Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Unique sequence arrangement of ribosomal genes in the palindromic rDNA molecule of Physarum polycephalum.

R-loop and restriction mapping procedures reveal the organization of coding regions at each end of the giant rDNA palindrome of Physarum polycephalum. A 19S coding region of 2.10 +/- 0.21 kb is located at each end of a very long central spacer (35.64 +/- 2.08 kb). An internal spacer of 1.66 +/- 0.12 kb lies distal to the 19S gene. The 5.8S rRNA coding region is located in this spacer. The 26S gene lies distal to the internal spacer. The 26S gene is unusual among those of eukaryotes in that it consists of 3 coding regions (alpha, beta and gamma) interrupted by 2 intervening sequences. The 26S alpha (most central) coding segment of 2.41 +/- 0.33 kb is separated from the 26S beta segment by an intervening sequence of 0.68 +/- 0.13 kb. The 26S beta segment (0.70 +/- 0.11 kb) is separated from the most distal 26S gamma segment (0.59 +/- 0.14 kb) by an intervening sequence of 1.21 +/- 0.14 kb. The 2 intervening sequences are present in at least 88% of ribsomal genes from active plasmodia, indicating that genes containing these sequences are transcribed. The rDNA termini contain a heterogeneous region which varies in length by +/- 300 base pairs.     

A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes

The organization of the 18S, 28S and 5.8S rRNA genes in the mouse has been elucidated by mapping with restriction endonucleases Eco RI, Hind III and Bam HI. Ribosomal DNA fragments were detected in electrophoretically fractionated digests of total nuclear DNA by in situ hybridization with radioiodinated rRNAs or with complementary RNA synthesized directly on rRNA templates. A map of the rDNA which includes 13 restriction sites was constructed from the sizes of rDNA fragments and their labeling by different probes The map indicates that the rRNA genes lie within remarkably large units of reiterated DNA, at least 44,000 base pairs long. At least two, and possibly four, classes of repeating unit can be distinguished, the heterogeneity probably residing in the very large nontranscribed spacer region. The 5.8S rRNA gene lies in the transcribed region between the 18S and 28S genes.     

Arrangement of the rRNA sequences in the rDNA of Lytechinus variegatus

The arrangement of the 26S RNA and 18S RNA sequences of the ribosomal DNA (rDNA) from the sea urchin Lytechinus variegatus was investigated by an electron microscopic analysis of R-loops formed between the ribosomal RNA genes and the mature ribosomal RNAs. Ninety-eight percent of observed molecules contained R-loops clearly seen as a three-stranded complex. The size of DNA complementary to mature cytoplasmic 18S and 26S ribosomal RNA (rRNA) was calculated by measuring the double-strand (ds) and single-strand (ss) part of the R-loops separately. The values for the 18S R-loop are 1.75±0.24 kb¹ (ss) and 1.56±0.23 kb (ds). The 26S R-loop is 3.34±0.39 kb (ss) and 3.33±0.33 kb (ds). These measurements agree fairly well with the rRNA sizes measured on denaturing sucrose density gradients: 3.23±0.22 kb for the 26S and 1.93±0.10 kb for 18S. The short spacer between the 18S and 26S R-loops is 1.03±0.24 kb and the longer spacer is 5.36±0.53 kb. In long molecules a repeating pattern was observed. The average length of an rDNA repeat unit is 11.33±0.64 kb when computed using double-strand R-loop measurements and 11.50±0.72 when computed using R-loop single-strand lengths.Abbreviations kb kilobases, 1000 bases of RNA or single-strand DNA, and kilobase pairs, 1000 base pairs of duplex DNA or DNA/RNA hybrid - EDTA ethylenediaminetetraacetate - SSC 0.15 M NaCl, 0.015 M sodium citrate - PIPES piperazine-N,N-bis (2-ethanesulfonic acid)-Na_1.4     

Dynamics of 5S rDNA in the tilapia (Oreochromis niloticus) genome: repeat units,inverted sequences,pseudogenes and chromosome loci

In higher eukaryotes, the 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units composed of a coding region and a non-transcribed spacer sequence (NTS). These tandem arrays can be found on either one or more chromosome pairs. 5S rDNA copies from the tilapia fish, Oreochromis niloticus, were cloned and the nucleotide sequences of the coding region and of the non-transcribed spacer were determined. Moreover, the genomic organization of the 5S rDNA tandem repeats was investigated by fluorescence IN SITU hybridization (FISH) and Southern blot hybridization. Two 5S rDNA classes, one consisting of 1.4-kb repeats and another one with 0.5-kb repeats were identified and designated 5S rDNA type I and type II, respectively. An inverted 5S rRNA gene and a 5S rRNA putative pseudogene were also identified inside the tandem repeats of 5S rDNA type I. FISH permitted the visualization of the 5S rRNA genes at three chromosome loci, one of them consisting of arrays of the 5S rDNA type I, and the two others corresponding to arrays of the 5S rDNA type II. The two classes of the 5S rDNA, the presence of pseudogenes, and the inverted genes observed in the O. niloticus genome might be a consequence of the intense dynamics of the evolution of these tandem repeat elements.     

