Cloning and analysis of a 6.8-kb rDNA intergenic spacer region of the European ash (Fraxinus excelsior L.)

The 6.8-kb rDNA intergenic spacer region of F. excelsior was isolated from a CsCl/actinomycin-D gradient and cloned into pUC18 for further characterization. We observed the presence of subrepeats delimited by HaeIII enzyme sites. These subrepeats were sub-cloned and 11 clones were sequenced. These corresponded to subrepeated elements of either 32 bp or 41 bp that shared a 23-bp common sequence in the 5 end. Within each family of subrepeats, the percentage of common nucleotides was 84.4% for the 5 32-bp subrepeats and 67.4% for the 640-bp subrepeats. Non-repeated HaeIII fragments of 450 bp and 650 bp were also sub-cloned. To compare homology at the IGS region between the rDNA spacers of F. excelsior and the three related species (F. oxyphylla, F. americana, F. ornus), we conducted Southern hybridization analyses using each member of the 32-bp and 40-bp subrepeat families and the unique 450-bp and 650-bp fragments as probes. These analyses indicated that (1) the American ash is more genetically distant from the other three species that the latter are from each other and (2) F. oxyphylla and F. excelsior are more closely related to each other than to F. ornus.     

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.     

Sequence subfamilies of satellite repeats related to rDNA intergenic spacer are differentially amplified on<Emphasis Type="Italic"> Vicia sativa</Emphasis> chromosomes

We cloned and sequenced the Vicia sativa 25S-18S rDNA intergenic spacer (IGS) and the satellite repeat S12, thought to be related to the spacer sequence. The spacer was shown to contain three types of subrepeats (A, B, and C) with monomers of 173 bp (A), 10 bp (B), and 66 bp (C), separated by unique or partially duplicated sequences. Two spacer variants were detected in V. sativa that differed in length (2990 and 3168 bp) owing to an extra copy of the subrepeat A. The A subrepeats were also shown to be highly homologous to the satellite repeat S12, which is located in large clusters on chromosomes 4, 5, and 6, and is not associated with the rDNA loci. Sequencing of additional S12 clones retrieved from a shotgun genomic library allowed definition of three subfamilies of this repeat based on minor differences in their nucleotide sequences. Two of these subfamilies could be discriminated from the rest of the S12 sequences as well as from the IGS A subrepeats using specific oligonucleotide primers that labeled only a subset of the S12 loci when used in the primed in situ DNA labeling (PRINS) reaction on mitotic chromosomes. These experiments showed that, in spite of the high overall similarity of the IGS A subrepeats and the S12 satellite repeats, there are S12 subfamilies that are divergent from the common consensus and are present at only some of the chromosomes containing the S12 loci. Thus, the subfamilies may have evolved at these loci following the spreading of the A subrepeats from the IGS to genomic regions outside the rDNA clusters.Electronic Supplementary Material Supplementary material is available in the online version of this article at Accession numbers: GenBank AY234364–AY234374. The monomer sequences and additional information about the family of IGS-like repeat S12 will also appear in the PlantSat database (Macas et al. 2002, ) under Accession name Vicia_sativa_IGS-like     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.     

Chromosomal polymorphism of ribosomal genes in the genus Oryza

The genes encoding for 18S–5.8S–28S ribosomal RNA (rDNA) are both conserved and diversified. We used rDNA as probe in the fluorescent in situ hybridization (rDNA-FISH) to localized rDNAs on chromosomes of 15 accessions representing ten Oryza species. These included cultivated and wild species of rice, and four of them are tetraploids. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci varies from one to eight among Oryza species. The rDNA locus located at the end of the short arm of chromosome 9 is conserved among the genus Oryza. The rDNA locus at the end of the short arm of chromosome 10 was lost in some of the accessions. In this study, we report two genome specific rDNA loci in the genus Oryza. One is specific to the BB genome, which was localized at the end of the short arm of chromosome 4. Another may be specific to the CC genome, which was localized in the proximal region of the short arm of chromosome 5. A particular rDNA locus was detected as stretched chromatin with bright signals at the proximal region of the short arm of chromosome 4 in O. grandiglumis by rDNA-FISH. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution. The possible mechanisms of cyto-evolution in tetraploid Oryza species are discussed.     

