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     

Denaturation mapping of the ribosomal DNA of Saccharomyces cerevisiae

Summary The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a T_m of 80.6°C and the second phase has a T_m of 87.3°C, corresponding to GC contents of 28 and 44%, respectively. The T_m of the nonribosomal nuclear DNA, called DNA, is 85.7°C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6×10⁶ dalton rDNA SmaI restriction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions.A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array.     

Phylogenetic relationships of coffee-tree species (Coffea L.) as inferred from ITS sequences of nuclear ribosomal DNA

 Phylogenetic relationships of Coffea species were estimated from the sequences of the internal transcribed spacer (ITS 2) region of nuclear ribosomal DNA. The ITS 2 region of 37 accessions belonging to 26 Coffea taxa and to three Psilanthus species was directly sequenced from polymerase chain reaction (PCR)-amplified DNA fragments. The level of variation was high enough to make the ITS 2 a useful tool for phylogenetic reconstruction. However, an unusual level of intraspecific variation was observed leading to some difficulty in interpreting rDNA sequence divergences. Sequences were analysed using Wagner parsimony as well as the neighbour-joining distance method. Coffea taxa were divided into several major groups which present a strong geographical correspondence (i.e. Madagascar, East Africa, Central Africa and West Africa). This organisation is well supported by cytogenetic evidence. On the other hand, the results were in contradiction with the present classification of coffee-tree taxa into two genera, namely Coffea and Psilanthus. Furthermore, additivity of parental rDNA types was not observed in the allotetraploid species C. arabica. Received: 25 July 1996 / Accepted: 18 October 1996     

Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Phylogenetic Analysis and Karyotype Evolution in the Genus Clivia(Amaryllidaceae)

Phylogenetic relationships of five taxa of Clivia, one probablenew species plus four recognized species, and three outgroupspecies were studied using sequences of the nuclear ribosomal5S non-transcribed spacer and the internal transcribed spacer(ITS) of 45S rDNA. Analysis of the data sets separately generatedsome well-supported groupings and congruent phylogenies. Cliviaminiata and C. gardenii are closely related. ‘Robust Gardenii’,the putative new species, is a sister clade of this group. Clivianobilis is distantly related to these three taxa and C. caulescensoccupies an intermediate position between the two groups. Chromosomelocations and distribution patterns of the 5S nuclear ribosomalgene in the species of Clivia were investigated using fluorescenceinsitu hybridization (FISH). In all species, only one pair of5S rDNA signals was observed. These were located on the shortarm of chromosome 8, at the position of the interstitial C-bands.The phylogenies obtained from the DNA sequences together withthe chromosome data accumulated here and previously publishedinformation on the location of the 45S rDNA sites have beenused to postulate evolutionary trends in Clivia chromosomes.Copyright 2001 Annals of Botany Company Clivia, chromosome evolution, 45S and 5S rDNA, ITS, FISH, molecular phylogeny     

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.     

Multiple Independent Losses of Photosynthesis and Differing EvolutionaryRates in the Genus Cryptomonas (Cryptophyceae): Combined Phylogenetic Analyses of DNA Sequences of the Nuclear and the Nucleomorph Ribosomal Operons

In this study, evidence for at least three independent losses of photosynthesis in the freshwater cryptophyte genus Cryptomonas is presented. The phylogeny of the genus was inferred by molecular phylogenetic analyses of the nuclear internal transcribed spacer 2 (nuclear ITS2), partial nuclear large subunit ribosomal DNA (LSU rDNA), and nucleomorph small subunit ribosomal DNA (SSU rDNA, NM). Both concatenated and single data sets were used. In all data sets, the colorless Cryptomonas strains formed three different lineages, always supported by high bootstrap values (maximum parsimony, neighbor joining and maximum likelihood) and posterior probabilities (Bayesian analyses). The three leukoplast-bearing lineages displayed differing degrees of accelerated evolutionary rates in nuclear and nucleomorph rDNA. Also an increase in A+T-content in highly variable regions of the nucleomorph SSU rDNA was observed in one of the leukoplast-bearing lineages.This article contains three online-only supplementary tables.Reviewing Editor: Dr. Yves Van de Peer     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

