Showing papers on "Ribosomal DNA published in 1993"

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Abstract: We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Identification of Single Specimens of the Anopheles Gambiae Complex by the Polymerase Chain Reaction

Abstract: A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.

Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms.

Abstract: To facilitate genus and species level identification of a broad range of bacteria without the requirement of presumptive identification, we have developed a unified set of primers and polymerase chain reaction conditions to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. Spacer regions within these loci show a significant level of length and sequence polymorphism across both genus and species lines. A generic pair of priming sequences was selected for the amplification of these polymorphisms from highly conserved sequences in the 16S and 23S genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions was used for the amplification of 16S-23S spacer regions for over 300 strains of bacteria belonging to eight genera and 28 species or serotypes, including Listeria, Staphylococcus, and Salmonella species and additional species related to these pathogenic organisms. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the species of bacteria within the test group. Unique elements in the amplification product patterns generally clustered at the species level, although some genus-specific characteristics were also observed. On the basis of the results obtained with our test group of 300 bacterial strains, amplification of the 16S-23S ribosomal spacer region is a suitable process for generating a data base for use in a polymerase chain reaction-based identification method, which can be comprehensively applied to the bacterial kingdom.

Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms

Abstract: Regions representing about 80% of the 16S rDNA sequences of 40 mycoplasmalike organism (MLO) strains from North America, Asia, and Europe were amplified by polymerase chain reaction (PCR) using a primer pair designed on the basis of an MLO 16S rRNA gene. This primer pair detected every MLO examined from infected periwinkle (Catharanthus roseus) and some other plants. No PCR products were obtained in samples containing DNA extracted from healthy plants. The partial 16S rDNA sequences amplified from these various MLOs were compared through restriction fragment length polymorphism (RFLP) analyses [...]

Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov.

Abstract: A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

Reclassification of American Rhizobium leguminosarum Biovar Phaseoli Type I Strains as

Abstract: A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16s ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nod4 and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16s ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16s rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal. The bacteria of the genus Rhizobiurn nodulate the roots of leguminous plants. Rhizobiurn species have been defined primarily on the basis of phenotypic characteristics such as host range, colony morphology, growth on selective media, and certain metabolic attributes. The bacteria that infect peas, clovers, and beans (Phaseolus vulgaris L.) have been clustered in a single species, Rhizobiurn legurninosarurn, which has three biovars (I?. legurninosarurn biovar viciae, R. legurninosarurn biovar trifolii, and R. legurninosarurn biovar phaseoli) (11). This subdivision is based mainly on the presence of different symbiotic plasmids coding for distinct nodulation specificities in a uniform chromosomal background. The uniformity of this background has been supported by different types of data; the symbionts of peas, clovers, and beans cannot reliably be distinguished on the basis of phenotypic characteristics (17). Under laboratory conditions, recombination of chromosomal genes among isolates belonging to the different biovars can be obtained easily (9, 13); symbiotic plasmids conferring different symbiotic capacities can be transferred among strains isolated from peas, clovers, and beans and remain functional (7, 10, 14). DNA-DNA hybridization levels are higher among isolates belonging to the three biovars than to isolates belonging to other related species (3); the nucleotide sequences of a 16s rDNA fragment from different isolates obtained from peas, clovers, and beans have been found to be identical (6, 25). This classification has been challenged by several lines of evidence. Heterogeneity in R. legurninosarurn biovar phaseoli has been identified by a variety of methods, including protein patterns in two-dimensional polyacrylamide gel electrophoresis (21), antibiotic resistance profiles, serological types, DNA-DNA hybridization data (8), plasmid profiles, and exopolysaccharide structures (16). The genetic structures of several R. leguminosarurn populations have been determined. Young’s analysis of three enzymes in a collection of strains belonging to the three biovars of R. legurninosarurn isolated in English fields showed that there is a considerable amount of sharing of enzyme mobility variants

The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences.

