of the NWISSP repeats was found in an intercalary location, supporting the hypothesis that dispersion prevents the repeats becoming homogeneous. Sequence, structural and chromosomal features of the parental satellites were faithfully inherited by N. arentsii. � Our study revealed that intergenomic homogenization of subtelomeric satellite repeats does not occur in N. arentsii allotetraploid. We propose that the sequence and structural divergence of subtelomeric satellites may render allopolyploid chromosomes less vulnerable to intergenomic exchanges.

Abstract: 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.

Abstract: The dynamics of genome modification that occurred from the initial hybridization event to the stabilization of allopolyploid species remains largely unexplored. Here, we studied inheritance and expression of rDNA loci in the initial generations of Brassica napus allotetraploids (2n�=�38, AACC) resynthesized from Brassica oleracea (2n�=�18, CC) and B. rapa (2n�=�20, AA) and compared the patterns to natural forms. Starting already from F1 generation, there was a strong uniparental silencing of B. oleracea genes. The epigenetic reprogramming was accompanied with immediate condensation of C-genome nucleolar organizer region (NOR) and progressive transgeneration hypermethylation of polymerase I promoters, mainly at CG sites. No such changes were observed in the A-genome NORs. Locus loss and gains affecting mainly non-NOR loci after the first allotetraploid meiosis did not influence established functional status of NORs. Collectively, epigenetic and genetic modifications in synthetic lines resemble events that accompanied formation of natural allopolyploid species.

Abstract: We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (<200,000 years old), with its diploid progenitors, N. sylvestris (maternal, S-genome donor) and N. tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenisation, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus the three genomes of N. sylvestris, N. tomentosiformis and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable and downsized respectively.

Abstract: Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.

Abstract: The polyploid species in Rosa section Caninae (2n=21, 28 or 35) are characterized by an unusual reproductive system known as odd (or asymmetric) meiosis. Only two chromosome sets form bivalents in meiosis, whereas the remaining chromosomes are transmitted as univalents through the female germline. Evolution of ribosomal rRNA genes (rDNA) does not seem to be significantly affected by interlocus homogenization in dogroses. As a consequence, most species contain several rDNA families falling into two main clades (beta and gamma) thought to be differentially distributed between bivalent and univalent chromosomes, respectively. Here, we have investigated expression of rRNA gene families in five pentaploid species (R. canina, R. rubiginosa, R. dumalis, R. sherardii and R. caesia, 2n=35) and in one tetraploid (R. mollis, 2n=28). Using extensive sequencing of ITS clones and cleaved amplified polymorphism sequence (CAPS) analysis, we found that the beta-family was constitutively expressed in all species. However, there was large variation in the expression patterns of families constituting the gamma-clade. In addition, a single family can be active in one species, whereas silenced in another. The data show that the families on bivalent-forming chromosomes dominate rDNA expression in all dogrose species. We hypothesize that genes on bivalent genomes are stably expressed, whereas those on univalent genomes undergo variable levels of epigenetic silencing. Nonetheless, mosaic expression of univalent genomes could contribute to phenotypic variation between the species. Heredity (2010) 104, 113-120; doi:10.1038/hdy.2009.94; published online 29 July 2009

Abstract: P>Allopolyploids represent natural experiments in which DNA sequences from different species are combined into a single nucleus and then coevolve, enabling us to follow the parental genomes, their interactions and evolution over time. Here, we examine the fate of satellite DNA over 5 million yr of divergence in plant genus Nicotiana (family Solanaceae). We isolated subtelomeric, tandemly repeated satellite DNA from Nicotiana diploid and allopolyploid species and analysed patterns of inheritance and divergence by sequence analysis, Southern blot hybridization and fluorescent in situ hybridization (FISH). We observed that parental satellite sequences redistribute around the genome in allopolyploids of Nicotiana section Polydicliae, formed c. 1 million yr ago (Mya), and that new satellite repeats evolved and amplified in section Repandae, which was formed c. 5 Mya. In some cases that process involved the complete replacement of parental satellite sequences. The rate of satellite repeat replacement is faster than theoretical predictions assuming the mechanism involved is unequal recombination and crossing-over. Instead we propose that this mechanism occurs with the deletion of large chromatin blocks and reamplification, perhaps via rolling circle replication.

Abstract: Background: Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. Results: Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in F-1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. Conclusions: Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.

Abstract: Background: In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in similar to 200 species representing the family diversity and other closely related groups. Results: Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Conclusions: Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units, their copy number and chromosomal organisation may occur within relatively short evolutionary time. We hypothesize that the 5S gene integration within the 35S unit might have repeatedly occurred during plant evolution, and probably once in Asteraceae.

Abstract: P>One little understood feature of polyploid speciation is the transition from polysomic to disomic inheritance, and much recent attention has focused on the role of pairing genes in this process. Using computer simulations we studied the effects of mutations, chromosomal inversions, chiasma, neofunctionalization, subfunctionalization and selection on the evolution of disomic inheritance in a polyploid over 10 000 generations. We show that: the evolution of pairing genes is not essential for the establishment of disomic inheritance, as genetic drift, coupled with a threshold for homologue pairing fidelity, is sufficient to explain the transition from polysomic to disomic inheritance; high rates of recombination increase the number of generations required for disomic inheritance to become established; both neofunctionalization and subfunctionalization speed up the transition to disomic inheritance. The data suggest that during polyploid species establishment, selection will favour reduced chiasma number and/or more focused distribution. The data also suggest a new role for subfunctionalization in that it can drive disomic inheritance. The evolution of subfunctionalization in genes across the genome will then act to maintain genes in syntenic blocks and may explain why such regions are so highly conserved.

Abstract: P>Allopolyploidy is a major driving force in plant evolution and can induce rapid structural changes in the hybrid genome. As major components of plant genomes, transposable elements are involved in these changes. In a previous work, we observed turnover of retrotransposon insertions in natural allotretraploid tobacco (Nicotiana tabacum). Here, we studied the early stages of allopolyploid formation by monitoring changes at retrotransposon insertion sites in the Th37 synthetic tobacco. We used sequence-specific amplification polymorphism (SSAP) to study insertion patterns of two populations of the Tnt1 retrotransposon in Th37 S4 generation plants, and characterized the nature of polymorphic insertion sites. We observed significant amplification of young Tnt1 populations. Newly transposed copies were amplified from maternal elements and were highly similar to Tnt1A tobacco copies amplified in response to microbial factors. A high proportion of paternal SSAP bands were not transmitted to the hybrid, corresponding to various rearrangements at paternal insertion sites, including indels or the complete loss of the Tnt1/flanking junction. These data indicate that major changes, such as retrotransposon amplification and molecular restructuring in or around insertion sites, occur rapidly in response to allopolyploidy.

Abstract: Typically in plants, the 5S and 35S ribosomal DNA (rDNA) encoding two major ribosomal RNA species occur at separate loci. However, in some algae, bryophytes and ferns, they are at the same locus (linked arranged). Southern blot hybridisation, polymerase chain reactions (PCR), fluorescent in situ hybridisation, cloning and sequencing were used to reveal 5S and 35S rDNA genomic organisation in Artemisia. We observed thousands of rDNA units at two-three loci containing 5S rDNA in an inverted orientation within the inter-genic spacer (IGS) of 35S rDNA. The sequenced clones of 26-18S IGS from Artemisia absinthium appeared to contain a conserved 5S gene insertion proximal to the 26S gene terminus (5S rDNA-1) and a second less conserved 5S insertion (5S rDNA-2) further downstream. Whilst the 5S rDNA-1 showed all the structural features of a functional gene, the 5S-rDNA-2 had a deletion in the internal promoter and probably represents a pseudogene. The linked arrangement probably evolved before the divergence of Artemisia from the rest of Asteraceae (> 10 Myrs). This arrangement may have involved retrotransposons and once formed spread via mechanisms of concerted evolution. Heterogeneity in unit structure may reflect ongoing homogenisation of variant unit types without fixation for any particular variant.