Molecular systematics of Solanum section Lycopersicum (Lycopersicon) using the nuclear ITS rDNA region

The phylogenetic relationships of all nine known tomato species of Solanum section Lycopersicum, together with other Solanum sections and species from several related genera, were investigated using parsimony analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA). Most parsimonious reconstructions divided the section Lycopersicum into three clades, reflecting their mating behaviour and fruit colour. Data from sequencing studies were congruent with those from morphological and other molecular investigations, and provided detailed information concerning species relationships. Received: 11 July 2000 / Accepted: 17 April 2001     

Identification of five Orius species in Japan by multiplex polymerase chain reaction

We developed multiplex polymerase chain reaction methods to identify five Orius (Heteroptera: Anthocoridae) species that occur commonly in Japan: Orius sauteri, Orius minutus, Orius strigicollis, Orius nagaii, and Orius tantillus. The method amplified internal transcribed spacer 1 of the nuclear ribosomal DNA by using five primers simultaneously and produced species-specific banding patterns upon agarose gel electrophoresis. Reliability of the method was tested for 350 individuals of 23 strains, and consistent results were obtained. Dichotomous keys are also provided for easy and quick species identification.     

Ribosomal RNA genes in species of theCynareae tribe (Compositae). II

Summary The structure of the ribosomal DNA has been analyzed in three species of theCynareae tribe using Southern blot hybridization.Silybum marianum possesses two types of ribosomal genes 12.3 and 13.4 kb long;Cirsium vulgare has at least four types of rDNA repeats 10.6, 10.5, 11.7 and 11.9 kb long;Carlina acaulis only one type of ribosomal genes 9.7 kb long. The rRNA genes of the three species studied showed an identical restriction mapping in the 18 S and 25 S regions. However species differentiation in length and/or nucleotide sequences are present in the external spacer and very probably in the internal transcribed spacer.By cytophotometric studies and byin vitro rRNA/DNA filter hybridization, the DNA amount/4 C nucleus and the rDNA percentage were calculated in nine species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Cynara cardunculus subsp.cardunculus (wild artichoke),Onopordon acanthium, O. illyricum, Galactites tomentosa, Carduus nutans, Silybum marianum, Cirsium vulgare andCarlina acaulis. The DNA amount/4 C nucleus in eight species are similar, ranging from 4.24 pg inGalactites tomentosa to 5.96 pg inCirsium vulgare, whileCarlina acaulis has a DNA amount/ 4C nucleus of 11.8 pg. The rDNA percentages range from 0.192% inOnopordon acanthium to 1.022% inSilybum marianum, whileCarlina acaulis has 0.038% of rDNA. This latter species is clearly distinct within theCynareae tribe.     

Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal dna sequences

Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (34–45%) and second (50–53%) internal transcribed spacers among the species was greater than in the 5.8S gene (3–5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern (s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.     

The organization of ribosomal genes in vertebrates

The organization of the repeat unit of the ribosomal genes has been determined in 15 different species of vertebrates. The EcoRI and BamHI restriction maps of the rDNA from single individuals of different species of fishes, amphibians, and reptiles have been analysed. Two rDNA clones from Xenopus laevis (representing one complete repeat unit) were used as probes in Southern blots to detect restriction fragments containing ribosomal genes. The results obtained indicate that the transcribed regions are highly conserved in length and sequence inside the same zoological class. These regions are less conserved when species from different classes are compared but a general trend has been observed. In contrast, the length and sequence of the spacer regions are very variable, even within the same zoological class. Different types of heterogeneity have been observed; examples range from a single type of ribosomal repeat unit within a species to the absence of any detectable regular tandem array of units.     

Development of Multiplex PCR to Detect Five Pythium Species Related to Turfgrass Diseases

The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species‐specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.     

