Showing papers on "Ribosomal DNA published in 1994"
TL;DR: The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected.
Abstract: Of the three primary phylogenetic domains--Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes)--Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota (one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, O. & Wheelis, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 4576-4579]) have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also of crenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.
796 citations
TL;DR: The phylogenetic relationships of the genus Sorghum and related genera were studied by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS) and it is indicated that S. arundinaceum race aethiopicum may be the closest wild relatives of cultivated sorghum.
Abstract: The phylogenetic relationships of the genus Sorghum and related genera were studied by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS). DNA was extracted from 15 Sorghum accessions, including one accession from each of the sections Chaetosorghum and Heterosorghum, four accessions from Parasorghum, two accessions from Stiposorghum, and seven representatives from three species of the section Sorghum (one accession from each of S. propinquum and S. halepense, and five races of S. bicolor). The maize (Zea mays) line, H95, and an accession from Cleistachne sorghoides were also included in the study. Variable nucleotides were used to construct a strict consensus phylogenetic tree. The analyses indicate that S. propinquum, S. halepense and S. bicolor subsp. arundinaceum race aethiopicum may be the closest wild relatives of cultivated sorghum; Sorghum nitidum may be the closest 2n=10 relative to S. bicolor, the sections Chaetosorghum and Heterosorghum appear closely related to each other and more closely related to the section Sorghum than Parasorghum; and the section Parasorghum is not monophyletic. The results also indicate that the genus Sorghum is a very ancient and diverse group.
708 citations
TL;DR: This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa.
Abstract: Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. Images
417 citations
TL;DR: The internal transcribed spacer of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence, but a statistical test suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila.
Abstract: The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.
345 citations
TL;DR: The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88 degrees C outflow from Octopus Spring, Yellowstone National Park, was examined and a single sequence type was examined closely related to Aquifex pyrophilus.
Abstract: The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88 degrees C outflow from Octopus Spring, Yellowstone National Park, was examined. Three phylogenetic types ("phylotypes"), designated EM 3, EM 17, and EM 19, were identified by cloning and sequencing the small subunit rRNA genes (16S rDNA) obtained by PCR amplification of mixed-population DNA. All three phylotypes diverge deeply within the phylogenetic domain Bacteria sensu Woese (C. R. Woese, O. Kandler, and M. L. Wheelis, Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). No members of the Archaea or Eucarya were detected. EM 3 comprises a unique lineage within the Thermotogales group, and EM 17 and EM 19 are affiliated with the Aquificales. A total of 35 clones were examined, of which the majority (26 clones) were of a single sequence type (EM 17) closely related to Aquifex pyrophilus. In situ hybridization with clone-specific probes attributes the majority sequence, EM 17, to the pink filaments. Images
344 citations
TL;DR: Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA, revealing them to be tandemly arranged.
Abstract: Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA Two amplified products of different molecular weights were obtained, cloned, and sequenced, revealing them to be tandemly arranged The nucleotide sequences differed between the two clones in the length of the nontranscribed spacer (NTS) and in three nucleotides of the coding sequence By means of fluorescence in situ hybridization the 5S rDNA was chromosomally located in the heterochromatic arm of the pair bearing the satellite, adjacent to the major ribosomal DNA locus (rDNA)
295 citations
TL;DR: Patterns of geographic distribution among intersterility groups suggest that several species lineages evolved quite early, with recently evolved groups restricted to the Northern Hemisphere and older lineages occurring throughout the world.
Abstract: Evidence from molecular systematic studies suggests that many mushroom species may be quite ancient. Gene phylogenies were developed to examine the relationship between reproductive isolation, genetic divergence, and biogeography in oyster mushrooms (Pleurotus). Sequence data were obtained for two regions of DNA from populations belonging to eight intersterility groups (biological species). Phylogenetic analysis of sequences from the 5' portion of the nuclear encoded large subunit rDNA demonstrates an ancient origin for four intersterility groups of broad geographic distribution (world-wide), with a more recent radiation of several intersterility groups that are restricted to the Northern Hemisphere. An expanded analysis using sequence data from the more variable rDNA internal transcribed spacer region also reveals a phylogenetically based pattern of genetic divergence associated with allopatric speciation among populations from different continents in the Northern Hemisphere. The ability of rDNA sequences to resolve phylogenetic relationships among geographically isolated populations within intersterility groups illustrates the importance of biogeography for understanding speciation in Pleurotus. Patterns of geographic distribution among intersterility groups suggest that several species lineages evolved quite early, with recently evolved groups restricted to the Northern Hemisphere and older lineages occurring throughout the world. Based on phylogenetic evidence, analysis of historical biogeography using area cladograms shows that multiple dispersal and vicariance events are responsible for patterns of speciation observed.
