Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.     

Isolation and characterization of rat ribosomal DNA clones

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.     

Organization of the ribosomal RNA gene cluster in Aspergillus nidulans

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.     

A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

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.     

Cloning and characterization of the rDNA repeat unit of Podospora anserina

Summary DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.     

Polymorphism of genes coding for nuclear 18S rRNA indicates genetic distinctiveness of anastomosis group 10 from other groups in the Rhizoctonia solani species complex.

DNA polymorphism in the 18S nuclear rRNA gene region was investigated by using 11 restriction endonucleases for 161 isolates of 25 intraspecific groups (ISGs) representing 11 reported anastomosis groups (AGs) of Rhizoctonia solani. A PCR-based restriction mapping method in which enzymatically amplified DNA fragments and subfragments were digested with one or two restriction enzymes was employed. Four types of DNA restriction maps of this region were constructed for these 25 ISGs. Map type I of the 18S rDNA region was represented by isolates of a majority of R. solani ISGs. Map types II and III, represented by ISG 2E and 9 isolates and 5C isolates, respectively, differed from map I by the absence of one (map type II) or two (map type III) restriction sites. Map type IV, represented by ISG 10A and B (or AG 10) isolates, showed significant restriction site variations, with five enzymes in this region compared with those of the remaining ISGs or AGs. Ten of the 25 restriction sites in the 18S rRNA gene region were informative and selected for analysis. Previously reported restriction maps of the 5.8S rRNA gene region, including the internal transcribed spacers, were aligned with each other, and 12 informative restriction sites were identified. These data were used alone and in combination to evaluate group relationships. Analyses derived from these data sets by maximum parsimony and likelihood methods showed that AG 10 isolates were distinct and distantly related to the majority isolates of the other AGs of this species complex.     

Characterisation of the genes for ribosomal RNA in flax

DNA coding for the 18S and 25S rRNAs in flax (Linum usitatissimum) has been purified and is arranged in tandem arrays with a repeat length of 8.6 kb. There is no detectable variation in the size of this repeat unit. Single repeat units have been cloned in the plasmid pAT 153. The coding sequences for the 25S, 18s and 5.85 rRNAs have been localised by hybridisation. The cloned rDNA has been used to compare two genotrophs, L1 ad S1, where the number of rRNA cistrons has been altered by growth under different environmental conditions. In terms of the size of the repeat unit and the position of a number of restriction enzyme sites the rDNAs from L1 and S1 were identical.     

Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals

The chromosomal locations of ribosomal DNA in wheat, rye and barley have been determined by in situ hybridization using high specific activity ¹²⁵I-rRNA. The 18S-5.8S-26S rRNA gene repeat units in hexaploid wheat (cv. Chinese Spring) are on chromosomes 1B, 6B and 5D. In rye (cv. Imperial) the repeat units occur at a single site on chromosome 1R(E), while in barley (cv. Clipper) they are on both the chromosomes (6 and 7) which show secondary constrictions. In wheat and rye the major 5S RNA gene sites are close to the cytological secondary constrictions where the 18S-5.8S-26S repeating units are found, but in barley the site is on a chromosome not carrying the other rDNA sequences. — Restriction enzyme and R-loop analyses showed the 18S-5.8S-26S repeating units to be approximately 9.5 kb long in wheat, 9.0 kb in rye and barley to have two repeat lengths of 9.5 kb and 10 kb. Electron microscopic and restriction enzyme data suggest that the two barley forms may not be interpersed. Digestion with EcoR1 gave similar patterns in the three species, with a single site in the 26S gene. Bam H1 digestion detected heterogeneity in the spacer regions of the two different repeats in barley, while in rye and wheat heterogeneity was shown within the 26S coding sequence by an absence of an effective Bam H1 site in some repeat units. EcoR1 and Bam H1 restriction sites have been mapped in each species. — The repeat unit of the 5S RNA genes was approximately 0.5 kb in wheat and rye and heterogeneity was evident. The analysis of the 5S RNA genes emphasizes the homoeology between chromosomes 1B of wheat and 1R of rye since both have these genes in the same position relative to the secondary constriction. In barley we did not find a dominant monomer repeat unit for the 5S genes.     

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp

A library of genomic DNA from the brine shrimp, Artemia, has been constructed with the Charon 4A phage vector, utilizing EcoRI passenger fragments. Screening this library with purified Xenopus laevis cloned rDNA genes has resulted in the identification and plaque purification of a recombinant containing a complete Artemia (18 S + 26 S) rDNA repeat unit. A physical map derived from the analysis of restriction endonuclease digests of the repeat unit, which measures 13.9 kilobase pairs, is similar to the map derived from genomic DNA. In common with several other species, the 26 S rRNA gene terminates with a HindIII recognition site.     

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.     

A restriction map of the ribosomal RNA genes and the short single-copy DNA sequence of the pearl millet chloroplast genome

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.     

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Fragments of rDNA3 from Drosophila melanogaster produced by the restriction endonuclease EcoRI were cloned in the form of recombinant plasmids in Escheriehia coli. Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single EcoRI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by EcoRI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.