Patterns of variation in the intergenic spacers of ribosomal DNA in Drosophila melanogaster support a model for genetic exchanges during X-Y pairing

Detailed analysis of variation in intergenic spacer (IGS) and internal transcribed spacer (ITS) regions of rDNA drawn from natural populations of Drosophila melanogaster has revealed contrasting patterns of homogenization although both spacers are located in the same rDNA unit. On the basis of the role of IGS regions in X-Y chromosome pairing, we proposed a mechanism of single-strand exchanges at the IGS regions, which can explain the different evolutionary trajectories followed by the IGS and the ITS regions. Here, we provide data from the chromosomal distribution of selected IGS length variants, as well as the detailed internal structure of a large number of IGS regions obtained from specific X and Y chromosomes. The variability found in the different internal subrepeat regions of IGS regions isolated from X and Y chromosomes supports the proposed mechanism of genetic exchanges and suggests that only the "240" subrepeats are involved. The presence of a putative site for topoisomerase I at the 5' end of the 18S rRNA gene would allow for the exchange between X and Y chromosomes of some 240 subrepeats, the promoter, and the ETS region, leaving the rest of the rDNA unit to evolve along separate chromosomal lineages. The phenomenon of localized units (modules) of homogenization has implications for multigene family evolution in general.     

Preferential homogenization between adjacent and alternate subrepeats in wheat rDNA.

DNA from the "non-transcribed spacer" (NTS) of two wheat ribosomal RNA gene (rDNA) clones was sequenced. The regions flanking the internal subrepeat arrays are highly conserved between the two clones; the nucleotide sequence differ by less than one-half percent. In contrast, the consensus sequences of the subrepeats in the two arrays differ by three percent. Mutations unique to each array, yet found in more than one subrepeat of the array, are preferentially found in adjacent and alternate subrepeats. The similarity of the DNA sequences of the flanking regions is consistent with a model of homogenization among rDNA gene units by intergenic conversion. We propose that a different mechanism, preferential conversion between neighboring subrepeats, is largely responsible for the homogenization of subrepeats within an array.     

Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.     

Cytogenetic analysis in Polypterus ornatipinnis (Actinopterygii, Cladistia, Polypteridae) and 5S rDNA

Polypteridae is a family of archaic freshwater African fish that constitute an interesting subject for the study of the karyological evolution in vertebrates, on account of their primitive morphological characters and peculiar relationships with lower Osteichthyans. In this paper, a cytogenetic analysis on twenty specimens of both sexes of Polypterus ornatipinnis the ornate "bichir", coming from the Congo River basin, was performed by using both classical and molecular techniques. The karyotypic formula (2n=36; FN=72) was composed of 26 M+10 SM. The Alu I banding, performed to characterize heterochromatin in this species, was mainly centromeric. Both the chromosome location of the ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA(3) staining and FISH. CMA(3) marked all centromerical regions and showed the presence of two GC rich regions on the p arm of the chromosome pair n°1 and on the q arm of the pair n°14. Staining with Ag-NOR marked the only telomeric region of the chromosome n°1 p arm. After PCR, the 5S rDNA in this species was cloned, sequenced and analyzed. In the 665bp 5S rDNA sequence of P.ornatipinnis, a conserved 120bp gene region for the 5S rDNA was identified, followed by a non-transcribed variable spacer (NTS) which included simple repeats, microsatellites and a fragment of a non-LTR retrotransposon R-TEX. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair n°14, previously marked by CMA(3). FISH with 18S rDNA marked the telomeric region of the p arm of the pair n°1, previously marked both by Ag-NOR and CMA(3). The (GATA)(7) repeats marked the telomeric regions of all chromosome pairs, with the exclusion of the n°1, n°3 and n°14; hybridization with telomeric probes (TTAGGG)(n) showed signals at the end of all chromosomes. Karyotype evolution in Polypterus genus was finally discussed, including the new data obtained.     

Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (llpl.l) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5S rDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.     

Repeats and subrepeats in the intergenic spacer of rDNA from the nematode Meloidogyne arenaria

Summary Ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria are heterogeneous in size and appear to contain 5S rRNA gene sequences. Moreover, in a recA ⁺ bacterial host, plasmid clones of a 9 kb rDNA repeat show deletion events within a 2 kb intergenic spacer (IGS), between 28S and 5S DNA sequences. These deletions appear to result from a reduction in the number of tandem 129 by repeats in the IGS. The loss of such repeats might explain how rDNA length heterogeneity, observed in the Meloidogyne genome, could have arisen. Each 129 by repeat also contains three copies of an 8 by subrepeat, which has sequence similarity to an element found in the IGS repeats of some plant rDNAs.     