Abstract: Interferon-alpha (IFN-alpha) is an important drug used in anti-melanoma therapy. However, metastases eventually reappear in almost 60% of melanoma patients, who have received adjuvant cytokine therapy suggesting that IFN-alpha can paradoxically promote disease progression in some cases, at least. In this study, we have investigated the possibility that a growth-promoting STAT3 protein might be activated by interferon-alpha in melanoma cells. We examined 24 primary cultures established from node metastases of melanoma patients who were monitored in a 5-year clinical follow-up. The patients differed in the course of disease and survival end-points. Using Western blot analyses, we show that interferon-alpha stimulated STAT3 phosphorylation at tyrosine (Y705) residue in 17% of cases. These over-reactive cell populations originated from patients who had the shortest disease-free intervals. A significant correlation was obtained between the length of survival end-points and a lack of STAT3 activation by IFN-alpha. No STAT3 induction was observed in normal melanocytes. The STAT1 activation at tyrosine (Y701) occurred at a similar frequency as that of STAT3 (17%) albeit in different patients, no clear correlation with the clinical status could be made. The interferon-alpha/beta receptors (IRFARs) were expressed irrespective to the signal transducers and activators of transcription (STATs) inducibility suggesting that signalling defects occur downstream from IRFAR. We propose that in some cases the application of IFN-alpha could increase the probability of disease progression via overactive STAT3. The tests for STAT3 inducibility prior to cytokine immnunotherapy in the clinic are therefore warranted. (c) 2009 Elsevier Ltd. All rights reserved.

Abstract: The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation (P < 0.001) between SOCS3 inducibility and growth inhibition by IFN-gamma. The correlations between sensitivity and STAT1 phosphorylation levels (pY-701) were less pronounced since there were cases of STAT1-pY induction (2/12) without apparent growth inhibitory effect. The data indicate defective STAT1 phosphorylation and SOCS3 induction in breast cancer cells. These defects likely contribute to reduced sensitivity of malignant cells to antiproliferative effect of IFN-gamma.

Abstract: 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.

Abstract: 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.

Abstract: Background: Polyploidy, frequently termed "whole genome duplication'', is a major force in the evolution of many eukaryotes. Indeed, most angiosperm species have undergone at least one round of polyploidy in their evolutionary history. Despite enormous progress in our understanding of many aspects of polyploidy, we essentially have no information about the role of chromosome divergence in the establishment of young polyploid populations. Here we investigate synthetic lines and natural populations of two recently and recurrently formed allotetraploids Tragopogon mirus and T. miscellus (formed within the past 80 years) to assess the role of aberrant meiosis in generating chromosomal/genomic diversity. That diversity is likely important in the formation, establishment and survival of polyploid populations and species. Methodology/Principal Findings: Applications of fluorescence in situ hybridisation (FISH) to natural populations of T. mirus and T. miscellus suggest that chromosomal rearrangements and other chromosomal changes are common in both allotetraploids. We detected extensive chromosomal polymorphism between individuals and populations, including (i) plants monosomic and trisomic for particular chromosomes (perhaps indicating compensatory trisomy), (ii) intergenomic translocations and (iii) variable sizes and expression patterns of individual ribosomal DNA (rDNA) loci. We even observed karyotypic variation among sibling plants. Significantly, translocations, chromosome loss, and meiotic irregularities, including quadrivalent formation, were observed in synthetic (S-0 and S-1 generations) polyploid lines. Our results not only provide a mechanism for chromosomal variation in natural populations, but also indicate that chromosomal changes occur rapidly following polyploidisation. Conclusions/Significance: These data shed new light on previous analyses of genome and transcriptome structures in de novo and establishing polyploid species. Crucially our results highlight the necessity of studying karyotypes in young (< 150 years old) polyploid species and synthetic polyploids that resemble natural species. The data also provide insight into the mechanisms that perturb inheritance patterns of genetic markers in synthetic polyploids and populations of young natural polyploid species.

Abstract: In pentaploid dogroses, Rosa section Caninae (2n = 5x = 35), the pollen transmits one basic genome (x = 7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x = 28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the b rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

Abstract: We have studied the inheritance of the epigenetic state of tobacco transgenes whose expression was post-transcriptionally silenced by an invertedly repeated silencer locus. We show that, in hybrids, the coding region of the target neomycin phosphotransferase (nptII) gene was almost exclusively methylated at CG configurations, and dense non-CG methylation occurred in the 3' untranslated region. Homologous sequences in the silencer locus were heavily methylated at both CG and non-CG motifs. After segregation of the silencer locus, the CG methylation but not the non-CG methylation of the target genes was transmitted to the progeny. In the segregants, we observed an overall increase of CG methylation in the target genes, associated with a re-distribution from the 3' end of the coding region towards the middle. This pattern was inherited with some fluctuation for at least two additional generations in the absence of a detectable T-DNA-derived small RNA fraction. Thus CG methylation is not cleared during meiosis and may be inherited over generations without RNA signals being present. These epi-allelic variants re-expressed the reporter gene immediately after segregation of the trigger, showing that relatively dense CG methylation (approximately 60-80%) imprinted on most of the coding region (> 500 bp) did not reduce expression compared with the parental non-methylated locus. We propose that the genic CG methylation seen in euchromatic regions of the genome may originate from ancient post-transcriptional gene silencing events as a result of adventitiously produced methylation-directing RNA molecules.

Abstract: LTR-retrotransposons contribute substantially to the structural diversity of plant genomes. Recent models of genome evolution suggest that retrotransposon amplification is offset by removal of retrotransposon sequences, leading to a turnover of retrotransposon populations. While bursts of amplification have been documented, it is not known whether removal of retrotransposon sequences occurs continuously, or is triggered by specific stimuli over short evolutionary periods. In this work, we have characterized the evolutionary dynamics of four populations of copia-type retrotransposons in allotetraploid tobacco (Nicotiana tabacum) and its two diploid progenitors Nicotiana sylvestris and Nicotiana tomentosiformis. We have used SSAP (Sequence-Specific Amplification Polymorphism) to evaluate the contribution retrotransposons have made to the diversity of tobacco and its diploid progenitor species, to quantify the contribution each diploid progenitor has made to tobacco's retrotransposon populations, and to estimate losses or amplifications of retrotransposon sequences subsequent to tobacco's formation. Our results show that the tobacco genome derives from a turnover of retrotransposon sequences with removals concomitant with new insertions. We have detected unique behaviour specific to each retrotransposon population, with differences likely reflecting distinct evolutionary histories and activities of particular elements. Our results indicate that the retrotransposon content of a given plant species is strongly influenced by the host evolutionary history, with periods of rapid turnover of retrotransposon sequences stimulated by allopolyploidy.

Abstract: In this paper, we have compared hypomethylating ability of classical beta-D-anomer of 5-aza-2'-deoxycytidine (5-aza-CdRf) and its alpha anomer in cell cultures. Alpha anomers of nucleosides generally exhibit low biological activity compared to their beta counterparts. It is reported that alpha anomer of 5-aza-CdRf efficiently hypomethylated genomic DNA in human T-lymphoblastoid CCRF-CEM cells. Satellite 2 and 18S rDNA were hypomethylated by alpha anomer at concentrations comparable to the beta form. However, the toxicity of the alpha anomer was 4-fold less than that of beta form. Contrast to CCRF-CEM the A549 lung carcinoma cells, possessing negligible level of methylation at repetitive loci, were highly resistant to 5-aza-CdRf treatment suggesting that global genomic methylation might be needed to mediate cytotoxic effect of the drug. Possible mechanisms of inhibition of DNA methylation by alpha anomer are discussed. In conclusion, alpha anomer of 5-aza-CdRf displaying lower host cytotoxicity than the classical beta form may be of potential use in epigenetic therapy. (c) 2006 Elsevier Ltd. All rights reserved.