293 citations
TL;DR: The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes, which is basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.
Abstract: Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.
280 citations
TL;DR: The sequence of Francisella novicida was virtually identical to the sequences of the F. tularensis strains, thereby supporting the hypothesis that these organisms are members of the same species.
Abstract: The 16S ribosomal DNAs (rDNAs) of two strains of Francisella tularensis and one strain of Francisella philomiragia were sequenced. On the basis of phylogenetic analysis data, the genus Francisella was placed in the γ subclass of the Proteobacteria. The most closely related organism was the intracellular bacterium Wolbachia persica. The sequenced 16S rDNA molecules of the Francisella species exhibited very high levels of similarity (98.5 to 99.9%). Two variable regions, comprising 390 to 450 nucleotides of the 16S rDNA molecules of 17 additional Francisella strains, including members of the species F. tularensis and F. philomiragia, were also sequenced. At most, six nucleotide differences were observed among the sequences of the F. tularensis strains. The sequence of Francisella novicida was virtually identical to the sequences of the F. tularensis strains, thereby supporting the hypothesis that these organisms are members of the same species. On the basis of the observed differences, primer pairs were designed to distinguish strains by using the PCR at the genus, species, and subspecies levels. This permitted sensitive identification of strains belonging to the genus Francisella and discrimination of the species F. tularensis and F. philomiragia.
268 citations
TL;DR: The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16S ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16S rDNA gene.
Abstract: The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16S ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16S rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyl-etically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to Acholeplasma laidlawii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16S rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16S rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16S rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16S rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.
262 citations
TL;DR: Results demonstrate that the PCR-RFLP analysis of rONA spacers provides an efficient alternative for typing pure fungal cultures and fruit bodies for the food industry and a versatile tool for strain fingerprinting of ectomycorrhizas in ecosystems.
TL;DR: The results from Krigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence.
Abstract: The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.
TL;DR: The presence of chromosomal arrays that are homogeneous for different variants in interbreeding populations of Drosophila melanogaster indicates that there is little recombination between the chromosomes while new mutations are being homogenized along the individual arrays.
TL;DR: Discovery of a close relative of rickettsias associated with sex ratio distortion in insects has implications for the evolution and population dynamics of this bacterial genus.
Abstract: A cytoplasmically inherited microorganism associated with male killing in the two-spot ladybird beetle, Adalia bipunctata, is shown to be closely related to bacteria in the genus Rickettsia. Sequencing of a PCR-amplified product of the 16S genes coding for rRNA (16S rDNA) shows the organism associated with male killing in ladybirds to share a common ancestry with the Rickettsias relative to other genera (e.g., Anaplasma, Ehrlichia, and Cowdria). The rickettsial 16S rDNA product is found in four strains of ladybird beetle showing male embryo lethality and is absent from two uninfected strains and an antibiotic-cured strain. In addition, a revertant strain that had naturally lost the male-killing trait failed to amplify the rickettsial 16S rDNA product. Use of PCR primers for a 17-kDa protein antigen which is found only in rickettsias also resulted in an amplified product from infected strains. Uninfected, cured, and revertant strains and insect species infected with related bacteria (cytoplasmic-incompatibility bacteria from Nasonia wasps) failed to amplify the product. Discovery of a close relative of rickettsias associated with sex ratio distortion in insects has implications for the evolution and population dynamics of this bacterial genus. Images
TL;DR: When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered.
Abstract: When PCR was used to recover small-subunit (SSU) rRNA genes from a hot spring cyanobacterial mat community, chimeric SSU rRNA sequences which exhibited little or no secondary structural abnormality were recovered. They were revealed as chimeras of SSU rRNA genes of uncultivated species through separate phylogenetic analysis of short sequence domains.