Abstract: Summary: Partial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of Ribosomal DNA in Winteraceae

Abstract: The internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA were sequenced and analyzed to address questions of generic relationships in Winteraceae. The molecular data generated a single tree that is congruent with one based on morphological data. The sequences of ITS 1 in the family range from 235 to 252 bases in size and of ITS 2 from 213 to 226 bases. The size of the 5.8S coding region is 164 bases. The range of ITS 1 and ITS 2 sequence divergence between pairs of genera within Winteraceae is relatively low in comparison to other plant families. Two types of ITS 1 and ITS 2 sequences were observed in the same individual for some taxa. Sequence variations between the two arrays are 4.70/-o 6.3% for ITS 1 and 5. 10/o-7.0% for ITS 2. Both arrays of sequences, however, generate the same phylogenetic relationships. Rates of nucleotide substitutions for the internal transcribed spacers are 3.2-5.2 x 10-'? substitution per site per year estimated in ITS 1 and 3.6-5.7 x 10-'? in ITS 2.

Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae

Abstract: The chromatin structure of yeast ribosomal DNA was analyzed in vivo by crosslinking intact cells with psoralen. We found that in exponentially growing cultures the regions coding for the 35S rRNA precursor fall into two distinct classes. One class was highly accessible to psoralen and associated with nascent RNAs, characteristic for transcriptionally active rRNA genes devoid of nucleosomes, whereas the other class showed a crosslinking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rRNA gene copies. By crosslinking the same strain growing in complex or minimal medium, we have shown that yeast cells can modulate the proportion of active (non-nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions which suggests that yeast can regulate rRNA synthesis by varying the number of active gene copies, in contrast to the vertebrate cells studied so far. Whereas intergenic spacers flanking inactive rRNA gene copies are packaged in a regular nucleosomal array, spacers flanking active genes show an unusual crosslinking pattern suggesting a complex interaction of regulatory factors and histones with DNA.

Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis

Abstract: In order to investigate the genetic diversity of streptomycetes in an acid forested soil sample from Mt. Coot-tha, Brisbane, Australia, cells were mechanically lysed within the soil matrix and genomic DNA was isolated and purified. 16S ribosomal (r)DNA was amplified by the polymerase chain reaction (PCR) method using one primer conserved for members of the domain Bacteria and a second designed specifically for streptomycetes and related taxa. PCR amplification products were cloned into phage vector M13 mp19 and the diversity of 16S rDNA genes was determined by sequence analysis and oligonucleotide probing of the resultant clone library. Comparison of partial 16S rDNA sequences with published sequences revealed that few sequences originated from streptomycetes. The majority of sequences belonged to members of the alpha subclass of Proteobacteria. Other clones were related to planctomycetes, actinomycetes, or represented novel lines of descent. Bacteria that are customarily isolated from soil of pH 4-7 such...

Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis.

Abstract: Using sequence data from the 28s ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of wellaccepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is ~40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.

Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA

Abstract: Summary: A method has been developed to amplify the 16S rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BclI restriction site in the 16S rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16S rDNA of the MLOs from BclI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16S rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16S rRNA genes using AluI endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups II to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by AluI restriction were also obtained by digesting the 16S rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16S rDNA of isolates within the same group exhibited 97·8 to 99·5% homology while those of different groups showed 89·6 to 92·0% homology.

Phylogenetic analysis of Rhizobia and Agrobacteria based on 16S rRNA gene sequences

Abstract: The phylogenetic relationships of members of the genera Rhizobium, Agrobacterium, Bradyrhizobium, and Azorhizobium were studied by direct sequencing of their amplified 16S rRNA genes. Comparative analysis of the sequence data confirmed that the genera Bradyrhizobium and Azorhizobium belong to distinct phylogenetic lineages. The genera Rhizobium and Agrobacterium were found to be phylogenetically heterogeneous, and several subgroupings in which Rhizobium and Agrobacterium species were intermixed were evident. The present findings show that the genus and species definitions of these organisms are in need of revision. Different possibilities for this are discussed in the light of sequencing data.

Molecular phylogenetics of Calycadenia (Compositae) based on its sequences of nuclear ribosomal DNA: chromosomal and morphological evolution reexamined