Physical mapping of the 18S.26S rRNA multigene family in common wheat: Identification of a new locus

In situ hybridization in conjunction with deletion mapping was used to map physically the 18S.26S multigene rRNA family in Triticum aestivum L. cv. Chinese Spring. Using in situ hybridization, we report a new locus in the 7DL arm of Chinese Spring and Aegilops squarrosa, and also confirm the nucleolus organizing region (Nor) locus in the short arm of chromosome 1A at the telomeric end in Chinese Spring. Based on in situ hybridization labeling patterns, we show that rDNA exists as condensed rDNA (heterochromatic) at each end and diffused rDNA within the secondary constriction region of the Nor-B1 (1B), Nor-B2 (6B) and Nor-D3 (5D) loci. In Nor-B1, 80% of the condensed rDNA domain lies in the proximal end and 20% in the distal end joined by diffuse rDNA threads. In Nor-B2, condensed rDNA is distributed evenly at each end joined by diffuse rDNA in the middle. In Nor-D3, the base of the satellite contains a greater concentration of condensed rDNA than the tip of the short arm. On the basis of these observations, we support the model that the usual state of rDNA is inactive (facultatively heterochromatic; Hilliker and Appels 1989). A small fraction of rDNA at a specific location (usually in the middle in wheat) exists as a diffuse region (active) in condensed chromosomes.by R. Appels     

Heterogeneity and organization of the ribosomal RNA genes ofCucurbita maxima

Thirty-six clones were recovered fromCucurbita maxima genomic DNA which had been enriched for rDNA and cleaved at the unique repeat unitHind III site. Twenty-nine of these, which contain complete rDNA units, were compared to a standard whose intergenic spacer (IGS) nucleotide sequence has been determined. Twenty-one are identical in length and restriction site pattern. Eight which differ from the standard in length do so because of addition or deletion of varying numbers of IGS subrepetitive units of two different classes, with four of the length variants being different in both of these classes. Seven clones were isolated which contain incomplete repeat units, six of which are composites of rDNA and non-rDNA material. They have been cleaved at the unique rDNAHind III site at one end and at a non-rDNAHind III site at the other. We consider it most likely that these are derived from the termini of repeat unit tandem arrays, although other explanations are possible. Twelve individual plants of two different cultivars were examined for heterogeneity of IGS length distribution. They all appear to be identical in this regard.     

Directed alterations of the X-chromosomal ribosomal gene repeats in mutant sc8 of Drosophila melanogaster

The patterns of the ribosomal DNA (rDNA) repeat units in seven Drosophila melanogaster inversional mutants have been studied. Among them, only the In(1)sc⁸ and its deletional derivative Df(1)mal¹² female rDNAs exibited significant reduction in the size of nearly all units, compared to the wild-type females (Canton S, Oregon R). Further investigation shows that each kind of repeat (insertion-free, insertion-containing) in the Xsc⁸ rDNA array is highly enriched with short (reduced to 4 kilobases) intergenic spacers (IGSs). We revealed two main types of rearrangements. Only part of the 4 kb IGSs display variable length deletions (0.2–0.6 kb) at the 5′ ends, within the so-called ‘1900’ base pair (bp) region, recognizable by restriction endonuclease AluI. The presence of additional 100–150 bp DNA in the start portion of this region has also been demonstrated. In contrast, the 3′ end spacer regions, corresponding to the external transcribed spacer, do not show any changes in size. These data indicate how reductions of approximately 1.1 kb DNAs in sc⁸ IGSs, carrying both the rearranged and non-rearranged ‘1900’ sequences, are achieved: the fixed decrease of a number of 240 bp AluI subrepeats, clustered in the central IGS portion, also contribute. None of the other similar inversional mutants examined has so many IGS variants. Therefore, alterations in the Xsc⁸ rRNA gene cluster seem not to be dependent on its inversional status. This revised version was published online in July 2006 with corrections to the Cover Date.     

Toward closing rice telomere gaps: mapping and sequence characterization of rice subtelomere regions

Despite the collective efforts of the international community to sequence the complete rice genome, telomeric regions of most chromosome arms remain uncharacterized. In this report we present sequence data from subtelomere regions obtained by analyzing telomeric clones from two 8.8 × genome equivalent 10-kb libraries derived from partial restriction digestion with HaeIII or Sau3AI (OSJNPb HaeIII and OSJNPc Sau3AI). Seven telomere clones were identified and contain 25–100 copies of the telomere repeat (CCCTAAA)_n on one end and unique sequences on the opposite end. Polymorphic sequence-tagged site markers from five clones and one additional PCR product were genetically mapped on the ends of chromosome arms 2S, 5L, 10S, 10L, 7L, and 7S. We found distinct chromosome-specific telomere-associated tandem repeats (TATR) on chromosome 7 (TATR7) and on the short arm of chromosome 10 (TATR10s) that showed no significant homology to any International Rice Genome Sequencing Project (IRGSP) genomic sequence. The TATR7, a degenerate tandem repeat which is interrupted by transposable elements, appeared on both ends of chromosome 7. The TATR10s was found to contain an inverted array of three tandem repeats displaying an interesting secondary folding pattern that resembles a telomere loop (t-loop) and which may be involved in a protective function against chromosomal end degradation.Electronic Supplementary Material Supplementary material is available for this article at