TL;DR: The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia were compared with reference strains belonging to different genera and species of the family Rhizobiaceae and it was confirmed that therhizobia that nodulate chickpeas cannot be assigned to a previously described species.
Abstract: The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.
TL;DR: Phylogenetic relationships among coprinoid and closely related taxa were studied using restriction site data to establish a phylogenetic tree of Coprinus.
Abstract: Phylogenetic relationships among coprinoid and closely related taxa were studied using restriction site data. Seven taxa in Coprinus (C. comatus, C. atramentarius, C. cinereus, C. micaceus, C. cord...
TL;DR: Genomic typing by the rapid PCR-RFLP method is shown to be useful for an independant identification of strains and of the conjugative Ti plasmids.
Abstract: Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB2 genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independant identification of strains and of the conjugative Ti plasmids.
TL;DR: Phylogenetic trees of the 10 grass species generated from the ITS sequence data were in general agreement with phylogenies based on molecular data from ribosomal RNA and chloroplast DNA of similar grass taxa.
Abstract: Entire sequences of the internal transcribed spacers (ITSs) and 5.8S subunit of nuclear ribosomal DNA (nrDNA) were obtained from nine grass species by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified DNA fragments. These sequences from subfamily Pooideae (Triticum aestivum, Crithodium monococcum, Sitopsis speltoides, Hordeum vulgare, Secale montanum, Avena longiglumis, Bromus inermis, Brachypodium distachyon) and subfamily Panicoideae (Sorghum bicolor) together with published ITS sequence of rice (Oryza sativa, Bambusoideae) were analyzed using Wagner parsimony (PAUP) and the neighbor-joining distance method to assess the phylogenetic utility of ITS sequences at various taxonomic levels. Among the aligned sequences that ranged from 588 to 603 nucleotides in length, 118 of 269 variable sites contained potential phylogenetic information. A member of Bromus, B. inermis, was the sister taxon to the Triticeae species. Brachypodium was more distantly related to Triticeae than was B...
TL;DR: The relatedness of a range of isolates of Colletotrichum species, selected to represent the major morphological forms of the genus, was studied, and it was concluded that isolates within this group may represent a single species, which should be referred to as C. orbiculare.
TL;DR: Intra‐and interspecific polymorphism within the species complex were further examined by DNA sequencing of forty‐eight ITS2 clones obtained by polymerase chain reaction from individuals of the five species, supporting the hypothesis that species of the complex evolved recently.
Abstract: The primary and secondary structure of the internal transcribed spacers of ribosomal DNA (ITS1 and ITS2) and their utility for phylogenetic analysis of closely related species were examined using the Anopheles gambiae complex as a model. Restriction mapping revealed an unusual architectural feature in the ITS1 of several members of an An. gambiae cryptic species complex. Multiple spacer lengths are prevalent in An. merus and An. melas and are due to variable numbers of a repeated 250 bp sequence. Secondary structure analysis indicated that the repeat forms a helix and loop that may be involved in rDNA processing. Intra- and interspecific polymorphism within the species complex were further examined by DNA sequencing of forty-eight ITS2 clones obtained by polymerase chain reaction from individuals of the five species. Interspecies variation in the approximately 426 bp ITS2 sequence ranged between 0.4% and 1.6%; intraspecies variation ranged from 0.07% in An. arabiensis to 0.43% in An. gambiae. Intraindividual variation ranged from 0% in four individuals to a high of 0.4% in one An. quadriannulatus specimen. None of the variants were shared between species. The low level of variation supports the hypothesis that species of the complex evolved recently.
TL;DR: Surprisingly, strain 2438 harbors an additional mutation in the ribosomal protein S12 (Lys-88-->Gln), which is equivalent to invariant position 913 of the Escherichia coli 16S rRNA gene, an A-->G transition of which has been shown previously to impair streptomycin binding and strePTomycin-induced misreading in vivo.
