An Unspliced Group I Intron in 23S rRNA Links Chlamydiales, Chloroplasts, and Mitochondria

Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU. 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae) – re-examination by FISH

Chromosomal polymorphism regarding the number of chromosomal NOR sites in the cyprinid fish Chondrostoma lusitanicum reported previously (Rodrigues & Collares-Pereira, 1996) was re-examined using fluorescence in situ hybridization (FISH) with a ribosomal DNA (rDNA) probe. All positive CMA3-bands contained ribosomal DNA documented by either two or four FISH positive signals in the respective karyotypes. This polymorphism suggests the occurrence of structural rearrangements of translocation type in rDNA region from one ancestral NOR-bearing chromosome pair ubiquitous among leuciscine cyprinid fishes to another pair. The absence of individuals heterozygous for this polymorphism is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular cytogenetics of the ribosomal (18S + 28S and 5S) DNA loci in primitive and advanced urodele amphibians.

In the present work we performed a cytogenetic analysis of the ribosomal (18S + 28S and 5S) loci in amphibian species belonging to the advanced family Salamandridae (genera Triturus, Salamandra, and Salamandrina) and in the primitive hynobiid Salamandrella keyserlingii (family Hynobiidae). In each analyzed karyotype the 5S rDNA sites appear to be stable, and definite in number, while an intraspecific variability both in number and chromosomal location of the 18S + 28S rDNA loci has been found in some Triturus species. In particular, an evolutionary trend toward a large intraspecific variability of the 18S + 28S rDNA loci has been found in the T. vulgaris species group. A structural analysis of the ribosomal repetition units demonstrates the occurrence of a length polymorphism within the 18S + 28S rDNA repeats in the examined species of the family Salamandridae; however, this polymorphism is rather limited, even in those Triturus species characterized by high intragenomic variability of the ribosomal sites. We show that in T. vulgaris meridionalis the variant repetition units actually segregate with individual chromosomes. This implies that they are not intermingled in the ribosomal clusters.     

Cloning and comparative characterization of the rIGS regulatory regions in humans and the pygmy chimpanzee Pan paniscus

Investigation of randomly cloned genomic and chromosome-specific sequences of ribosomal DNA (rDNA) from different organisms show that different regions of this long repeat unit evolve at different rates. This proves to be true not only with regard to evolutionary variability of transcribed and nontranscribed intergenic (spacer) regions of rDNA. The intergenic spacer (rIGS) of human ribosomal DNA contains both highly variable and more conservative regions with putative regulatory functions. In the present study a comparative analysis of some segments of the rIGS pre-promoter (regulatory) region in human and pygmy chimpanzee (Pan paniscus) was carried out. For these purposes, the corresponding DNA fragments were amplified in PCR with oligonucleotide primers specific to human rIGS and sequenced. Our results show that at the background of substantial structural similarity of these regions in man and chimpanzee, i.e., the presence of highly homologous sequences and similar repetitive units, there are substantial differences between them. These differences are associated with point mutations, insertions, deletions, and complex structural rearrangements.     

Cloning and Comparative Characterization of the rIGS Regulatory Regions in Human and Pygmy Chimpanzee Pan paniscus

Investigation of randomly cloned genomic and chromosome-specific sequences of ribosomal DNA (rDNA) from different organisms show that different regions of this long repeat unit evolve at different rates. This proves to be true not only with regard to evolutionary variability of transcribed and nontranscribed intergenic (spacer) regions of rDNA. The intergenic spacer (rIGS) of human ribosomal DNA contains both highly variable and more conservative regions with putative regulatory functions. In the present study a comparative analysis of some segments of the rIGS pre-promoter (regulatory) region in human and pygmy chimpanzee (Pan paniscus) was carried out. For these purposes, the corresponding DNA fragments were amplified in PCR with oligonucleotide primers specific to human rIGS and sequenced. Our results show that at the background of substantial structural similarity of these regions in man and chimpanzee, i.e., the presence of highly homologous sequences and similar repetitive units, there are substantial differences between them. These differences are associated with point mutations, insertions, deletions, and complex structural rearrangements.     

