rDNA Repeat Unit Research Articles

Requirements for activity of the yeast mitotic recombination hotspot HOT1: RNA polymerase I and multiple cis-acting sequences.

When inserted at novel locations in the yeast genome, the Saccharomyces cerevisiae recombination hotspot HOT1 stimulates mitotic exchange in adjacent sequences. HOT1 is derived from the rDNA repeat unit, and the sequences required for the recombination-stimulatory activity closely correspond to the rDNA transcription enhancer and initiation site, suggesting there is an association between high levels of RNA polymerase I transcription and increased recombination. To directly test whether RNA polymerase I is essential for HOT1 activity, a subunit of RNA polymerase I was deleted in a strain in which rRNA is transcribed by RNA polymerase II. HOT1 is completely inactive in this strain. Deletion analysis and site-directed mutagenesis were used to further define the sequences within the rDNA enhancer required for HOT1 activity. These studies show that the enhancer contains at least four distinct regions that are required for hotspot activity. In most cases mutations in these regions also decrease transcription from this element, further confirming the association of recombination and transcription.

Electrophoretically-detected allozyme variation reveals only moderate differentiation between Chinese and Philippine Schistosoma japonicum

A number of discrete strains of Schistosoma japonicum have been described of which those from China and the Philippines are the most important in terms of public health significance. The strains have previously been shown to differ in a number of biological characteristics and isoenzyme analysis has established that Chinese and Philippine S. japonicum are, especially, quite distinct. As few direct genetic differences have been identified, with nuclear and mitochondrial genetic markers being shown earlier to be highly conserved between the two forms (Bowles et al., 1993), enzyme electrophoresis was performed to re-evaluate whether the two strains are as genetically diverse as field and laboratory maintained isolates previously examined using this approach. Some allelic differences were evident between Chinese and Philippine S. japonicum but the genetic distance (a measure of genetic relatedness), D = 0.272, between the two strains was, however, considerably smaller than the earlier obtained estimates of D > 0.4–0.575. In addition to the enzyme studies, restriction patterns, obtained using 4-base cutting restriction enzymes and hybridisation with DNA probes comprising the entire ribosomal DNA (rDNA) repeat unit of S. mansoni, were indistinguishable between the Chinese and Philippine forms of S. japonicum, thereby re-emphasising their remarkable similarity at the genetic level.

Biological and genetic analysis of a longitudinal collection of Giardia samples derived from humans

Duodenal aspirates from children investigated for diarrhoea have been examined for the presence of Giardia over an eleven year period, and where possible, in vitro or in vivo Giardia cultures in mice were established. Based on biochemical characteristics of electrophoretic karyotype, RFLP analysis and rDNA hybridization studies of 40 stocks at least two major varieties, or demes, of Giardia have infected the population of South East Queensland and environs during this period. These demes carried different rDNA repeat units and differed markedly in both the electrophoretic karyotype pattern and the molar representation of chromosome bands. From 1983 to 1991 only one deme was documented. The first evidence of a new deme seen in local children occurred in 1991 and was followed by a predominance of this deme in 1993. These 40 stocks do not represent all positive samples. Other stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks were derived from children with similar chronic symptoms it appears that at least two demes of Giardia are pathogenic.

Use of virulence and length variability within the rDNA repeat unit to distinguish isolates ofPuccinia graminis f. sp. triticirace QCC

Use of virulence and length variability within the rDNA repeat unit to distinguish isolates of<i>Puccinia graminis f. sp. tritici</i>race QCC

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications.

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified.

Cytoplasmic DNAs and nuclear rDNA restriction fragment length polymorphisms in commercial witloof chicories

Restriction fragment length polymorphisms of cytoplasmic DNAs and nuclear rDNA were analyzed in several Cichorium intybus genotypes, comprising four white inbred lines, eight red witloof experimental lines, and a number of F1 hybrids derived from two white parents. Chloroplast and mitochondrial restriction patterns led to the distinction between two different cytoplasms, called I and II. Southern hybridization using a nuclear rDNA probe revealed that all the lines possessed two types of rDNA repeat units. The shortest unit was 10 kb and was common to all lines. The largest rDNA repeat unit was 10.5 kb in lines I and 10.4 kb in lines II. In addition, a sequence heterogeneity between the 10.5 and 10.4-kb rDNA repeat units was revealed by Sac I digestion. A 10-kb rDNA unit was successively cloned, mapped, and used as a probe to check the genetic purity of F1 hybrid seeds between line I and II white parents. We found a 30% average percentage of impurities, originating both from selfing and full-sib crossing, in different open-pollinated hybrid samples.