Abstract: Phylogenetic patterns within Calycadenia were estimated from 1 8-26S nuclear ribosomal DNA sequences of the internal transcribed spacer (ITS) region in 19 representatives of all species in Calycadenia, including Osmadenia (C.) tenella, and in two outgroup species. In pairwise comparisons among the Calycadenia and Osmadenia sequences, divergence ranged from 0 to 11.2% of nucleotides in ITS 1 and from 0 to 8.6% in ITS 2. Of 62 nucleotide sites with potential phylogenetic information, 51.6% were in ITS 1, 46.8% were in ITS 2, and 1.6% were in the 5.8S subunit. A highly resolved, strict consensus tree from Wagner parsimony analysis of these data agrees well with morphological and cytological evidence. This tree suggests that: 1) the monotypic Osmadenia tenella is the sister-group to Calycadenia; 2) the base chromosome number in Calycadenia is n = 7, from which other numbers were derived; 3) species with multiple T-glands on cylindrical bracts and chromosome numbers of n = 5 or 6 (or 7 in C. oppositifolia) form a monophyletic group derived from an n = 7 species similar or identical in genomic structure to C. hooveri or C. villosa; 4) C. spicata (n = 4) is the product of an independent dysploid reduction from n = 7; 5) C. multiglandulosa and C. pauciflora, sensu Keck, are not monophyletic taxa; and 6) loss of chromosomal homology between Calycadenia species, as reflected by meiotic chromosomal association in hybrids, is positively correlated with time since evolutionary divergence. These results offer little evidence of homoplasy in chromosomal and phenotypic characters in Calycadenia and provide further support for the phylogenetic utility of plant ITS sequences.

Ribosomal Dna Sequences of Encephalitozoon Hellem and Encephalitozoon Cuniculi: Species Identification and Phylogenetic Construction

Abstract: . A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. the amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. the sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos.

Abstract: We have analysed the replication of the chromosomal ribosomal DNA (rDNA) cluster in Xenopus embryos before the midblastula transition. Two-dimensional gel analysis showed that replication forks are associated with the nuclear matrix, as in differentiated cells, and gave no evidence for single-stranded replication intermediates (RIs). Bubbles, simple forks and double Ys were found in each restriction fragment analysed, showing that replication initiates and terminates without detectable sequence specificity. Quantification of the results and mathematical analysis showed that the average rDNA replicon replicates in 7.5 min and is 9-12 kbp in length. This time is close to the total S phase duration, and this replicon size is close to the maximum length of DNA which can be replicated from a single origin within this short S phase. We therefore infer that (i) most rDNA origins must be synchronously activated soon in S phase and (ii) origins must be evenly spaced, in order that no stretch of chromosomal DNA is left unreplicated at the end of S phase. Since origins are not specific sequences, it is suggested that this spatially and temporally concerted pattern of initiation matches some periodic chromatin folding, which itself need not rely on DNA sequence.

Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, Pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction.

Abstract: Summary: The sequence of a 292 bp segment of the DNA encoding 16S rRNA (corresponding to positions 44-337 of the Escherichia coli 16S rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16S rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16S rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Sequence-based differentiation of strains in the Mycobacterium avium complex.

Abstract: The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.

Monophyly of aneuploid astragalus (fabaceae): evidence from nuclear ribosomal dna internal transcribed spacer sequences

Abstract: Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 1 8S-26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.

Physical mapping of rDNA loci in Brassica species.

Abstract: The number of major rDNA loci (the genes coding for 18S–5.8S–26S rRNA) was investigated in the economically important Brassica species and their wild relatives by in situ hybridization of an rDNA probe to metaphase chromosomes and interphase nuclei. The diploid species B. nigra (B genome) has two major pairs of rDNA loci, B. oleracea (C genome) has two major pairs and one minor pair of loci, while B. campestris (A genome) has five pairs of loci. Among the three tetraploid species arising from these three diploid ancestors, B. carinata (BBCC genomes) has four loci, B. juncea (AABB genomes) has five major pairs and one minor pair of loci, and B. napus (AACC genomes) has six pairs of loci, indicating that the number of loci has been reduced during evolution. The complexity of the known rDNA restriction fragment length polymorphism patterns gave little indication of number of rDNA loci. It is probable that chromosome rearrangements have occurred during evolution of the amphidiploid species. The data will be u...

The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes.