Abstract: This paper establishes relationships between two aspects of ribosomal DNA (rDNA) biology: epigenetic silencing of rDNA loci; and homogenization leading to concerted evolution. Here, we examined rDNA inheritance and expression patterns in three natural Nicotiana allopolyploids (closest living descendants of diploid parents are given), N. rustica (N. paniculata x N. undulata), N. tabacum (N. sylvestris x N. tomentosiformis) and N. arentsii (N. undulata x N. wigandioides), and synthetic F-1 hybrids and allopolyploids. The extent of interlocus rDNA homogenization decreased in the direction N. arentsii > N. tabacum > N. rustica. The persistence of parental rDNA units in one of the subgenomes was associated with their transcription inactivity and likely heterochromatization. Of synthetic hybrids and polyploids only N. paniculata x N. undulata showed strong uniparental transcriptional silencing of rDNA triggered already in F-1. Epigenetic patterns of expression established early in allopolyploid nucleus formation may render units susceptible or resistant to homogenization over longer time-frames. We propose that nucleolus-associated transcription leaves rDNA units vulnerable to homogenization, while epigenetically inactivated units, well-separated from the nucleolus, remain unconverted.

Abstract: Relationships among genomes are often revealed by the occurrence of common or related satellite DNA (satDNA) types. A typical satDNA characterized by specific sites for one (or more) restriction endonuclease(s) is called 'restriction satellite DNA'. Restriction satDNA comprises - in addition to transposons and retrotransposable elements - often highly repeated genome components present in most higher plants. Large arrays of satDNA elements are concentrated at subtelomeric and/or centromeric regions (intermingled with other retrotransposon-derived elements), however, they can be also located as large intercalating blocks along the chromosome. The head-to-tail tandemly arranged repeat units (monomers) of satDNA mostly exhibit lengths of 160 to 180 bp or 320 to 370 bp, but other lengths were also found in plants. In particular, in interspecific hybrids between more distantly related species, which often exist only after polyploidization, the individual repetitive DNA of the crossing partners contribute to recombination and rearrangement processes in the hybrids, thereby stimulating genome evolution. Here, we concentrate on the possible origin, molecular evolution, organization and distribution of highly repeated satDNA in various higher plants with emphasis on hybrids and allopolyploids.

Abstract: The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the evolution of an IFN-resistant state in vitro using melanoma cell lines. We found that the cells became less sensitive to antiproliferative effect of IFN-gamma after prolonged cultivation enabling us to isolate sensitive and resistant subclones of the parental line. We investigated transcription of signal transducer and activator of transcription (STAT) 1-6 and suppressor of cytokine signalling (SOCS) 1-3 genes, and phosphorylation of STAT 1 protein. The resistant subline (termed WM 1158R) differed from the sensitive subline (WM 1158S) by a constitutive expression of SOCS 3, lack or weak SOCS 1-3 activation following IFN-gamma, and short duration of cytokine activatory signal. Similar correlations were observed in additional melanoma lines differing in IFN sensitivities. At the protein level, IFN-gamma induced strong and prolonged STAT 1 activation at serine 727 (S727) in WM 1158R while in WM 1158S cells phosphorylation of this amino acid was much less pronounced. On the other hand, phosphorylation of tyrosine 701 (Y701) was stimulated regardless of the sensitivity phenotype. In conclusion, constitutive expression of SOCS 3 is correlated with attenuation of its induction following IFN treatment. These results suggest that progression of melanoma cells from IFN sensitivity to IFN insensitivity associates with changes in SOCS expression.

Abstract: We analyzed nuclear ribosomal DNA (rDNA) transcription and chromatin condensation in individuals from several populations of Tragopogon mirus and T. miscellus, allotetraploids that have formed repeatedly within only the last 80 years from 7: dubius and T. porrifolius and T. dubius and T. pratensis, respectively. We identified populations with no (2), partial (2), and complete (4) nucleolar dominance. It is probable that epigenetic regulation following allopolyploidization varies between populations, with a tendency toward nucleolar dominance by one parental homeologue. Dominant rDNA loci are largely decondensed at interphase while silent loci formed condensed heterochromatic regions excluded from nucleoli. Those populations where nucleolar dominance is fixed are epigenetically more stable than those with partial or incomplete dominance. Previous studies indicated that concerted evolution has partially homogenized thousands of parental rDNA units typically reducing the copy numbers of those derived from the 1: dubius diploid parent. Paradoxically, despite their low copy number, repeats of 7: dubius origin dominate rDNA transcription in most populations studied, i.e., rDNA units that are genetic losers (copy numbers) are epigenetic winners (high expression).

Abstract: o Analyses of selected bacterial artificial chromosomes (BACs) clones suggest that the retrotransposon component of angiosperm genomes can be amplified or deleted, leading to genome turnover. Here, Nicotiana allopolyploids were used to characterize the nature of sequence turnover across the whole genome in allopolyploids known to be of different ages. Using molecular-clock analyses, the likely age of Nicotiana allopolyploids was estimated. Genomic in situ hybridization (GISH) and tandem repeat characterization were used to determine how the parental genomic compartments of these allopolyploids have diverged over time. Paternal genome sequence losses, retroelement activity and intergenomic translocation have been reported in early Nicotiana tabacum evolution (up to 200 000 yr divergence). Here it is shown that within 1 million years of allopolyploid divergence there is considerable exchange of repeats between parental chromosome sets. After c. 5 million years of divergence GISH fails. This GISH failure may represent near-complete genome turnover, probably involving the replacement of nongenic sequences with new, or previously rare sequence types, all occurring within a conserved karyotype structure. This mode of evolution may influence or be influenced by long-term diploidization processes that characterize angiosperm polyploidy-diploid evolutionary cycles.

Abstract: Background Aims One of the classic examples of an allopolyploid is Iris versicolor, 'Blue Flag' (2n = 108), first studied by Edgar Anderson and later popularized by George Ledyard Stebbins in cytogenetics and evolutionary textbooks. It is revisited here using modem molecular and cytogenetic tools to investigate its putative allopolyploid origin involving progenitors of I. virginica (2n = 70) and L setosa (2n = 38). Methods Genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) and Southern hybridization with 5S and 18-26S ribosomal DNA (rDNA) probes were used to identify the parental origin of chromosomes, and to study the unit structure, relative abundance and chromosomal location of rDNA sequences. Key Results GISH shows that L versicolor has inherited the sum of the chromosome complement from the two progenitor species. In L versicolor all the 18-26S rDNA units and loci are inherited from the progenitor of I. virginica, those loci from the L setosa progenitor are absent. In contrast 5S rDNA loci and units from both progenitors are found, although one of the two 5S loci expected from the L setosa progenitor is absent. Conclusions These data confirm Anderson's hypothesis that L versicolor is an allopolyploid involving progenitors of I. virginica and L setosa. The number of 18-26S rDNA loci in L versicolor is similar to that of progenitor I. virginica, suggestive of a first stage in genome diploidization. The locus loss is targeted at the L setosa-origin subgenome, and this is discussed in relation to other polyploidy systems.

Abstract: 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.

Abstract: We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.

Abstract: Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.

Abstract: In plants, 5S ribosomal DNA (5S rDNA) is typically found in hundreds of copies of tandemly arranged units. Nucleotide database searches revealed that the majority of 5S genes (> 90%) have repeat lengths that are not simple multiples of a plant nucleosomal unit, ranging in plants from 175-185 bp. To get insight into the chromatin structure, we have determined positions of nucleosomes in the Nicotiana sylvestris and Nicotiana tomentosiformis 5S rDNA units with repeat lengths of about 430 and 645 bp, respectively. Mapping experiments carried out on isolated nucleo somal DNA revealed many (> 50) micrococcal nuclease cleavage sites in each class of repeats. Permutation analysis and theoretical computer prediction showed multiple DNA bend sites, mostly located in the nontranscribed spacer region. The distance between bend sites, however, did not correspond to the average spacing of nucleosomes in 5S chromatin (similar to 180 bp). These data indicate that 5S rDNA does not have fixed nucleosomal positioning sites and that units can be wrapped in a number of alternative nucleosome frames. Consequently, accessibility of transcription factors to cognate motifs might vary across the tandem array, potentially influencing gene expression.

