Showing papers on "Ribosomal DNA published in 1989"

Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers (cytochrome b/12S ribosomal DNA/control region/evolutionary genetics/molecular phylogenies)

Abstract: With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments ofmtDNA from more than 100 animal species, including mammals, birds, amphib- ians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nano- gram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility ofthese primers offers opportunities for phylogenetic and population research.

Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA

Abstract: Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.

Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA.

Abstract: The sequence of the large subunit ribosomal RNA (LsuRNA) gene of the dinoflagellateProrocentrum micans has been determined. The inferred rRNA sequence [3408 nucleotides (nt)] is presented in its most probable secondary structure based on compensatory mutations, energy, and conservation criteria. No introns have been found but a hidden break is present in the second variable domain, 690 nt from the 5′ end, as judged by agarose gel electrophoresis and primer extension experiments.Prorocentrum micans LsuRNA length and G+C content are close to those of ciliates and yeast. The conserved portions of the molecule (1900 nt) have been aligned with corresponding sequences from various eukaryotes, including five protista, one metaphyta, and three metazoa. An extensive phylogenetic study was performed, comparing two phenetic methods (neighbor joining on difference matrix, and Fitch and Margoliash on Knuc values matrix) and one cladistic (parsimony). The three methods led to similar tree topologies, except for the emergence of yeast that groups with ciliates and dinoflagellates when phenetic methods are used, but emerges later in the most parsimonious tree. This discrepancy was checked by statistical analyses on reduced trees (limited to four species) inferred using parsimony and evolutionary parsimony methods. The data support the phenetic tree topologies and a close relationship between dinoflagellates, ciliates, and yeast.

Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome.

Abstract: Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.

Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA.

Abstract: Nucleotide sequences of the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA) from two dicot plants, carrot and broad bean, were determined. These sequences were compared with those of rice, a monocot plant, and other eukaryotic organisms. Both types of ITS region in some species of Angiospermae were the shortest among all eukaryotes so far examined and showed a wide range of variation in their G+C content, in contrast to a general trend toward very high G+C content in animals. Phylogenetic relationships of plants with animals and lower eukaryotes were considered using the nucleotide sequences of carrot and broad bean 5.8S rDNA that were determined in the present study, together with that of wheat 5.8S rRNA, which has been reported previously.

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns.

Abstract: SUMMARY: The rRNA gene restriction patterns of 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindlII and Eco RI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilisinserted in a plasmid vector, pBR322. Fourty-four distinct Hindlll patterns and 44 distinct EcoRl patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed within some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcusxylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g. S. aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.

Ribosomal-dna variation and distribution in rudbeckia missouriensis.

Abstract: Ribosomal DNA (rDNA) has been used to describe both the genetic structure within plant populations and the phylogenetic relationships among species of plants

Organization and evolution of the 5S ribosomal RNA gene family in wheat and related species

Abstract: Variation in restriction fragments in nullisomic–tetrasomic and ditelosomic lines of Triticum aestivum 'Chinese Spring' and disomic and ditelosomic substitutions of chromosomes of diploid species Lophopyrum elongatum, T. monococcum ssp. aegilopoides, T. tauschii, and T. umbellulatum for 'Chinese Spring' chromosomes were used to identify chromosomal loci of 5S rRNA genes (5S DNA) in wheat and related species. These loci are on wheat chromosome arms 1BS, 1DS, 5AS, 5BS, and tentatively 5DS, T. m. aegilopoides chromosomes 1A and 5A, T. tauschii chromosomes 1D and 5D, and T. umbellulatum chromosome 5U. In diploid L. elongatum a locus was detected on chromosome arm 1ES. In most genomes, the locus on chromosome 1 contains 5S DNA subfamily with short spacers and the locus on chromosome 5 contains 5S DNA subfamily with long spacers. Only a few genomes were found to be potential exceptions to this rule. Concerted evolution of the 5S DNA loci was examined in several genomes. It appeared that homogenization of spacer...

Extraction of DNA from Basidiomycetes for ribosomal DNA hybridizations

Abstract: We describe a method for extracting high molecular weight DNA from milligram amounts of fungus specimens. DNA extracted from 1 – 100 mg of fresh, air-dried, or lyophilized tissue was sufficient for 1–50 Southern hybridizations. Typical yields were 1 – 10 nanograms (ng) of DNA per milligram of starting material for fresh and air-dried samples, and 50–500 ng/mg for lyophilized samples. Of the 78 samples from 63 species of saprobic and ectomycorrhizal Basidiomycetes, all but one yielded measurable amounts of nucleic acids. The DNA was used for restriction enzyme ribosomal DNA hybridizations. While most species showed similarities in the genic regions, significant size variation was seen in the intergenic regions, in most cases allowing for distinction between samples at the species level.

Use of mitochondrial and ribosomal DNA polymorphisms to classify clinical and soil isolates of Histoplasma capsulatum.

Abstract: We have developed an improved scheme for the classification of environmental and clinical isolates of Histoplasma capsulatum that is based on analysis of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA). Strains were initially divided into mtDNA groups according to restriction digests of whole-cell DNA and Southern hybridization with cloned mtDNA probes. Strains within a mtDNA class could be further grouped by polymorphisms in rDNA. The majority of soil and clinical isolates from the United States had identical mtDNA patterns; however, rDNA polymorphisms were common in both types of isolates. The combination of mtDNA and rDNA typing described in this report will be useful in resolving questions concerning the epidemiology of H. capsulatum infections.

Variation in ribosomal dna among biological species of armillaria, a genus of root-infecting fungi.

Abstract: Armillaria is a genus of root-infecting fungi composed of several biological species in North America and Europe. To examine relatedness among biological species, ribosomal DNA (rDNA) from one isolate was cloned and rDNAs from 30 isolates were mapped for eight restriction enzymes. The positions of the large (26S) and small (18S) rRNA cistrons were found by Northern hybridizations of total cellular RNA with rDNA subclones and by alignment of maps with conserved restriction sites present in rRNA genes of other fungi. Nine restriction-site and two length poly- morphisms were observed. Eight North American (Roman numerals) and five European (species epithets) biological species could be placed in six classes with respect to rDNA maps (rDNA class 1: I and A. ostoyae; class 2: 11; class 3: A. borealis; class 4: V, IX, and X; class 5: 111, VII, A. lutea, and A. cepistipes; and class 6: VI and A. mellea). Most, but not all, polymorphisms were in intergenic regions.

Ribosomal RNA genes in B chromosomes of Crepis capillaris detected by non-radioactive in situ hybridization.

Abstract: A non-radioactive in situ hybridization method using biotin-labelled rDNA has made it possible to localize rRNA genes not only at the secondary constriction in both homologous chromosomes No. 3 of Crepis capillaris but also in the B chromosomes occurring in the plants employed. Very clear dot-like rDNA signals at the telomeres of both arms were observed in all B chromosomes. Histochemical silver staining, which is indicative of transcriptional activity of rRNA gene clusters, resulted in both darkly-staining nucleolar constrictions of chromosomes No. 3 and silver deposits at the telomeres of Bs. We conclude that the B chromosomes of C. capillaris are isochromosomes with active rRNA genes located near both telomeres.

A system for the analysis of yeast ribosomal DNA mutations.

Abstract: To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus.

Abstract: A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.

Recombinational Controls of rDNA Redundancy in Drosophila

Abstract: Throughout this review we are guided by several basic questions. First, to what extent do recombinational processes act to control rDNA copy number in wild-type Drosophila? Second, if any of these processes is induced by an rDNA deficiency and therefore can be considered as a compensatory mechanism, then by what mechanism(s) is an rDNA deficiency sensed and then corrected? Third, we explore whether the systems controlling rDNA copy number in flies represent mechanisms unique to rDNA

Unique organization of Leptospira interrogans rRNA genes.

Abstract: We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.

Circular DNA of Entamoeba histolytica encodes ribosomal RNA.

Abstract: . The presence of repeated DNA sequences encoding RNA in Entamoeba histolytica has been reported. In the present study we demonstrate by agarose gel electrophoresis, DNase digestion and electron microscopic analysis that these genes are located on extrachromosomal circular DNA molecules with an approximate size of 26 kb. Detection of replication intermediates suggests the episomal nature of these molecules. Amplified, extrachromosomal rRNA genes appear to be a common feature among the lower eukaryotes, occurring more commonly as linear molecules and less commonly as circles. Entamoeba histolytica is 1 of the few organisms studied in which rRNA genes are located predominantly on extrachromosomal circles.

Patterns of variation in the rDNA cistron within and among world populations of a mosquito, Aedes albopictus (Skuse).

Abstract: A restriction map was constructed of the ribosomal cistron in a mosquito, Aedes albopictus (Skuse). The 18s, 28s and nontranscribed spacer (NTS) regions were subcloned and used to probe for intraspecific variation. Seventeen populations were examined throughout the world range of the species. No variation was detected in the coding regions but extensive and continuous variation existed in the NTS. The NTS consisted of two nonhomologous regions. The first region contained multiple 190-bp AluI repeats nested within larger XhoI repeats of various sizes. There was a large number of length variants in the AluI repeat region of the NTS. No repeats were found in the second region and it gave rise to relatively fewer variants. An analysis of NTS diversity in individual mosquitoes indicated that most of the diversity arose at the population level. Discriminant analysis was performed on spacer types in individual mosquitoes and demonstrated that individuals within a population carried a unique set of spacers. In contrast with studies of the NTS in Drosophila populations, there seems to be little conservation of spacers in a population. The importance of molecular drive relative to drift and selection in the generation of local population differentiation is discussed.

Genetic variability for pathogenicity, isozyme, ribosomal DNA and colony color variants in populations of Rhynchosporium secalis.

Abstract: Samples of Rhynchosporium secalis were collected from two experimental barley populations known to carry a diverse array of alleles for resistance to this fungal pathogen. Classification of 163 isolates for four putative isozyme systems, a colony color dimorphism and 20 ribosomal DNA restriction fragment length variants revealed 49 different multilocus phenotypes (haplotypes). The six most common haplotypes differed significantly in pathogenicity. Genetic analyses of the data indicated that effective population sizes of the fungus were very large, that the effects of genetic drift were small, and that negligible recombination occurred in the populations studied. Frequency dependent selection was suggested as an explanation for the maintenance of variation in pathogenicity in the fungus.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species.

Abstract: Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1–3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A — H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B — H. leporinum; Map C — H. vulgare, H. spontaneum, H. agriocrithon; Map D — H. chilense, H. bogdanii; and Map E — remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

Evidence for unlinked rrn operons in the Planctomycete Pirellula marina.

Abstract: Southern hybridization of rRNAs to chromosomal BamHI-digested DNA of the eubacterium Pirellula marina revealed the presence of two sets of 16S and 23S rRNA genes. The two copies of the 23S rRNA genes, located on 11- and about 13-kilobase (kb) inserts, were isolated from a lambda bacteriophage Charon 35 library. The 11-kb fragment was cloned directly into pBR322, while a 5.4-kb BamHI-PstI rDNA subfragment of the approximately 13-kb insert was cloned into pUC18. Both recombinant plasmids, pPI1100 and pPI540, were characterized by restriction enzyme mapping and Southern hybridization with the large rRNA species. Restriction fragments from both inserts were subcloned into phage M13 mp18 and mp19. Correlation of genomic hybridization data with physical characterization of recombinant plasmids showed that, in contrast to the general organization of rrn operons in eubacteria, the 16S rRNA genes of P. marina are separated by at least 8.5 (pPI540) and 4.4 (pPI1100) kb, respectively, from the closely linked 23S-5S rRNA genes. Comparison of the flanking regions from both 23S-5S rRNA genes with published consensus sequences of structural elements indicates the presence of putative transcription signals, i.e., a single Pribnow box, discriminator, antitermination boxes A, B, and C, and a Rho-independent terminator.

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila

Abstract: The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.