Abstract: When spread chromatin is visualized by electron microscopy, active rRNA genes have a characteristic Christmas tree appearance: From a DNA "trunk" extend closely packed "branches" of nascent transcripts whose ends are decorated with terminal "balls." These terminal balls have been known for more than two decades, are shown in most biology textbooks, and are reported in hundreds of papers, yet their nature has remained elusive. Here, we show that a rRNA-processing signal in the 5'-external transcribed spacer (ETS) of the Xenopus laevis ribosomal primary transcript forms a large, processing-related complex with factors of the Xenopus oocyte, analogous to 5' ETS processing complexes found in other vertebrate cell types. Using mutant rRNA genes, we find that the same rRNA residues are required for this biochemically defined complex formation and for terminal ball formation, analyzed electron microscopically after injection of these cloned genes into Xenopus oocytes. This, plus other presented evidence, implies that rRNA terminal balls in Xenopus, and by inference, also in the multitude of other species where they have been observed, are the ultrastructural visualization of an evolutionarily conserved 5' ETS processing complex that forms on the nascent rRNA.

Phylogenetic Analysis of Two Ribosomal Dna Regions Indicates Multiple Independent Losses of a Sexual Talaromyces State Among Asexual Penicillium Species in Subgenus Biverticillium

Abstract: Nucleotide sequences were obtained from the mitochondrial small subunit ribosomal DNA, and the nuclear ribosomal DNA region containing the internal transcribed spacers and 5.8S ribosomal RN...

Physical mapping of four sites of 5S rDNA sequences and one site of the α-amylase-2 gene in barley (Hordeum vulgare).

Abstract: The 5S rDNA sequences have been mapped on four pairs of barley (Hordeum vulgare L.) chromosomes using in situ hybridization and barley monotelotrisomic lines. The 5S rDNA sequences are located, genetically and physically, on the short arm of chromosome 1 (7I) and the long arms of chromosomes 2 (2I) and 3 (3I). The 5S rDNA sequence is also located on the physically long arm of chromosome 4 (4I). Only one site on chromosome 2(2I) has previously been reported. The characteristic locations of the 5S rDNA sequences make them useful as molecular markers to identify each barley chromosome. The physical position of the low-copy α-amylase-2 gene was determined using in situ hybridization; the location of this gene on the long arm of chromosome 1 (7I) was confirmed by reprobing the same preparation with the 5S rDNA probe. The results show that there is a discrepancy between the physical and genetic position of the α-amylase-2 gene.Key words: genetic mapping, physical mapping, barley, mapping, 5S DNA, α-amylase, in ...

Identification of Mycobacterium species by using amplified ribosomal DNA restriction analysis.

Abstract: A rapid procedure for the identification of cultured Mycobacterium isolates, based on the combination of enzymatic amplification and restriction analysis, is described. The 16S rRNA genes (rDNA) of 99 strains belonging to 18 different species of the genus Mycobacterium were enzymatically amplified. Amplified rDNA restriction analysis with the enzymes CfoI, MboI, and RsaI was carried out. The combination of the amplified rDNA restriction analysis patterns obtained after restriction with CfoI and MboI enabled differentiation between Mycobacterium asiaticum (number of strains = 4), M. avium (n = 22), M. chelonae (n = 5), M. flavescens (n = 1), M. fortuitum (n = 6), M. gordonae (n = 6), M. intracellulare (n = 13), M. marinum (n = 7), M. nonchromogenicum (n = 1), M. simiae (n = 5), M. terrae (n = 5), the M. tuberculosis complex (n = 11), and 2 of 4 strains of M. xenopi. Further restriction with RsaI was necessary to differentiate between the species M. kansasii (n = 5), M. scrofulaceum (n = 4), and the 2 other M. xenopi strains. The M. avium-M. intracellulare complex was characterized by a specific MboI pattern, and M. avium and M. intracellulare strains could further be differentiated by restriction with CfoI. The whole procedure, including sample preparation prior to the polymerase chain reaction, can be carried out within 8 h, starting from a pure culture. Images

Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae.

Abstract: Based on morphological and immunological studies, we hypothesize that there are two lineages within the Sclerotiniaceae, a family of plant-infecting ascomycetes in the order Helotiales: 1) genera p...

Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions

Abstract: SUMMARY: To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. difficile strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable in vivo, since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing C. difficile typing methods; it has universal applicability, it is objective and is moderately rapid.

Ribosomal RNA genes are interspersed throughout a heterochromatic chromosome arm in Atlantic salmon.

Abstract: The ribosomal RNA genes (rDNA) have been mapped by fluorescent in situ hybridization (FISH) using four rDNA probes (rDNA/FISH) to Atlantic salmon chromosomes. Signals appeared over the whole heterochr