Abstract: Polyploids have significantly influenced angiosperm evolution. Understanding the genetic consequences of polyploidy is advanced by studies on synthetic allopolyploids that mimic natural species. In Nicotiana, Burk (1973) and Kostoff (1938) generated synthetic tobacco (N. tabacum) using the parents female N. sylvestris X male N. tomentosiformis. We previously reported rapid genetic changes in the Burk material. Kostoff's material has 24 chromosomes of N. sylvestris origin (S-genome), 24 of N. tomentosiformis origin (T-genome), and a large intergenomic translocation, but not an additive distribution of ribosomal DNA (rDNA) families as expected from the parental contribution. Our new synthetic tobacco lines TR1 and TR2 are chromosomally balanced with no intergenomic translocations and are either sterile or have highly reduced fertility, supporting the nuclear cytoplasmic hypothesis that allopolyploid fertility is enhanced by intergenomic translocations.,Two plants of TR1 (TR1-A, TR1-B) have the expected number, structure, and chromosomal distribution of rDNA families. in contrast to Burk's and Kostoff's synthetic tobaccos and to synthetic polyploids of Arabidopsis. Perhaps allopotyploids must pass through meiosis before genetic changes involving rDNA become apparent, or the genetic changes may occur stochastically in different synthetic allopolyploids. The lack of fertility in the first generation of our synthetic tobacco lines may have uses in biopharmacy.

Abstract: Nicotiana tabacum (tobacco, 2n = 4x = 48) is a natural allotetraploid combining two ancestral genomes closely related to modern Nicotiana sylvestris and Nicotiana tomentosiformis. Here we examine the immediate consequences of allopolyploidy on genome evolution using 20 S-4-generation plants derived from a single synthetic, S-0 plant made by Burk in 1973 (Th37). Using molecular and cytogenetic methods we analysed 14 middle and highly repetitive sequences that together total approximate to 4% of the genome. Two repeats related to endogenous geminiviruses (GRD5) and pararetroviruses (NtoEPRV), and two classes of satellite repeats (NTRS, A1/A2) were partially or completely eliminated at variable frequency (25-60%). These sequences are all from the N. tomentosiformis parent. Genomic in situ hybridization revealed additivity in chromosome numbers in two plants (2n = 48), while a third was aneuploid for an N. tomentosiformis-origin chromosome (2n = 49). Two plants had homozygous translocations between chromosomes of the S- and T-genomes. The data demonstrate that genetic changes in synthetic tobacco were fast, targeted to the paternal N. tomentosiformis-donated genome, and some of the changes showed concordance with changes that presumably occurred during evolution of natural tobacco.

Abstract: We investigated concerted evolution of rRNA genes in multiple populations of Tragopogon mirus and T. miscellus, two allotetraploids that formed recurrently within the last 80 years following the introduction of three diploids (T. dubius, T. pratensis, and T. porrifolius) from Europe to North America. Using the earliest herbarium specimens of the allotetraploids (1949 and 1953) to represent the genomic condition near the time of polyploidization, we found that the parental rDNA repeats were inherited in roughly equal numbers. In contrast, in most present-day populations of both tetraploids, the rDNA of T. dubius origin is reduced and may occupy as little as 5% of total rDNA in some individuals. However, in two populations of T mirus the repeats of T dubius origin outnumber the repeats of the second diploid parent (T porrifolius), indicating bidirectional concerted evolution within a single species. In plants of T. miscellus having a low rDNA contribution from T. dubius, the rDNA of T. dubius was nonetheless expressed. We have apparently caught homogenization of rDNA repeats (concerted evolution) in the act, although it has not proceeded to completion in any allopolyploid population yet examined.

Abstract: The signal transducers and transcription activators (STATs) and their endogenous inhibitors of the suppressors of cytokine signalling (SOCS) family are major proteins harmonizing the transmission of external signals from the surface membrane to target genes in the nucleus. To correlate the induction of SOCS 3 by interferons (IFNs) on messenger RNA and protein levels with STAT 1 phosphorylation in human malignant melanoma cell lines we used a unique collection of 18 established malignant melanoma cell lines and six human non-malignant normal cells (two melanocytes, two skin keratinocytes and two fibroblasts). IFN-gamma induced SOCS 3 in 83% of melanoma cell lines, whereas IFN-alpha stimulated SOCS 3 expression in only 11% of cases. Similarly, melanocytes showed strong induction of SOCS 3 by IFN-gamma and, to a lesser extent, by IFN-alpha. In most cases, SOCS 3 expression was paralleled by STAT I phosphorylation at tyrosine residues (Y701). In several lines, however, SOCS 3 was not induced despite STAT 1 phosphorylation and, in a few lines, SOCS 3 induction occurred without detectable STAT 1 phosphorylation, indicating that STAT 1 might not be an exclusive inducer of SOCS 3. Similarly, non-malignant cells displayed STAT 1 activation and high levels of SOCS 3 expression after IFN-gamma (but not IFN-alpha) treatment. In conclusion, in contrast to IFN-alpha, IFN-gamma appeared to induce SOCS 3 apparently at the transcription level and exhibited higher cytotoxic effects regardless of the cell origin.

Abstract: (.) Here, we analyze long-term evolution in Nicotiana allopolyploid section Repandae ( the closest living diploids are N. sylvestris, the maternal parent, and N. obtusifolia, the paternal parent). We compare data with other more recently formed Nicotiana allopolyploids. (.) We investigated 35S and 5S nuclear ribosomal DNA ( rDNA) chromosomal location and unit divergence. A molecular clock was applied to the Nicotiana phylogenetic tree to determine allopolyploid ages. (.) N. tabacum and species of Repandae were c. 0.2 and 4.5 Myr old, respectively. In all Repandae species, the numbers of both 35S and 5S rDNA loci were less than the sum of those of the diploid progenitors. Trees based on 5S rDNA spacer sequences indicated units of only the paternal parent. (.) In recent Nicotiana allopolyploids, the numbers of rDNA loci equal the sum of those of their progenitors. In the Repandae genomes, diploidization is associated with locus loss. Sequence analysis indicates that 35S and 5S units most closely resemble maternal and paternal progenitors, respectively. In Nicotiana, 4.5 Myr of allopolyploid evolution renders genomic in situ hybridization ( GISH) unsuitable for the complete resolution of parental genomes.

Abstract: The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Abstract: Epigenetic changes accompanying plant cell dedifferentiation and differentiation are reported in 35S ribosomal DNA ( rDNA) of tobacco (Nicotiana tabacum). There was a reduction of CG and CNG methylation in both intergenic and genic regions of the rDNA cistron in fully dedifferentiated callus and root compared to leaf. The rDNA hypomethylation was not random, but targeted to particular rDNA gene families at units that are clustered within the tandem array. The process of hypomethylation was initiated as early as 2 weeks after the callus induction and established epigenetic patterns were stably maintained throughout prolonged culture. However, regenerated plants and their progeny showed partial and complete remethylation of units, respectively. Nuclear run-on assays revealed a 2-fold increase of primary (unprocessed) ribosomal RNA transcripts in callus compared to leaf tissue. However, the abundance of mature transcripts in callus was elevated by only about 25%. Fluorescence in situ hybridization analysis of interphase nuclei showed high levels of rDNA chromatin condensation in both callus and leaf, with substantially less decondensed rDNA than is observed in meristematic root-tip cells. It is likely that the regions of the rDNA locus showing decondensation correspond to the clusters of hypomethylated units that occur in the tandem array at each locus. The data together indicate that the establishment of pluripotency and cell proliferation occurring with callus induction is associated with enhanced ribosomal RNA gene expression and overall rDNA hypomethylation, but is not associated with material-enhanced relaxation of chromatin structure (decondensation) at rDNA loci.