Genotyping of a Miso and Soy Sauce Fermentation Yeast,Zygosaccharomyces rouxii,Based on Sequence Analysis of the Partial 26S Ribosomal RNA Gene and Two Internal Transcribed Spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

A multigene phylogeny of theGibberella fujikuroi species complex: Detection of additional phylogenetically distinct species

Phylogenetic relationships within theGibberella fujikuroi species complex were extended to newly discovered strains using nucleotide characters obtained by sequencing polymerase chain reaction (PCR) amplified DNA from 4 loci used in a previous study [nuclear large subunit 28S rDNA, nuclear ribosomal internal transcribed spacer (ITS) region, mitochondriaal small subunit (mtSSU) ribosomal DNA, and β-tubulin] together with two newly sampled protein-encoding nuclear genes, translation elongation factor EF-1α and calmodulin. Sequences from the ribosomal ITS region were analyzed separately and found to contain of two highly divergent, nonorthologous ITS2 types. Phylogenetic analysis of the individual and combined datasets identified 10 new phylogenetically distinct species distributed among the following three areas: 2 within Asia and 4 within both Africa and South America. Hypotheses of the monophyly ofFusarium subglutinans and its two formae speciales, f. sp.pini and f. sp.ananas, were strongly rejected by a likelihood analysis. Maximum parsimony results further indicate that the protein-encoding nuclear genes provide considerably more phylogenetic signal that the ribosomal genes sequenced. Relative apparent synapomorphy analysis was used to detect long-branch attraction taxa and to obtain a statistical measure of phylogenetic signal in the individual and combined datasets.     

EVOLUTION OF RIBOSOMAL DNA: FIFTY MILLION YEARS OF RECORDED HISTORY IN THE FROG GENUS RANA

Evolution of nuclear ribosomal DNA (rDNA) arrays of frogs of the genus Rana was examined among 32 species that last shared a common ancestor approximately 50 million years ago. Extensive variation in restriction sites exists within the transcribed and nontranscribed rDNA spacer regions among the species, whereas rDNA coding regions exhibit comparatively little interspecific variation in restriction sites. The most parsimonious phylogenetic hypothesis for the evolution of the group was constructed based on variation in restriction sites and internal spacer lengths among the 32 species of Rana and one species of Pyxicephalus (examined for outgroup comparison). This analysis suggests that R. sylvatica of North America is more closely related to the R. temporaria group of Eurasia than to other North American Rana. The hypothesized phylogeny also supports the monophyly of the R. boylii group, the R. catesbeiana group, the R. palmipes group, the R. tarahumarae group, and the R. pipiens complex. Furthermore, the restriction site data provide information about the evolution within and among these species groups. This demonstrates that restriction site mapping of rDNA arrays provides a useful molecular technique for the examination of historical evolutionary questions across considerable periods of time.     

The evolution of genome size and rDNA in diploid species of Chenopodium s.l. (Amaranthaceae)

The evolution of genome size and ribosomal DNA (rDNA) locus organization was analysed in 23 diploid species of Chenopodium s.l., all of which share the same base chromosome number of x = 9. Phylogenetic relationships among these species were inferred from plastid and nuclear ribosomal internal transcribed spacer (nrITS) DNA sequences. The molecular phylogenetic analyses assigned all analysed species of Chenopodium s.l. to six evolutionary lineages, corresponding to the recent new generic taxonomic treatment of Chenopodium s.l. The distribution of rDNA loci for four species is presented here for the first time using fluorescence in situ hybridization (FISH) with 5S and 35S rDNA probes. Most of the 23 analysed diploid Chenopodium spp. possessed a single subterminally located 35S rDNA locus, except for three species which possessed two 35S rDNA loci. One or two 5S rDNA loci were typically localized subterminally on chromosomes, rarely interstitially. Analyses of rDNA locus numbers in a phylogenetic context resulted in the reconstruction of one locus each of 35S rDNA and 5S rDNA, both in subterminal positions, as the ancestral state. Genome sizes determined using flow cytometry were relatively small (2C value < 2.8 pg), ranging from 0.734 pg in C. schraderianum to 2.721 pg in C. californicum (nearly four‐fold difference), and were often conserved within major phylogenetic lineages, suggesting an adaptive value. The reconstructed ancestral genome size was small for all evolutionary lineages, and changes have probably coincided with the divergence of major lineages. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 218–235.