Abstract: We report on the identification of mutations associated with streptomycin resistance in Mycobacterium tuberculosis. Two isolates (3656 and 3976) showed a wild-type ribosomal protein, S12, but exhibited a single point mutation at 16S rRNA position 491 (C-->T) or 512 (C-->T), respectively. Sequence analysis of a third isolate (2438) revealed a single base change at 16S rRNA position 904 (A-->G). This position is equivalent to invariant position 913 of the Escherichia coli 16S rRNA gene, an A-->G transition of which has been shown previously to impair streptomycin binding and streptomycin-induced misreading in vivo. Surprisingly, strain 2438 harbors an additional mutation in the ribosomal protein S12 (Lys-88-->Gln).
TL;DR: Two distinct small‐subunit ribosomal RNA genes (SSU rDNAs) were recently found in the toxic dinoflagellate Alexandrium fundyense Balech and a restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers.
Abstract: Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni.
The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.
TL;DR: The majority of thermophilic strains of the genus Bacillus were found to cluster in two groups represented by B. stearothermophilus and B. pallidus, and the as yet undescribed taxon 'B. flavothermus' warrants species status.
Abstract: Sixteen thermophilic strains of the genus Bacillus, representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus. The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae, and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon ‘B. flavothermus’ warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.
TL;DR: PCR amplification of the ITS region, digestion with the endonudeasc HinfI and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibriHosa from a large number of other basidiomyictes.
Abstract: SUMMARY
Sitka spruce mycorrhizas, macroscopically identified as being formed by Tylospora fibritiosa Donk, were sampled from a young and on old plantation and the mycobionts were isolated into pure culture. DNA was extracted and fragments of the ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). The primers were directed to conserved regions of fungal rDXA and hybridize to a wide range of fungi. One amplified region includes the internal spacer (ITS) region which has a low degree of conservation. The JTS amplification products, which were approximately 600 base pairs (bp), were digested with a variety of restriction endonucleases in order to detect restriction fragment length polymorphisms (RFLPs). The RFLPs clearly separated T. fibrillosa from other ectomycorrhizal species but there were only minor differences between the T. fibrillosa isolates. PCR amplification of the ITS region, digestion with the endonudeasc HinfI and examination of the RFLPs produced proved to be a rapid method by which to distinguish T. fibriHosa from a large number of other basidiomyictes. The method was also applied to DNA extracted, from single mycotrhizal root tips. The imergenic spacer region (1GS) of the rDNA is more variable than the ITS region in several fungal species. The 5’end of the 25S and the intergenic region between the 25S and the 5S genes were amplified and analyzed as above. Polymorphisms between T. fibritiosa isolates within this region were limited and RFLPs were not useful m discriminating between isolates, suggesting a low genetic variability in this species.
TL;DR: Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed and showed the majority of isolates clustered with B. alcalophilus DSM 485T forming a distinct phylogenetic group within the radiation of the genus Bacillus and related taxa.
Abstract: Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485T and Bacillus cohnii DSM 6307T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus. The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.
TL;DR: RFLP analysis of the 5.8S rDNA and flanking internal transcribed spacers regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.
Abstract: The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.
01 Aug 1994
TL;DR: The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.
Abstract: A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.
TL;DR: Morels (Morchella spp.) are one of the most highly prized edible mushrooms found in the wild of North America as discussed by the authors, and the number of species has been disputed.
Abstract: Morels (Morchella spp.) are one of the most highly prized edible mushrooms found in the wild of North America. Because environmental conditions can cause members of the genus to vary morphologically, the number of species has been disputed. Some au? thors classify the genus into three to five species, while others argue for as many as 50 species. DNA from lines of Morchella and a closely related genus (Verpa) was isolated. The large subunit of the ribosomal DNA repeat was amplified using the polymerase chain re? action and then digested with restriction enzymes. Re? striction fragment length polymorphisms were found among the lines investigated and used to separate the lines into genotypic classes. More genetic variation may occur between geographically isolated popula? tions of the same species than between two putatively distinct species. The phylogenetic tree developed from restriction data suggests that the black morels (M. an- gusticeps, M. elata, and M. conica) and the yellow morels (M. esculenta, M. crassipes, and M. deliciosa) are separate taxonomic groups.
TL;DR: It is concluded that ITS1 is organized into two functionally and structurally distinct halves, suggesting two independent processing pathways for 26S rRNA formation.
Abstract: Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.