Abstract: Nicotiana rustica (2n = 4x = 48) is a natural allotetraploid composed of P and U genomes which are closely related to genomes of diploid species N. paniculata and N. undulata. Genomic in situ hybridization (GISH) also confirms that the diploid parents, or close relatives, are the ancestors of N. rustica. In order to study genetic interactions between ancestral genomes in the allotetraploid, we isolated three families of repetitive sequences, two from N. paniculata ( NPAMBE and NPAMBO) and one from N. undulata (NUNSSP). Southern blot hybridization revealed that the sequences are digested with a range of restriction enzymes into regular ladder patterns indicating a tandem arrangement of high copy repeats possessing monomeric units of about 180 bp. The three-tandem sequences belong to a larger Nicotiana tandem repeat family called here the HRS-60 family. Members of this family are found in all Nicotiana species studied. Fluorescence in situ hybridization ( FISH) analysis localized the satellite repeats to subtelomeric regions of most chromosomes of N. paniculata and N. undulata. The pattern of sequence distribution on the P- and U-genomes of N. rustica was similar to the putative parents N. paniculata and N. undulata respectively. However, NPAMBO repeats appear to be reduced and rearranged in N. rustica that may suggest evolution within the P genome. GISH and FISH with the tandem repeat probes failed to reveal intergenomic translocations as might be predicted from the nucleocytoplasmic interaction hypothesis. Copyright (C) 2005 S. Karger AG, Basel.

Abstract: A horizontal transmission of a geminiviral DNA sequence, into the germ line of an ancestral Nicotiana, gave rise to multiple repeats of geminivirus-related DNA, GRD, in the genome. We follow GRD evolution in Nicotiana tabacum (tobacco), an allotetraploid, and its diploid relatives, and show GRDs are derived from begomoviruses. GRDs occur in two families: the GRD5 family's ancestor integrated into the common ancestor of three diploid species, Nicotiana kawakamii, Nicotiana tomentosa and Nicotiana tomentosiformis, on homeologous group 4 chromosomes. The GRD3 family was acquired more recently on chromosome 2 in a lineage of N. tomentosiformis, the paternal ancestor of tobacco. Both GRD families include individual members that are methylated and diverged. Using relative rates of synonymous and nonsynonymous nucleotide substitutions, we tested for evidence of selection on GRD units and found none within the GRD3 and GRD5 families. However, the substitutions between GRD3 and GRD5 do show a significant excess of synonymous changes, suggesting purifying selection and hence a period of autonomous evolution between GRD3 and GRD5 integration. We observe in the GRD3 family, features of Helitrons, a major new class of putative rolling-circle replicating eukaryotic transposon, not found in the GRD5 family or geminiviruses. We speculate that the second integration event, resulting in the GRD3 family, involved a free-living geminivirus, a Helitron and perhaps also GRD5. Thus our data point towards recurrent dynamic interplay between geminivirus and plant DNA in evolution.

Abstract: Tragopogon mirus and T. miscellus (both 2n = 4x = 24) are recent allotetraploids derived from T. dubius X T. porrifolius and T dubius X T. pratensis (each 2n = 2x = 12), respectively. The genome sizes of T. mirus are additive of those of its diploid parents, but at least some populations of T. miscellus have undergone genome downsizing. To survey for genomic rearrangements in the allopolyploids, four repetitive sequences were physically mapped. TPRMBO (unit size 160 base pairs [bp]) and TGP7 (532 bp) are tandemly organized satellite sequences isolated from T pratensis and T porrifolius, respectively. Fluorescent in situ hybridization to the diploids showed that TPRMBO is a predominantly centromeric repeat on all 12 chromosomes, while TGP7 is a subtelomeric sequence on most chromosome arms. The distribution of tandem repetitive DNA loci (TPRMBO, TGP7, 18S-5.8S-26S rDNA, and 5S rDNA) gave unique molecular karyotypes for the three diploid species, permitting the identification of the parental chromosomes in the polyploids. The location and number of these loci were inherited without apparent changes in the allotetraploids. There was no evidence for major genomic rearrangements in Tragopogon allopolyploids that have arisen multiple times in North America within the last 80 yr.

Abstract: An similar to135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentsiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as in 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 Al/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

Abstract: Genomic in situ hybridisation was used to confirm that Nicotiana rustica (2n = 4x = 48) is an allotetraploid between N. paniculata ( 2n = 2x = 24, maternal P-genome donor) and N. undulata ( 2n = 2x = 24, paternal U-genome donor), their progenitors or species closely related to them. Fluorescent in situ hybridisation showed that N. paniculata has one 5S and two 18-5.8-26S rDNA loci whereas N. undulata has an additional 18-5.8-26S rDNA locus. N. rustica has the sum of the loci found in these putative parents. The sizes of the 185.8-26S rDNA loci indicate that the number of rDNA units on the U-genome chromosomes has amplified; perhaps this is associated with a concomitant reduction in the number of units on P-genome chromosomes. Restriction fragment length polymorphism analysis of the intergenic spacer IGS) of the 18-5.8-26S rDNA units in N. rustica and the two progenitor diploids revealed that about 80% of IGS sequences in N. rustica are of an N. undulata type and 20% of N. paniculata type. These data indicate that interlocus sequence homogenisation has caused the replacement of many N. paniculata-type IGSs in N. rustica with an N. undulata-type of sequence. It is probable that subsequent to this replacement there has been sequence divergence at the 50 end of the IGS. As in tobacco, an allotetraploid between N. sylvestris and N. tomentosiformis, the direction of the IGS interlocus conversion is towards the paternal genome donor.

Abstract: Changes in the distribution of methylcytosine residues along a transgene locus of tobacco (Nicotiana tabacum) in relation to the type of gene silencing were studied in parental plant leaves, calli, and regenerated plants derived thereof. Parental-silenced HeLo1 (hemizygous for locus 1) plants show posttranscriptional silencing of the residing nptII (neomycin phosphotransferase II) transgene and cytosine methylation restricted to the 3' end and center part of the transcribed region. Here, we report that with an increasing number of cell cycles, DNA methylation changes gradually, and methylation is introduced into the promoter during cell culture and more slowly in vegetatively propagated plants. After 24 months of callus in vitro cultivation, an epigenetic variant, designated locus 1E, was obtained in which cytosine methylation of symmetrical (CG and CNG) sites was almost complete within the 5' end of the nptII-transcribed region and the 35S promoter. Further, methylation of nonsymmetrical sites appeared de novo in the promoter, whereas this type of methylation was significantly reduced in the 3' end of the transcribed region when compared with locus 1. The newly established epigenetic patterns were stably transmitted from calli into regenerated plants and their progeny. The protein and steady-state RNA levels remained low in locus 1E, whereas with nuclear run-on assays, no detectable amounts of primary transcripts were found along the nptII gene, indicating that the methylated promoter became inactivated. The results suggest that a switch between posttranscriptional and transcriptional gene silencing could be a mechanism leading to irrevocable shut down of gene expression within a finite number of generations.

Abstract: Unidirectional gene conversion of rDNA units has occurred in the evolution of natural tobacco (Nicotiana tabacum). In this paper we report the use of the synthetic tobacco line Th37, 4n (N. sylvestris X N. tomentosiformis), to study early rDNA evolution associated with allopolyploidy. At least three classes of newly amplified rDNA unit variants were identified (17/20 plants). Their presence was often accompanied by near-complete elimination of N. tomentosiformis-donated rDNA units (15/20 plants). Novel rDNA units were of N. tomentosiformis-type and contained rearranged subrepeats in the intergenic spacer. The maternal N. sylvestris-derived units were unchanged, except for some alteration in the ratio of individual gene family members. A cytogenetic analysis revealed rDNA sites on N. sylvestris-derived chromosomes S 10, S 11, and S 12 and N. tomentosiformis-derived chromosomes T3 and in some cases T4. An rDNA locus does not occur on N. tomentosiformis chromosome 4. The locus on chromosome T4 of some hybrids correlates with the occurrence of the novel units that probably amplified at the locus. Combined with an analysis of tobacco cultivars, the data indicate that an initial burst of rDNA evolution associated with allopolyploidy was followed by a slower process that led towards reduced complexity and a decreased number of rDNA variants.

Abstract: To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.