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae)

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.     

Structure of melon rDNA and nucleotide sequence of the 17–25S spacer region

Summary Restriction enzyme and hybridization analysis of melon nuclear DNA suggests a homogenous rDNA population with a repeat unit of 10.2 kb. Several full length Hind III rDNA repeat units were cloned and one of these is described in detail. The regions coding for 25S, 17S and 5.8S rRNAs were located by crossed-contact hybridization and R-loop mapping. Introns were not observed. The nucleotide sequence of the internal transcribed spacer and flanking regions was determined and compared with the corresponding region from rice rDNA by dot matrix analysis. In addition, the extent of gross sequence homology between cloned melon and pea rDNA units was determined by heteroduplex mapping.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Molecular Cytogenetics ofMusaSpecies, Cultivars and Hybrids: Location of 18S-5.8S-25S and 5S rDNA and Telomere-like Sequences

The physical sites of 18S-5.8S-25S and 5S rRNA genes and telomericsequences in theMusaL. genome were localized by fluorescentinsituhybridization on mitotic chromosomes of selected lines.A single major intercalary site of the 18S-5.8S-25S rDNA wasobserved on the short arm of the nucleolar organizing chromosomein each genome. AA and BB genome diploids had a single pairof sites, triploids had three sites while a tetraploid hybridhad four sites. The probe is useful for quick determinationof ploidy, even using interphase nuclei from slowly growingtissue culture material. Variation in the intensity of signalswas observed among heterogeneousMusalines indicating variationin the number of copies of the 18S-5.8S-25S rRNA genes. Eightsubterminal sites of 5S rDNA were observed in Calcutta 4 (AA)while Butohan 2 (BB) had six sites; some were weaker in bothgenotypes. Triploid lines showed six to nine major sites of5S rDNA of widely varying intensity and near the limit of detection.The diploid hybrids had five to nine sites of 5S rDNA whilethe tetraploid hybrid had 11 sites. The telomeric sequence wasdetected as pairs of dots at the ends of all the chromosomesanalysed but no intercalary sequences were seen. The molecularcytogenetic studies ofMusausing repetitive and single copy DNAprobes should yield insight into the genome and its evolutionand provide data forMusabreeders, as well as generating geneticmarkers inMusa.Copyright 1998 Annals of Botany Company Genome evolution, nucleolar organizing regions, telomeres,in situhybridization, genetic markers, banana, plantain.     

Study of a cloned ribosomal operon region coding for 5.8S rRNA in the loach Misgurnus fossilis L

A fragment of the loach (Misgurnus fossilis L.) ribosomal operon containing 5.8S rDNA and adjacent regions of the internal transcribed spacer (ITS-1, and ITS-2) was sequenced. The 5'-terminal sequencing in 5.8S rDNA was corrected by analysing the primary structure of the loach 5.8S rRNA. This RNA was shown to be presented by three types of molecules; one of these was shorter by 4 nucleotides at the 5'-end because of the processing site being shifted in the rRNA precursor. The two other types differed in the 5'-terminal nucleotide (UMP or AMP). In the cloned fragment under study, the sequence of 5.8S rDNA has TMP at the 5'-terminus. The known nucleotide sequences of 5.8S rRNAs were compared in eukaryotes; as a result, conservative regions were revealed at the sites of molecule modification. All the 5.8S rRNAs of the vertebrates studied were found to have coincidences in the localization of nucleotide substitutions and other mutations (inversions and deletions). The authors propose a model for the secondary structure of ITS-1 and ITS-2 in the region of 5.8S rRNA processing.     

The structure of the yeast ribosomal RNA genes. 4. Complete sequence of the 25 S rRNA gene from Saccharomyces cerevisae.

The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.     

Cloning in a cosmid vector of complete 37 kb and 25 kb ribosomal DNA repeat units from the chicken

DNA fragments of up to 40 kb containing rRNA-coding sequences have been isolated from a chicken liver DNA library prepared in the cosmid pHC79. Characterization of the cloned DNA by R-loop and restriction mapping has shown that there are two size classes of repeat unit, one of 37 kb and one of 25 kb, the larger of which is a family of units which vary slightly in size. These two classes were shown to be present in the DNA of a single chicken. The size of the internal transcribed spacer in the chicken was measured to be 4.4 kb from analysis of R-loops and heteroduplexes between chicken and Xenopus laevis rDNAs. No introns were observed in either the 18 S or the 28 S coding sequences. The number of copies of the chicken rDNA unit was measured by titration against the cloned sequences to be 202±51 per haploid genome.     

Sequence arrangement of the 16S and 26S rRNA genes in the pathogenic haemoflagellate Leishmania donovani.

Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 X 10(7) Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, U3% middle repetitive DNA with an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be 166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively. The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order given: (a) A 16S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short sequence that may code for a 5.8S rRNA, (C) 26S gene with a length of 4.31 Kb which contains an internal gap region of length 0.581 Ib, (d) An external spacer of average length 5.85 Kb.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.