Restriction fragment length polymorphisms in the ribosomal RNA gene complex of isolates of the entomopathogenic fungus Metarhizium anisopliae

Restriction fragment length polymorphisms (RFLPs) in DNA coding for ribosomal RNA (rDNA) were analysed from different species and isolates of Metarhizium. M. anisopliae var. anisopliae, M. anisopliae var. majus, M. album and M. flavoviride could be differentiated by the presence of restriction enzyme sites in the rDNA repeat unit. Isolates of M. anisopliae could be differentiated according to their geographical origin after cluster analysis of rDNA RFLPs, but there was no apparent correlation between RFLPs and original insect host. Some M. anisopliae var. majus isolates were shown to be heterozygous diploids.

Rapid detection of polymorphism in yams (Dioscorea sp) through amplification by polymerase chain reaction and rDNA variation

AbstractRibosomal DNA (rDNA) from rice (Oriza Sativa) was used to examine polymorphism in 12 yam (Dioscorea sp) cultivars. Restriction enzyme digests of total yam DNA was probed with the pRR217 probe containing the entire repeat unit of rDNA from rice. The polymerase chain reaction was also used to amplify genomic DNA of six of the 12 cultivars studied using random primers. The amplification patterns of D rotundata‐cayenensis cv tau suggested that ‘tau’ is more closely related to D rotundata sp than it is to the D cayenensis sp. The results showed polymorphisms among the different yam cultivars.

Molecular characterization and identification of Saprolegnia by restriction analysis of genes coding for ribosomal RNA.

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.

A new rDNA repeat unit in human Giardia.

The rDNA repeat unit from a new human Giardia duodenalis strain shows significant differences from the previously reported G. duodenalis rDNA repeat. Twelve base-pair changes occurred in 490 bp of the SSrRNA gene and new restriction enzyme sites occurred in the LSrRNA gene. The overall length of the rRNA genes is the same but the spacer is 76 bp longer than previously reported. A boundary within the spacers of the two different rDNA units divides a region of 50% homology near the LSrRNA gene from a region of 80% homology toward the SSrRNA gene. This boundary region includes two copies of a 78 bp repeat.

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.

Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush).

Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5' external spacer region (5' ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5' ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3-6 kb from the 18S. Sequencing of a 609-b segment of the 5' ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5' ETS using the polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Evolutionary conservation of the transcribed spacer sequences of the rDNA repeat unit in three species of the genus Aspergillus.

We have cloned and sequenced the two intervening transcribed spacers in the rDNA repeat unit of three Aspergillus species--A. nidulans, A. awamori and A. wentii. The A. wentii and A. awamori spacers are almost identical and share a high degree of homology with the A. nidulans spacers. All spacers have a high G-C content (66%-76%) and the potential of forming complex secondary structures, which may indicate that they play a role in the maturation of pre-rRNA molecules.

Ribosomal DNA, peroxidase and extensin variation in genomic DNA of Medicago laciniata (L.) Miller from Australia and Africa

Fifty-four Medicago laciniata (L.) Miller accessions from diverse locations in eastern Australia, Africa and Israel were characterized for genotypic differences by restriction endonuclease cleavage of genomic DNA and probing with radio-labelled DNA sequences to detect restriction fragment length polymorphisms (RFLP). Digestion of the genomic DNA with the restriction endonuclease, Bam HI, and probing with the complete ribosomal DNA (rDNA) repeat unit of soybean (pGmrl) and its subclones revealed that the variable region in M. laciniata spans the Bam H1 sites of the intergenic spacer region between the 18s and 26s DNA. The length variation in the rDNA repeat unit clearly distinguished South African from Australian accessions. The rDNA variants from SA1847 (Morocco) and SA7750 (Tunisia) were the same as the Australian accessions and slightly larger than the rDNA spacer variant unique to SA3428 (Israel). When Eco R1 digested M. laciniata genomic DNA was probed with peroxidase and extensin cDNA clones, all Australian accessions were again characterized by the same RFLP genotype with further differentiation between African and Israeli accessions. An accession of unknown origin, SA3412, possessed the same RFLP genotype as the Australian accessions for all endonuclease and probe combinations. For the African accessions, differences in RFLP genotype were more frequent than differences in phenotype (leaflet laciniation and colour). However, of three South African accessions with the same RFLP genotype, SA26844, SA26847, and SA26848, SA26847 had more pronounced leaflet laciniation.