Abstract: It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a relatively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported here demonstrate that cell death can be prevented if 50 muM CdSO4 is removed from the growth medium during the lag phase, suggesting that an irreversible apoptotic trigger is delivered within 24 h, between the third and fourth days of cadmium treatment. The post-cadmium recovery phase was characterized by DNA repair at the level of 50-200 kb and increased telomerase activity. Analysis of high-molecular-weight DNA by pulsed-field-gel electrophoresis revealed that the majority of DNA strand breaks was repaired within 48 h after cadmium withdrawal. Telomerase activity increased 2.5-fold in the recovery phase, but elevated levels were also found in cell extracts from apoptotic cells suggesting that telomerase might be associated with DNA repair, but it is not capable of inhibiting ongoing apoptosis. Limited exposure of TBY-2 cells to cadmium elicits non-random DNA damage of relatively high magnitude that can be repaired. It is proposed that plants might have developed a highly efficient DNA repair system to cope with transient genotoxic stress.

Abstract: Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 by unit length and the other a 646 by unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 by and 644 by respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris 5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.

Abstract: Bisulfite genomic sequencing was used to localise 5-methylcytosine residues (mC) in 5S rRNA genes of Arabidopsis thaliana and Secale cereale. The maps of mC distribution were compared with the previously published map of the corresponding region in Nicotiana tabacum. In all three species, the level of methylation of 5S rRNA genes was generally higher than the average for the entire genome. The ratio of 5S rDNA methylation to average overall methylation was 44%/30-33% for N. tabacum, 27%/4-6% for A. thaliana and 24%/20-22% for S. cereale. With the exception of one clone from S. cereale, no methylation-free 5S rDNA was detected. The level of methylation at different sequence motifs in 5S rDNA was calculated for N. tabacum/A. thaliana/S. cereale, and this analysis yielded the following values (expressed as a percentage of total Q: mCG 90%/78%/85'Vo, mCWG 89%/41%/ 53%, mCmCG 72%/32%/16%, mCCG 4%/2%/0%, mCHH 15%/6%/1%, where W = A or T, and H = A or C or T. Non-symmetrical methylation was almost negligible in the large genome of S. cereale but relatively frequent in N. tabacum and A. thaliana, suggesting that the strict correlation between genome size and cytosine methylation might be violated for this type of methylation. Among non-symmetrical motifs the mCWA triplets were significantly over-represented in Arabidopsis, while in tobacco this preference was not as pronounced. The differences in methylation levels in different sequence contexts might be of phylogenetic significance, but further species in related and different taxa need to be studied before firm conclusions can be drawn.

Abstract: Nicotiana tabacum (tobacco, Solanaceae) has two 5S ribosomal DNA (rDNA) families, one of unit length similar to646 bp and the other similar to430 bp, that differ in the length of the 5S rDNA non-transcribed spacer (NTS). The long 5S rDNA family, found on the T genome of tobacco and in Nicotiana tomentosiformis, contains a GC-rich subregion that is absent in the short family. We designed primers for this subregion and generated a probe that we used against a range of Nicotiana and related Solanaceous species. We demonstrated the presence of the GC-rich subregion in a range of Nicotiana species, but it was absent in Nicotiana sylvestris, Nicotiana longiflora, and two closely related genera, Petunia and Solanum. These data suggest that this subregion of the NTS is likely to have evolved with the genus Nicotiana. The absence of the subregion in N. sylvestris and N. longiflora is likely to have arisen by a deletion event in the evolution of section alatae. We demonstrate patterns of evolution in the 5S rDNA unit cluster in relation to a phylogenetic reconstruction of species relationships in section tomentosae. Nicotiana glutinosa diverged early from the section and contains a 5S rDNA family based on a 550-bp unit. After this divergence, 430- and 650-bp rDNA unit families evolved. The 650-bp family is found in all species of tomentosae (except N. glutinosa) and in tobacco. The 430-bp family within tomentosae includes the GC-rich subregion and is thus unrelated to the 430-bp family in N. sylvestris. Nicotiana setchellii is unusual in that it has three 5S rDNA loci, including one locus that is exceptionally large. This species, unique to tomentosae, has a very abundant 900-bp unit family. It is possible that this 900-bp family occurs on this one large locus. In N. tomentosa and N. kawakamii, the 650-bp family is predominant, whereas N. tomentosiformis and N. otophora have only the 650-bp family. There is no clear relationship between the number of 5S families and the number of 5S rDNA loci. Certainly the replacement of 5S rDNA units, perhaps by gene conversion, has occurred repeatedly in the evolution of genus Nicotiana.

Abstract: Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show hat the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N, tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.

Abstract: During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that dithered from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-by direct repeat derived from the R8.3 sequence, the 9-by duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-by direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 by in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.

Abstract: Highly repeated satellite DNA (stDNA) of citric plants was characterized by cloning and sequencing 10-14 repeats of each plant (Citrus limon, C. sinensis, C. ichangensis, Poncirus trifoliata). The monomers are mostly 181 bp in length with a GC-content between 60% and 68% (significantly higher than the average GC-content of the citrus group genomes). Similarity among the repeats indicates that they belong to a satellite family that underwent species-specific modifications, which are reflected in the phylogenetic relationships. Curvature provoked by dA-stretches of the repeats analyzed by gel shifts revealed structural conservation, even though the nucleotide sequences vary among species, thereby probably supporting the heterochromatic structure of stDNA. We show that the species-specific modification of the satellite consensus involves changes in the position and number of dA tracts. The molecule shapes of satellite oligomeres predicted by computer modelling indicate a superhelical structure of the tandem repeats which is in a good agreement with the satellite sequence dendrogram. The contribution of DNA bending elements to the evolution of plant satellite repeats is discussed.

Abstract: DNA methylation of chloroplast genome has been studied in a large variety of angiosperm species using restriction enzyme analysis of three genomic loci (totally encompassing about 10% of chloroplast genome) and bisulfite genomic sequencing of tobacco ribulose bisphosphate carboxylase/oxygenase (large subunit) gene (rbcL). Except for CCWGG (W = A or T) sites that were partially refractory to the cleavage with methylation sensitive EcoRII in all loci. no cytosine methylation was found at the CCGG (dMspI/HpaII) and several other restriction sites tested. However, EcoRII was unable to completely digest an unmethylated CCWGG site in the cloned rbcL gene on plasmid. Further a bisulfite genomic sequencing performed on EcoRII-restricted DNA failed to show any 5-methylcytosine either within or outside inspected EcoRII sites along the 3 ' end of rbcL coding region. In conclusion our results do not support evidence for methylated cytosine residues in plant chloroplast genomes and we suggest that results obtained with EcoRII should be interpreted with great care especially when small differences in methylation levels are analysed. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Abstract: Tobacco plants containing a transgene locus with two chimeric neomycin phosphotransferase II (nptII) genes in tail-to-tail orientation (locus 1) show posttranscriptional gene silencing. The silenced nptII transgenes of locus 1 can downregulate the expression of homologous nptII transgenes in hybrid plants. The 3' region of the silenced nptII genes located in the center of the inverted repeat locus 1 is extensively methylated. Moreover, 3' segments of in trans-inactivated transgenes also become methylated, suggesting cross-talk between homologous posttranscriptionally silenced genes. Our results are in accordance with the hypothesis that this cross-talk can be mediated by specially featured RNAs. (C) 2000 Federation of European Biochemical Societies.

Abstract: 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'-(m)C(m)CG-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'-(m)C(m)CG-3' sites with DHPA is a gradual non-random process proceeding in the direction (m)C(m)CG --> C(m)CG --> 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.

Abstract: We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sires on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome II. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.

