Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units

We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana 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 sites 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 11. 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. Received: 17 November 1999; in revised form: 3 February 2000 / Accepted: 3 February 2000     

Difference in strength of autonomously replicating sequences among repeats in the rDNA region of Saccharomyces cerevisiae

The rDNA region of Saccharomyces cerevisiae contains 100-200 tandemly repeated copies of a 9 kb unit, each with a potential replication origin. In the present studies of cloned fragments from the region involved in the regulation of replication of rDNA, we detected differences in autonomously replicating sequence (ARS) activity for clones from the same yeast strain. One clone, which showed very low ARS activity, carried a point mutation, a C instead of T, in position 9 of the essential 11 bp consensus ARS as compared to clones carrying the normal 10-of-11-bp match to the consensus. The mutation could be traced back to genomic rDNA where it represents about one-third of the rDNA units in that strain. Differences in ARS activity have implications for understanding the regulation of replication of rDNA, and the ratio of active to inactive ARS in the rDNA region may be important for potential generation of extrachromosomal copies.     

Babesia bovis, Babesia bigemina, Babesia canis, Babesia microti and Babesia rodhaini: comparison of ribosomal RNA gene organization.

The three ribosomal DNA (rDNA) units have been cloned from an Australian isolate of Babesia bigemina. The organization of the units is very similar to that reported for a Mexican isolate of B. bigemina. In Babesia canis four rDNA units have been identified. Both Babesia rodhaini and Babesia microti contain two different rDNA units. A small number of different rDNA units appears to be a common feature of this group of Protozoa. Restriction enzyme analysis of the rDNA units form these species and B. bovis suggests that the genus Babesia as currently defined does indeed include two distinct groups of organisms namely, B. bovis, B. bigemina and B. canis and B. rodhaini and B. microti.     

Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae.

Naturally occurring strains of Candida albicans are opportunistic pathogens that lack a sexual cycle and that are usually diploids with eight pairs of chromosomes. C. albicans spontaneously gives rise to a high frequency of colonial morphology mutants with altered electrophoretic karyotypes, involving one or more of their chromosomes. However, the most frequent changes involve chromosome VIII, which contains the genes coding for ribosomal DNA (rDNA) units. We have used restriction fragment lengths to analyze the number and physical array of the rDNA units on chromosome VIII in four normal clinical strains and seven morphological mutants derived spontaneously from one of the clinical isolates. HindIII does not cleave the rDNA repeats and liberates the tandem rDNA cluster from each homolog of chromosome VIII as a single fragment, whereas the cleavage at a single site by NotI reveals the size of the single rDNA unit. All clinical strains and morphological mutants differed greatly in the number of rDNA units per cluster and per cell. The four clinical isolates differed additionally among themselves by the size of the single rDNA unit. For a total of 25 chromosome VIII homologs in a total of 11 strains considered, the variability of chromosome VIII was exclusively due to the length of rDNA clusters (or the number of rDNA units) in approximately 92% of the cases, whereas the others involved other rearrangements of chromosome VIII. Only slight variations in the number of rDNA units were observed among 10 random C. albicans subclones and 10 random Saccharomyces cerevisiae subclones grown for a prolonged time at 22 degrees C. However, when grown faster at optimal temperatures of 37 and 30 degrees C, respectively, both fungi accumulated higher numbers of rDNA units, suggesting that this condition is selected for in rapidly growing cells. The morphological mutants, in comparison with the C. albicans subclones, contained a markedly wider distribution of the number of rDNA units, suggesting that a distinct process may be involved in altering the number of rDNA units in these mutants.     

Rapid evolution of parental rDNA in a synthetic tobacco allotetraploid line

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 × 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 S10, S11, and S12 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.     

Molecular Characterization of Two Types of rDNA Units in a Single Strain of Candida albicans

ABSTRACT. Strains of the opportunistic fungal pathogen Candida albicans vary in the presence or absence of a self‐splicing group I intron ribozyme (Ca.LSU) in the 25S rRNA gene on chromosome R. Strains of C. albicans typically either lack or contain this ribozyme. However, some strains have both intron‐containing and intronless rRNA genes (rDNA). Pulsed‐field gel electrophoresis analysis of undigested and restricted DNA showed at least six different karyotypes among eight independent colonies of such a heteroallelic strain. In each case, the variation was in chromosome R, and was due to changes in the number of rDNA units. In strains with only one type of rDNA, chromosome R also varied considerably. Polymerase chain reaction amplification spanning the rDNA unit demonstrated that intron‐containing rDNA units are tandemly arrayed, and are immediately adjacent to intronless units in the same cluster. Both types of units were present in the rDNA clusters of both R chromosomes. Possible explanations of these results are loss of Ca.LSU group I intron through purifying selection and/or a relaxation of the commonly accepted concerted evolution of the rDNA units.     

Comparison of the active and inactive chromatin structures of genes transcribed by RNA polymerases I and II

The coding sequences of the yeast 35S rDNA gene and of the yeast galactokinase gene both show clear staphylococcal nuclease nucleosome profiles under conditions in which the gene is inactive (galactokinase) or less active (rDNA). Under conditions of more active expression, the galactokinase gene shows marked smearing in the digestion profiles. The rDNA gene shows a qualitatively similar change in digestion patterns. There is a typical nucleosomal DNase I ladder on the coding sequences of both genes, regardless of the state of activity. In contrast to the coding sequences, the rDNA upstream region chromatin shows a nonnucleosomal profile. The nonnucleosomal character is more pronounced when the gene is more active. On the galactokinase upstream region chromatin, there is a nucleosomal structure, with some minor modifications, when the gene is inactive and a clear nonnucleosomal structure when the gene is expressed.     

Nuclear rDNA in Euglena gracilis: paucity of chromosomal units and replication of extrachromosomal units

Copy number of chromosomal rDNA units was investigated in two Euglena gracilis wild-type strains. It was established by dot blot analysis that these strains possess about four integrated units per haploid genome. This is the first example of a photosynthetic cell with only a few chromosomal ribosomal genes. In addition to these units, Euglena has 800 to 4000 extrachromosomal rDNA units. Electron microscopy revealed that these free rDNA circles bear a replication origin, and intermediates of replication show a D-loop structure.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA0 is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

Nucleolar rDNA folds into condensed foci with a specific combination of epigenetic marks

Arabidopsis thaliana 45S ribosomal genes (rDNA) are located in tandem arrays called nucleolus organizing regions on the termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, a crucial structural element of the ribosome. The current model of rDNA organization suggests that inactive rRNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while the nucleolus delineates active genes. We challenge the perspective that all intranucleolar rDNA is active by showing that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones that also contain the repressive H3K9me2 histone mark. By using plant lines containing a low number of rDNA copies, we further found that the condensed foci relate to the folding of rDNA, which appears to be a common mechanism of rDNA regulation inside the nucleolus. The H3K9me2 histone mark found in condensed foci represents a typical modification of bulk inactive rDNA, as we show by genome-wide approaches, similar to the H2A.W histone variant. The euchromatin histone marks H3K27me3 and H3K4me3, in contrast, do not colocalize with nucleolar foci and their overall levels in the nucleolus are very low. We further demonstrate that the rDNA promoter is an important regulatory region of the rDNA, where the distribution of histone variants and histone modifications are modulated in response to rDNA activity.