Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae).

To study the relationship between uniparental rDNA (encoding 18S, 5.8S and 26S ribosomal RNA) silencing (nucleolar dominance) and rRNA gene dosage, we studied a recently emerged (within the last 80 years) allotetraploid Tragopogon mirus (2n=24), formed from the diploid progenitors T. dubius (2n=12, D-genome donor) and T. porrifolius (2n=12, P-genome donor). Here, we used molecular, cytogenetic and genomic approaches to analyse rRNA gene activity in two sibling T. mirus plants (33A and 33B) with widely different rRNA gene dosages. Plant 33B had ~400 rRNA genes at the D-genome locus, which is typical for T. mirus, accounting for ~25% of total rDNA. We observed characteristic expression dominance of T. dubius-origin genes in all organs. Its sister plant 33A harboured a homozygous macrodeletion that reduced the number of T. dubius-origin genes to about 70 copies (~4% of total rDNA). It showed biparental rDNA expression in root, flower and callus, but not in leaf where D-genome rDNA dominance was maintained. There was upregulation of minor rDNA variants in some tissues. The RNA polymerase I promoters of reactivated T. porrifolius-origin rRNA genes showed reduced DNA methylation, mainly at symmetrical CG and CHG nucleotide motifs. We hypothesise that active, decondensed rDNA units are most likely to be deleted via recombination. The silenced homeologs could be used as a 'first reserve' to ameliorate mutational damage and contribute to evolutionary success of polyploids. Deletion and reactivation cycles may lead to bidirectional homogenisation of rRNA arrays in the long term.

Dancing together and separate again: gymnosperms exhibit frequent changes of fundamental 5S and 35S rRNA gene (rDNA) organisation.

In higher eukaryotes, the 5S rRNA genes occur in tandem units and are arranged either separately (S-type arrangement) or linked to other repeated genes, in most cases to rDNA locus encoding 18S-5.8S-26S genes (L-type arrangement). Here we used Southern blot hybridisation, PCR and sequencing approaches to analyse genomic organisation of rRNA genes in all large gymnosperm groups, including Coniferales, Ginkgoales, Gnetales and Cycadales. The data are provided for 27 species (21 genera). The 5S units linked to the 35S rDNA units occur in some but not all Gnetales, Coniferales and in Ginkgo (∼30% of the species analysed), while the remaining exhibit separate organisation. The linked 5S rRNA genes may occur as single-copy insertions or as short tandems embedded in the 26S-18S rDNA intergenic spacer (IGS). The 5S transcript may be encoded by the same (Ginkgo, Ephedra) or opposite (Podocarpus) DNA strand as the 18S-5.8S-26S genes. In addition, pseudogenised 5S copies were also found in some IGS types. Both L- and S-type units have been largely homogenised across the genomes. Phylogenetic relationships based on the comparison of 5S coding sequences suggest that the 5S genes independently inserted IGS at least three times in the course of gymnosperm evolution. Frequent transpositions and rearrangements of basic units indicate relatively relaxed selection pressures imposed on genomic organisation of 5S genes in plants.

Mobilization of retrotransposons in synthetic allotetraploid tobacco.

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.

Frequent silencing of rDNA loci on the univalent-forming genomes contrasts with their stable expression on the bivalent-forming genomes in polyploid dogroses (Rosa sect. Caninae).

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.

Making a functional diploid: from polysomic to disomic inheritance.

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.

Reduced inducibility of SOCS3 by interferon gamma associates with higher resistance of human breast cancer lines as compared to normal mammary epithelial cells.

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

Interferon-alpha treatment may negatively influence disease progression in melanoma patients by hyperactivation of STAT3 protein.

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 immunotherapy in the clinic are therefore warranted.

The asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae) is associated with a skewed distribution of rRNA gene families in the gametes.

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 beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae).

BACKGROUND: In studies looking at individual polyploid species, the most common patterns of genomic change are that either genome size in the polyploid is additive (i.e. the sum of parental genome donors) or there is evidence of genome downsizing. Reports showing an increase in genome size are rare. In a large-scale analysis of 3008 species, genome downsizing was shown to be a widespread biological response to polyploidy. Polyploidy in the genus Nicotiana (Solanaceae) is common with approx. 40 % of the approx. 75 species being allotetraploid. Recent advances in understanding phylogenetic relationships of Nicotiana species and dating polyploid formation enable a temporal dimension to be added to the analysis of genome size evolution in these polyploids. METHODS: Genome sizes were measured in 18 species of Nicotiana (nine diploids and nine polyploids) ranging in age from <200,000 years to approx. 4.5 Myr old, to determine the direction and extent of genome size change following polyploidy. These data were combined with data from genomic in situ hybridization and increasing amounts of information on sequence composition in Nicotiana to provide insights into the molecular basis of genome size changes. KEY RESULTS AND CONCLUSIONS: By comparing the expected genome size of the polyploid (based on summing the genome size of species identified as either a parent or most closely related to the diploid progenitors) with the observed genome size, four polyploids showed genome downsizing and five showed increases. There was no discernable pattern in the direction of genome size change with age of polyploids, although with increasing age the amount of genome size change increased. In older polyploids (approx. 4.5 million years old) the increase in genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization signal was still detected in species exhibiting genome downsizing. The possible significance of these results is discussed.

Development of IFN-gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cells.

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.

Molecular cytogenetics and tandem repeat sequence evolution in the allopolyploid Nicotiana rustica compared with diploid progenitors N. paniculata and N. undulata.

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.

Preferential elimination of repeated DNA sequences from the paternal, Nicotiana tomentosiformis genome donor of a synthetic, allotetraploid tobacco.

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 S4-generation plants derived from a single synthetic, S0 plant made by Burk in 1973 (Th37). Using molecular and cytogenetic methods we analysed 14 middle and highly repetitive sequences that together total approximately 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.

Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin.

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.

Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana.

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

The origin and evolution of geminivirus-related DNA sequences in Nicotiana.

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.

Ribosomal DNA evolution and gene conversion in Nicotiana rustica.

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 18-5.8-26S 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 5' 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.

Distribution of 5-methylcytosine residues in 5S rRNA genes in Arabidopsis thaliana and Secale cereale.

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 C): mCG 90%/78%/85%, 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.

Evolution of 5S rDNA unit arrays in the plant genus Nicotiana (Solanaceae).

Nicotiana tabacum (tobacco, Solanaceae) has two 5S ribosomal DNA (rDNA) families, one of unit length approximately 646 bp and the other -430 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.

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species.

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 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S 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.

Hypomethylation of CNG targets induced with dihydroxypropyladenine is rapidly reversed in the course of mitotic cell division in tobacco.

We followed the mitotic transmission of an experimentally induced hypomethylated state of several tobacco repetitive sequences in callus culture and plants. The initial hypomethylation was induced by a hypomethylation drug, dihydroxypropyladenine (DHPA), the competitive inhibitor of cellular S-adenosylhomocysteine hydrolase, which is known to preferentially inhibit methylation at CNG and non-symmetrical motifs while having a negligible effect on methylation at CG motifs. The deprivation of this drug resulted in an almost immediate remethylation of cytosines at CNG motifs ( MspI and EcoRII sites) leading us to conclude that, the hypomethylation effect of dihydroxypropyladenine is rather transient and differs from that of 5-azacytidine which often induces heritable changes in methylation patterns. The results suggest that de novo methylation of CNG motifs is a rapid and meiotically independent process on DNA sequences with pre-existing CG methylation.