Abstract: This work explores the influence of cadmium on a suspension cell culture of Nicotiana tabacum (TBY-2) by examining cell morphology, viability and DNA integrity. Changes in these parameters were strikingly dependent on concentration of cadmium in the culture medium: a concentration of 50-100 mmol m(-3) CdSO4 induced apoptotic changes including DNA fragmentation into oligonucleosomal units, while 1 mol m(-3) Cd2+ showed strong cytotoxicity, but no fragmentation of DNA, Low cadmium concentrations (below 10 mmol m(-3)) affected neither cell viability nor DNA integrity. A detailed kinetic study showed a significant delay in the onset of apoptosis after the application of high concentrations of cadmium. From days 0-3 after the application of 50 mmol m(-3) CdSO4, the morphology of the cells, their viability and growth were indistinguishable between control and treated cells, and 'domain' DNA fragmentation into 50-200 kb fragments was observed at the DNA level. After this (days 4-7), there was a characteristic and rapid decrease in cell viability, distinct changes in cell. morphology and oligonucleosomal fragmentation. The results suggest that chronic exposure of plant cells to cadmium can trigger programmed cell death.

Abstract: The effect of DNA methylation upon posttranscriptional gene silencing (PTGS) has been investigated in transgenic tobacco Lines showing PTGS and methylation of the neomycin phosphotransferase II (nptII) reporter genes. Application of the hypomethylation drugs dihydroxypropyladenine or 5-azacytidine resulted in approximately 30% reduced methylation of cytosines located in a non-symmetrical context in the 3' untranslated region of the nptII transgenes. The hypomethylation was accompanied by up to 12-fold increase in NPTII protein levels, suggesting that methylation of non-symmetrical motifs may account for an increased degree of PTGS, Models for the possible role of DNA methylation in PTGS are discussed. (C) 2000 Federation of European Biochemical Societies.

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Abstract: Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase.

Abstract: This paper describes the effects of hypomethylation with 5-azacytidine (5azaC) and dihydroxypropyladenine (DHPA) on protonemata of the moss Funaria hygrometrica Hedw. Following treatment with 5azaC or DHPA, hypomethylation of the EcoR II (CCA(T)GG) and Pst I (CTGCAG) sites in ribosomal DNA (rDNA) was confirmed using restriction enzyme analysis and Southern hybridization. Hypomethylation of the genome had profound effects on protonemal differentiation. Whilst apical cell organization and cell dimensions and shape remained unchanged, there was a marked retardation in both cytoplasmic and nuclear differentiation. Developmental abnormalities included: late and erratic side branch formation, some loss of the distinction between chloronema and caulonema, formation of aberrant buds, and loss of the potential to form brood and tmema cells after 5azaC treatment which in general had more profound effects than DHPA. Cytologically, caulonema cells were less highly polarized or unpolarized, and tended to retain small round chloroplasts, whilst nuclei in the hypomethylated protonema endoreduplicated to lower levels and tended to remain more spherical. Increased nucleolar volumes and loss of intranucleolar rDNA heterochromatin following hypomethylation might be the result of increased transcriptional activity of ribosomal RNA (rRNA) genes and drug-induced DNA decondensation, respectively. Growing hypomethylated protonema at 25 degrees C induced extremely atypical cells and development. This temperature sensitivity and aberrations in development overall can be attributed to changes in the normal patterns of gene expression brought about by hypomethylation of gene promoter or regulator regions.

Abstract: A method based on polymerase chain reaction (PCR) amplification of fungal 18S rDNA sequences was tested for the detection of fungi in blood samples. In order to increase sensitivity and specificity, PCR products were hybridized to the radioactively labelled fragment of 18S rDNA gene. Blood from 28 patients with haematological malignancies was taken immediately after death and the results of PCR analysis were compared with results of autopsy examination. To the best of our knowledge, no study of such a design has been published previously. PCR analysis turned out to be very sensitive (92%) and specific (92%) as well as capable of detecting various kinds of fungal infections (localized as well as generalized).

Abstract: We explored the possibility that the cytosine DNA methylation might be regulated by S-adenosyl-L-methionine (AdoMet) and S-adenosyl-L-homocysteine (AdoHcy) pools in plant cells. In order to change the AdoHcy/AdoMet ratio (methylation index), (S)-9-(2,3-dihydroxypropyl)adenine was employed, a selective reversible inhibitor of cellular S-adenosyl-L-homocysteine hydrolase. Micromolar concentrations of the inhibitor increased dramatically (more than 1000-fold) intracellular AdoHcy levels (and concominantly the AdoHcy/AdoMet ratio) in tobacco TBY-2 cells. No toxic effect of the drug was observed and the cells displayed only marginal inhibition of growth at high AdoHcy levels. At near equal intracellular concentrations of AdoHcy and AdoMet, a significant reduction of cytosine methylation in transcribed (5SrDNA) and non-transcribed (HRS60, NTRS) sequences was observed. Interestingly, the CpCpG and CpApG trinucleotide targets appeared to be most sensitive to changes in the methylation index. Methylation of cytosine residues at CpG sites was not affected even at AdoHcy/AdoMet ratio of > 10. These results support the possible regulation of DNA methylation via AdoHcy/AdoMet metabolic pathways in plant cells.

Abstract: This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). IRS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for IRS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.

Abstract: Programmed cell death (PCD) may be triggered by a variety of environmental stimuli, In this report we show that low temperature treatment of tobacco BY-2 cells results in specific chromatin changes, The early stage was characterised by chromatin condensation associated with specific endonucleolytic cleavage of the genome into fragments of 50-100 kbp in size, Later, after 2 weeks of the cold treatment, a ladder of nucleosomal units (178 bp) and their multiples occurred, Chromatin changes mere accompanied by a general decrease in cell viability, However, the cell culture retained about 11% of living cells even after prolonged incubation in the cold suggesting the presence of a cold-resistant population of cells, The results support the view that PCD was activated by the cold stress, We suggest that cold-stressed tobacco BY-2 culture might be a useful system for investigation of PCD in plant cells. (C) 1997 Federation of European Biochemical Societies.

Abstract: Plants have to cope with a number of environmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/IHpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress.

Abstract: The methylation status of ribosomal gene (rRNA) clusters have been investigated in a large variety of angiosperm species. Here we have analysed methylation in ribosomal gene (rRNA) clusters using MspI, HpaII, BstNI, EcoRII and CfoI restriction enzymes in combination with Southern hybridization to the 25S rDNA probe. It was shown that cytosine methylation at CpG dinucleotides and CpNpG trinucleotides occurred in all plant genomes examined. Methylation of rDNA units at CpG dinucleotides (studied with CfoI) was high in all species tested with approx. 40-70% of units being completely or nearly completely methylated. In contrast, the extent of the CpNpG methylation (studied with MspI and EcoRII) varied significantly between species; the percentage of the rDNA fraction entirely methylated at CpNpG trinucleotides ranged from less than 1% to almost 90% depending on the genome studied. Larger interspecies than within species variation was also observed among several non-transcribing repetitive sequences. In a small genome of A. thaliana, the CpNpG methylation appeared to be highly compartmentalized into the repetitive fraction. The methylation of trinucleotides was abundant in large A+T-rich genomes and it is proposed that the CpA(T)pG trinucleotides may help to maintain a high density of methylatable targets in plant repeated sequences. (C) 1997 Elsevier Science B.V.

Abstract: 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 100 kb in N, otophora, and from 40 to 160 kb in N. tabacum. The patterns of rDNA repeats (18S, 5.8S, 25S RNA) appeared to be highly homogeneous and species-specific; 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.

Abstract: Telomerase is a ribonucleoprotein enzyme which elongates the G-rich strand of telomeric DNA to compensate for the progressive reduction in its length due to incomplete replication of chromosome ends, which in human somatic cells leads to cell cycle arrest upon shortening of telomeres to a critical length, To examine the possible involvement of telomerase in metabolism of plant genetic material, we used cells of Nicotiana tabacum strain TBY-2, a stable long-term culture which has kept a constant pattern of restriction fragments from chromosome termini during its 6 month period of cultivation in our laboratory, In a direct assay for telomerase, a 5' end-labeled plant telomeric oligonucleotide (5')(TTTAGGG)(6)(3') was elongated in a TBY-2 cell extract, showing a pausing pattern which is a characteristic feature of telomerases from other organisms, The elongation was inhibited by RNase A pretreatment of the extract, We conclude that plant cells possess telomerase which is used for maintenance of their telomeres.

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Abstract: In this report, we have analyzed the function of two Spl sites present in the epithelium-specific MUC1 promoter, Using promoter-reporter gene (CAT) constructs, we found that mutagenesis of either of the Sp1 binding motifs at -576/-568 and -99/-90, reduced transcription in MUC1-expressing epithelial cell lines, However, abolition of the binding site at -99/-91 by mutagenesis also resulted in increased transcriptional activity in non-epithelial cell lines, suggesting involvement of the site in tissue-specific expression, In vitro binding assays revealed a novel binding motif at -101/-89 (AGGGGGCGGGGTT), which overlaps but differs from the Sp1 consensus motif by having an adenine residue in the 5'-flanking sequence, The 5'-flanking sequence appeared to be important for binding of an Sp1-unrelated factor (SpA) but not for binding of Sp1, Site-directed mutagenesis of the motif into a site able to bind Sp1, but unable to bind SpA, resulted in an increased level of transcription of the CAT reporter gene in all cell lines tested, suggesting a repressive effect of the novel factor on transcription, The ratio between the Sp1 and SpA binding activity in nuclear extracts correlated with both promoter activity and the levels of endogenous transcription in different breast cancer cell lines, Our results are consistent with the idea that the relative activities of the two factors may be involved in the upregulation of expression of the MUC1 gene seen in breast and other carcinomas.

Abstract: PCR and primers derived from the telomeric repeat (CCCTAAA)(n) and from the tobacco subtelomeric tandemly repetitive sequence HRS60 (EMBL X12489) were used to amplify the region linking the two loci. A 131 bp PCR product was obtained both from total tobacco DNA and from the DNA fraction enriched for telomeres. Its sequence only consists of the telomeric primer and the attached region of the HRS60 repetitive unit up to the end of the sequence complementary to the HRS60 primer. The site of direct continuity between the two sequences is formed by a (dA)(7) tract.

Abstract: We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)(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 pi electrophoresis (PFGE) nf 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.

Abstract: Recently a highly repetitive DNA sequence family (GRS) from tobacco was described in our laboratory. These sequences were found to be localized predominantly in the pericentromeric heterochromatin of tobacco chromosomes. To test the hypothesis that these sequences play an important role in the formation of heterochromatin, we investigated the DNA curvature of the GRS sequences and its possible impact to the chromatin structure at these loci. Application of the nearest-neighbour wedge model of intrinsic DNA curvature for the GRS1 family member predicted two loci of curvature: a major bend at the 5' end of the sequence and a minor bend of opposite direction at the centre of the GRS1. The presence of the major and the minor loci of DNA curvature was studied experimentally using permutation analysis and site-directed mutagenesis. The experimental results were consistent with the computer predictions. We gave evidence that the described DNA curvature is also present in the entire GRS family. Genomic statistical sequencing showed the conservation of the major bend sequence determinants in the members of the GRS family. To investigate the chromatin structure at the GRS sequences, we determined the nucleosome positioning in vivo at these sequences using thermal cycle primer extension. A relation between the curvature pattern and the histone octamer position was observed: tile major bend is excluded from the nucleosome surface to the linker region, while the minor bend is distributed along the core DNA. The suggestion is made that the sequences in the minor locus of curvature define the rotational setting of the nucleosome, and a possible role of the major bend as a factor, which defines the translational setting, is discussed.

Abstract: 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 mic 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.

Abstract: Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture using the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyladenine (DHPA) and ethionine (Ethi), and methylation-sensitive restriction endonucleases HpaII, MspI, HhaI, EcoRII, scrFI, and Fnu4HI. Surprisingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triplets, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylhomocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CAG/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase suggests the possibility that plant-specific DNA methylation at CNG targets might be modulated by alterations of the SAH/S-Adenosylmethionine ratio in plant cells.

Abstract: The 5'-sequences flanking the human MUC1 gene have been analyzed for their ability to direct expression of a reporter gene (the chloramphenicol acetyltransferase gene (CAT)) in cell lines that normally express or do not express the MUC1 gene. A construct containing 2.9 kilobase pairs of MUC1 5'-flanking sequence sequence showed expression of CAT in breast and pancreatic cell lines but not in the non-epithelial cell lines HT-1080, SK23, and HTB96. Deletion analysis showed that maximum expression was obtained in ZR-75 (breast cancer line) and HPAF (pancreatic cancer line) with only 743 base pairs of 5'-flanking sequence. Sequences within 1.6 kilobase pairs of the transcriptional start site showed enhancing activity in a vector carrying an enhancerless SV40 promoter. Analysis of proximal 5'-sequences in a promoterless CAT vector carrying the SV40 enhancer showed that sequences between -60 and -150 were crucial for tissue-specific expression. An Spl site at -99/-90 and an E box (E-MUC1) at -84/-64 in this region were shown by mutational analysis to play a role in the regulation of transcription. Gel shift analysis with oligonucleotides and nuclear extracts of ZR-75 showed protein binding to both of these sites. Sp1 binding activity was similar in ZR-75 and HT1080 cells, whereas binding of factors to the E-MUC1 oligonucleotide revealed quantitative and qualitative differences between epithelial and non-epithelial cells.

Abstract: Increased synthesis of RecA protein is induced in E. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined in E. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). Purified E. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of the RecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5 - 50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes.

Abstract: Five fusions between mouse embryonic cells and syngeneic adult peritoneal macrophages were performed. The resulting hybrids as well as both parental cells (6 cultures of embryonal cells and 6 cultures or adult macrophages) were grown in vitro under the same culture conditions. All populations of explanted macrophages died during the second month in primary culture and five populations of cultured embryonic cells were lost within six months under in vitro conditions as well. One embryonic cell line survived and acquired transformed and/or malignant phenotype: When inoculated into either newborn or adult syngeneic mice, progressive growth of tumors with 100% take (6/6), histologically classified as poorly differentiated fibrosarcoma with areas of metaplastic bone and osteoid, was observed. Two out of five wild hybrid strains died within six months of cell culture. The resulting three hybrid cultures adapted themselves to in vitro conditions and finally permanent lines were established with all features of transformed phenotype in vitro and with the capacity to grow as undifferentiated fibrosarcomas with 100% take (6/6) when inoculated into syngeneic mice either sc. or i.p. Cytogenetic studies were performed and phenotypic characteristics of these lines were explored as well. Biological assays performed for the presence of oncogenic viruses were negative and none of the malignant cell lines showed positive staining with the monoclonal antibody specific for large T-antigen. It is suggested that cell fusion of two normal partners may switch on the cascade of abnormal processes which may culminate in neoplastic conversion. Cell fusion might play also a significant role in the so called "spontaneous" transformation.

Abstract: An attempt was made to induce production of antibodies against C-type endogenous rat retrovirus BP 6. Syngenic Lewis rats were immunized with viable tumor cells, induced with benzo(a)pyrene, continuously producing BP 6 virus. By use of immunoblotting technique, neither short- nor long-term immunization did cause formation of any detectable amounts of antibody against structural proteins of the virus. On the other hand, in immunoblotting, antibodies arised in rats immunized with purified mouse leukemia virus have been found to cross-react with endogenous rat retrovirus.

Abstract: To study the possible involvement of a murine leukemia virus (MuLV) related agent in human cancer, an extensive immunoblotting analysis of human sera (cancer, autoimmune as well as control normal ones) for the presence of antibodies to MuLV structural proteins was performed. Out of 350 sera, 89 reacted with gag precursor Pr65, 72 reacted with major viral core protein p30 and five with the matrix protein p15. Antibody reactivity to the env-encoded glycoprotein gp70 was detected in 7 cases out of 16 sera tested. There were no significant differences between pathological and normal sera concerning the patterns and the frequency of the reactivity. Sera from patients with various malignancies (mainly with breast cancer) generally displayed more intensive signals to MuLV p30 than normal sera. Epitope mapping revealed that MuLV p30-reactive antibodies recognize an antigenic determinant(s) located at the carboxyterminus of the protein.

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