Cloning of the rDNA repeat unit from a British entomopathogenic nematode (Steinernematidae) and its potential for species identification

SUMMARYThe entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Schistosoma japonicum

Geographical isolates of S. japonicum, and particularly isolates from China and the Philippines, were examined at the molecular level for genetic divergence. Sequences from both nuclear and mitochondrial genomes were selected as markers of evolutionary divergence and S. mekongi and S. mansoni were included in the study for comparison purposes. Restriction fragment length polymorphism (RFLP) and PCRRFLP analysis of the rDNA repeat unit and sequence analysis of the second internal transcribed spacer region (ITS2) within the rDNA repeat and the cytochrome c oxidase I (COI) gene of the mitochondrial genome were performed. No intra-specific variation in S. japonicum was found in the rDNA repeat and only very slight variation was detected within the COI sequence. A survey of the entire genome, using random amplified polymorphic DNA (RAPD) analysis, again showed that Chinese and Philippine S. japonicum are remarkably similar at the DNA sequence level. We were thus unable to obtain direct molecular evidence in support of previous findings, particularly those based on isoenzyme analysis, that a very high level of intra-specific variation exists in S. japonicum.

Coffee berry disease pathogen in Africa: genetic structure and relationship to the group species Colletotrichum gloeosporioides

Restriction fragment length polymorphisms of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the coffee berry disease pathogen in Africa, Colletotrichum kahawae , were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. kahawae and C. gloeosporioides as probes. These analyses revealed homogeneity among C. kahawae isolates. The estimated sizes of the rDNA repeat unit and the mtDNA of C. kahawae were 8·4–9·18 kb and 60 kb, respectively. Polymorphisms were observed in rDNA and mtDNA when C. kahawae was compared with C. gloeosporioides from coffee, avocado and mango. However, an avocado isolate JIA1, from Australia, had an identical rDNA restriction pattern to C. kahawae when digested with Bam H I and C. kahawae showed greater than 96% similarity to two C. gloeosporioides avocado isolates (918 and 1072) from New Zealand in mtDNA restriction fragment pattern. Random amplified polymorphic DNA analysis also grouped the C. kahawae isolates together. Nucleotide sequence of the internally transcribed spacer 1 region of the rDNA repeat unit of C. kahawae and C. gloeosporioides isolates from Coffea spp. differed by only two to three bases (98·8–98·2 % homology). Results obtained confirmed the close genetic relationship of C. kahawae to the group species C. gloeosporioides .

Restriction site polymorphism in rDNA repeat unit ofHordeum vulgare for some restriction endonucleases

Restriction fragment length polymorphism in rDNA repeat unit for eight restriction endonucleases was studied in two genotypes of barley (Hordeum vulgare L.). Both genotypes contained restriction sites for EcoRI, EcoRV, PvuII and Sau3239I in rDNA repeat unit and contained two different rDNA repeat unit length variants 9.7 kbp and 9.1 kbp.

A rapid molecular technique to distinguish Fusarium species

The nuclear DNA (nDNA) of different isolates of three closely related, toxin-producing Fusarium species, F. moniliforme, F. nygamai and F. napiforme , was compared to ascertain the sensitivity of a molecular method to distinguish these three species. The nDNA of these strains was digested with the restriction enzyme Eco RI and Southern analysis performed with the 6·3 kb ribosomal DNA (rDNA) repeat unit of Neurospora crassa as probe. Distinct polymorphic fragment patterns, which distinguished between the different Fusarium species, were obtained.

Ribosomal and mitochondrial DNA polymorphisms in Colletotrichum gloeosporioides isolated from tropical fruits

DNA polymorphisms in 38 isolates of Colletotrichum gloeosporioides infecting avocado, banana, mango and papaya were examined using ribosomal DNA (rDNA) from Saccharomyces carlsbergensis and mitochondrial DNA (mtDNA) purified from C. gloeosporioides total DNA as probes. The average size of the rDNA repeat unit of the isolates was 9·2 kb, although there was considerable size variation. Isolates obtained from different hosts never had the same rDNA or mtDNA RFLP patterns. With the exception of those from mango, isolates could not easily be grouped but could be distinguished in relation to their host source within geographical localities. There was no RFLP that could be used diagnostically on a worldwide basis to identify isolates from avocado, banana or papaya. In contrast, isolates obtained from mango fruits in the eastern and western hemisphere had the same rDNA and very similar mtDNA restriction fragment-banding patterns.