Ribosomal genes in notothenioid fishes: Focus on the chromosomal organisation

This mini-review makes a survey and a summary of some major issues concerning the chromosomal organisation of ribosomal genes in fish genomes, by using Notothenioidei as the model. The increasing body of information, published during the last two decades on the chromosomal mapping of the two ribosomal genes classes (45S rDNA and 5S rDNA) in notothenioids, makes it possible to recognise the main evolutionary trends across the phylogeny of the group. As one of the major features, the rDNA clusters are organised in a single chromosomal locus in most of the species. This locus is located at different positions along the chromosomes in the basal groups (non-Antarctic Clade), whereas it maintains a strongly conserved location in the cold-adapted species (Antarctic Clade). Important structural changes, leading to the co-localisation of the two ribosomal gene classes, occurred early in the notothenioid phylogeny, perhaps in the common ancestor of the Eleginopidae and Nototheniidae. The cytogenetic evidences indicate that an increased amount of ribosomal genes, organised in two large chromosomal loci, is present in the giant Antarctic fish Dissostichus mawsoni. This gain in rRNA genes is an important genomic change, having possible implications for the fitness of this notothenioid fish that combines large size, pelagic lifestyle and cold-adaptation.     

Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes.

Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.     

Cloning of tomato nuclear ribosomal DNA. rDNA organization in leaves and suspension-cultured cells

The rDNA fragments of EcoRI digested tomato nuclear DNA were detected by cross-hybridization with cloned Aspergillus nidulans rDNA. Two fragments coding for 18S and 25S rRNA, respectively were cloned and characterized. They were used to study structural organization of rRNA genes of a tomato cell suspension culture by hybridization with specifically cleaved nuclear DNA. The repeating units found were variable in restriction sites and in size with a basic unit of 8.6 kbp. Additionally, the analysis clearly demonstrates that nuclear DNA isolated from tomato leaves (Lycopersicon esculentum cv. Lukullus) and cultured cells derived therefrom bear significant differences in the structural organization of their ribosomal DNA.     

The ribosomal DNA plasmids of entamoeba

In most organisms, the nuclear ribosomal RNA (rRNA) genes are highly repetitive and arranged as tandem repeats on one or more chromosomes. In Entamoeba, however, these genes are located almost exclusively on extrachromosomal circular DNA molecules with no clear evidence so far of a chromosomal copy. Such an uncommon location of rRNA genes may be a direct consequence of cellular physiology, as suggested by studies with Saccharomyces cerevisiae mutants in which the rDNA is extrachromosomal. In this review, Sudha Bhattacharya, Indrani Som and Alok Bhattacharya summarize current knowledge on the structural organization and replication of the Entamoeba rDNA plasmids. Other than the rRNAs encoded by these molecules, no protein-coding genes (including ribosomal protein genes) are found on any of them. They are unique among plasmids in that they do not initiate replication from a fixed origin but use multiple sites dispersed throughout the molecule. Further studies should establish the unique biochemical features of Entamoeba that lead to extrachromosomal rDNA.     

Karyotype,chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

Karyotype and chromosomal characteristics, i.e. number and location of ribosomal DNA (rDNA) clusters, and sequence variation of the ribosomal internal transcribed spacer 2 (ITS2) were studied in a monozoic (unsegmented) tapeworm, Caryophyllaeides fennica (Caryophyllidea), using conventional and Ag-staining, fluorescent in situ hybridization (FISH) with 18S rDNA probe, and PCR amplification, cloning and sequencing of the complete ribosomal ITS2 spacer. The karyotype of this species was composed of ten pairs of metacentric (m) chromosomes (2n = 20). All chromosomes except the pair No. 2 displayed DAPI-positive heterochromatin in centromeric regions. In addition, two distinct interstitial DAPI-positive bands were identified on chromosome pair No. 7. FISH with 18S rDNA probe revealed four clusters of major ribosomal genes situated in the pericentromeric region of the short arms in two pairs of metacentric chromosomes Nos. 8 and 9. Hybridization signals were stronger in the pair No. 8, indicating a higher amount of rDNA repeats at this nucleolar organizer region (NOR). Analysis of 15 ITS2 rDNA sequences (five recombinant clones from each of three individuals) showed 13 structurally different ribotypes, distinguished by 26 nucleotide substitutions and variable numbers and combinations of short repetitive motifs that allowed sorting the sequences into four ITS2 variants. These results contribute to recently published evidence for the intraindividual ribosomal ITS sequence variability in basal tapeworms with multiple rDNA loci and imply that both phenomena may be mutually linked.     

Chromosomal localization of 5S and 45S ribosomal DNA in species of Linum L. section Linum (syn=Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton