Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.     

Nucleotide sequence of Citrus limon 26S rRNA gene and secondary structure model of its RNA

The complete nucleotide sequence of Citrus limon 26S rDNA has been determined. The sequence has been aligned with large ribosomal RNA (L-rRNA) sequences of Escherichia coli, Saccharomyces cerevisiae and Oryza sativa. Nine extensive expansion segments in dicot 26S rRNA relative to E. coli 23S rRNA have been identified and compared with analogous segments of monocot, yeast, amphibian and human L-rRNAs. A secondary structure model for lemon 26S rRNA has been derived based on the refined model of E. coli 23S rRNA. It has been compared with other eukaryotic L-rRNAs models in terms of location of functionally important regions. Origin and evolution of L-rRNA expansion segments are discussed.     

Phylogeography and genetic relationships of North African <Emphasis Type="Italic">Bufo mauritanicus</Emphasis> Schlegel, 1841 estimated from mitochondrial DNA sequences

Genetic variation within the North African toad Bufo mauritanicus was estimated by sequencing partial 12S rRNA and 16S rRNA mitochondrial regions from widespread populations in Morocco, Algeria and Tunisia. Unlike many other wide ranging species from this area, B. mauritanicus demonstrated very low levels of intraspecific variation. The minimal intraspecific genetic variation may be due to a relatively recent, possibly post-glacial, expansion into its current range. Further phylogeographic studies of other North African species are needed to assess if this is a common biogeographical phenomenon. Phylogenetic analyses support immunological data that B. mauritanicus is part of a clade of predominantly sub-Saharan Bufo, recently assigned to a new genus Amietophrynus. Two different lineages within this clade, B. mauritanicus and the B. pardalis group, appear to have reverted from 20 chromosomes to the more typical 22 chromosomes found in most other Bufonids. However, the alternative hypothesis that the Bufo species with 20 chromosomes form a monophyletic lineage cannot be rejected.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

Organization of three rRNA (rrn) operons from Sphingobium chungbukense DJ77

The nucleotide sequences of all three rRNA operons (rrnA, rrnB, and rrnC) of Sphingobium chungbukense DJ77 were determined. The three rrn operons have the same gene order (16S rRNA-tRNA^Ile-tRNA^Ala-23S rRNA-5S rRNA-tRNA^fMet). The nucleotide sequences were identical over a 5,468 bp region spanning the 16S rRNA gene to the 5S rRNA gene. Variability was observed in the 5S rRNA-tRNA^fMet spacer sequence of rrnB. The tRNA^fMet gene sequences were identical except for two bases (T₅₇₉₄ and A₅₈₇₁ in rrnB, T₅₉₄₂ and A₅₉₅₆ in rrnA, but C₅₉₄₂ and G₅₉₅₆ in rrnC). Comparative sequence analyses of ribosomal RNA operons from DJ77 with those of the class Alphaproteobacteria, to which the genus Sphingobium belongs, reveal close evolutionary relationships with other members of the order Sphingomonadales.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

Polymorphism of mtDNA regions in brown hare (Lepus europaeus) populations from Vojvodina (Serbia and Montenegro)

It is well known that there is heterogeneity of mitochondrial DNA (mtDNA) within and among natural populations of same species. The polymorphism level of particular regions of mtDNA gives valuable results in detection of population genetic structure. The aim of this paper was to detect polymorphism of three mtDNA regions: cytochrome oxidase I (COI), Control region, and 12S/16S rRNA, by the mtDNA RFLP-PCR method, in three Lepus europaeus populations from Vojvodina province (Serbia and Montenegro). Polymorphism was detected within the two regions, COI and Control region, while 12S/16S rRNA region was monomorphic in all 77 individuals. Eight haplotypes were detected in the brown hare population in Vojvodina, and three were unique for the Srem brown hare population.     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

Phylogenetic analysis of Spiroplasmas from three freshwater crustaceans (Eriocheir sinensis, Procambarus clarkia and Penaeus vannamei) in China

Disease epizootics in freshwater culture crustaceans (crab, crayfish and shrimp) gained high attention recently in China, due to intensive developments of freshwater aquacultures. Spiroplasma was identified as a lethal pathogen of the above three freshwater crustaceans in previous studies. Further characterization of these freshwater crustacean Spiroplasma strains were analyzed in the current study. Phylogenetic position was investigated by analysis of partial nucleotide sequences of 16S ribosomal RNA (rRNA), gyrB and rpoB genes, together with complete sequencing of 23S rRNA gene and 16S–23S rRNA intergenetic spacer regions (ISRs). Phylogenetic analysis of these sequences showed that the above-mentioned three freshwater crustacean Spiroplasma strains were identical and had a close relationship with Spiroplasma mirum. Furthermore, the genomic size, serological studies and experimental infection characteristics confirmed that three freshwater crustacean Spiroplasma strains are a single species other than traditional S. mirum. Therefore, these data suggest that a single species of Spiroplasma infects all three investigated freshwater crustaceans in China, and is a potential candidate for a new species within the Spiroplasma genus. These results provide critical information for the further investigations in fresh aquaculture epizootics related to tremor diseases, caused by this infectious agent.     

Comparison of primary and secondary 26S rRNA structures in twoTetrahymena species: Evidence for a strong evolutionary and structural constraint in expansion segments

Summary We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for twoTetrahymena species,T. thermophila andT. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the twoTetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes.The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to presesrve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.     

Structure and evolution of 5S rRNA genes and pseudogenes in the genusAspergillus

Summary We have cloned and determined the nucleotide sequence of 18 DNA fragments hybridizing to 5S rRNA from twoAspergillus species-A. wentii andA. awamori. Four of the analyzed sequences were pseudogenes. The gene sequences of these two species were very similar and differed fromAspergillus nidulans at both constant and microheterogeneous sites.     

Speciation of two sympatric coastal fish species, Girella punctata and Girella leonina (Perciformes, Kyphosidae)

Girella punctata and Girella leonina are sympatric sister species showing extensive distributional overlap in shallow rocky reefs in the Pacific Ocean south of the Japanese Islands. Differences between the two species in external morphological characters, such as number of pored lateral line scales, colour of opercular flap and shape of caudal fin, are congruent with genetic divergence. Nucleotide identity between the two species in the 3.3 kbp region of partial mitochondrial DNA containing the D-loop region, in 12S and 16S ribosomal RNA (rRNA) and transfer RNA genes is 95%. To estimate divergence time, Bayesian analysis was conducted using a dataset comprising concatenated nucleotide sequences from the two rRNA genes of three girellid and nine other fish species. Using the Elopomorpha – Clupeocephala split (265 million years ago (mya)) as a calibration point, divergence between G. punctata and G. leonina is estimated as having occurred 6.0±1.4 mya. Speciation is suggested to have been caused by geographical isolation associated with formation of the Japanese Islands, which resulted in disjunction of Girella habitat.     

Chromosomal Localization and Sequence Variation of 5S rRNA Gene in Five Capsicum Species

Chromosomal localization and sequence analysis of the 5S rRNA gene were carried out in five Capsicum species. Fluorescence in situ hybridization revealed that chromosomal location of the 5S rRNA gene was conserved in a single locus at a chromosome which was assigned to chromosome 1 by the synteny relationship with tomato. In sequence analysis, the repeating units of the 5S rRNA genes in the Capsicum species were variable in size from 278 bp to 300 bp. In sequence comparison of our results to the results with other Solanaceae plants as published by others, the coding region was highly conserved, but the spacer regions varied in size and sequence. T stretch regions, just after the end of the coding sequences, were more prominant in the Capsicum species than in two other plants. High G-C rich regions, which might have similar functions as that of the GC islands in the genes transcribed by RNA PolII, were observed after the T stretch region. Although we could not observe the TATA like sequences, an AT rich segment at -27 to -18 was detected in the 5S rRNA genes of the Capsicum species. Species relationship among the Capsicum species was also studied by the sequence comparison of the 5S rRNA genes. While C. chinense, C. frutescens, and C. annuum formed one lineage, C. baccatum was revealed to be an intermediate species between the former three species and C. pubescens.     

Regions of Intermolecular Complementarity in Escherichia coli 16S rRNA,mRNA, and tRNA Molecules

The results of computer analysis of complementarity regions in the sequences of E. coli 16S rRNA, mRNAs and tRNAs are reported in this article. The potential regions of intermolecular RNA–RNA hybridization, or clinger fragments, in 16S rRNA, which are complementary to the sites frequently occurring in mRNAs and tRNAs, were found. Major clinger fragments on 16S rRNA are universal for genes that belong to different functional groups. Our results show there are adaptations of the structural organization of the 16S rRNA molecule to messenger and transport RNA sequences. RNA interaction with clinger fragments may contribute to upregulation of the translation process through increasing the local concentration of mRNAs and tRNAs in the vicinity of the ribosome and their proper positioning, as well as decrease the efficiency of translation through nonspecific mRNA–16SrRNA interactions.