The intergenic spacer region of the ribosomal RNA gene of Penicillium marneffei shows almost no DNA sequence diversity

The fungus Penicillium marneffei causes fatal systemic infections and is endemic in many parts of South-East Asia, especially Thailand. The intergenic spacer (IGS) region, the most variable region of rRNA genes, was found to be highly conserved among 58 P. marneffei strains. IGS analysis might not be suitable for molecular epidemiological analysis of P. marneffei infections.     

Sequence diversity of the intergenic spacer region of the rRNA gene of Cryptococcus albidus isolated from the skin of patients with atopic dermatitis and healthy individuals

The yeast species Cryptococcus albidus var. albidus was found to more often colonize the skin surface of patients with atopic dermatitis (77.0%, 47/61) than that of  healthy subjects (37.5%, 15/40). The intergenic spacer 1 region of the rRNA gene of this species consists of four sequence types: I, II, III and IV. Types I and II were predominant among healthy subjects and atopic dermatitis patients, respectively.     

Identification ofArmillaria species from hokkaido by analysis of the intergenic spacer (IGS) region of ribosomal DNA using PCR-RFLP

The intergenic spacer (IGS) region, which is located between the 3′ end of 26S ribosomal DNA (rDNA) and the 5′ end of 5S rDNA, of sixArmillaria species from Hokkaido was investigated using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP). Restriction with onlyAlu I could distinguishA. mellea subsp.nipponica from the other species. WithAlu I andDde I,A. ostoyae andA. gallica could be distinguished from the other species. Digestion withAlu I resulted in two patterns (types A and B) ofA. singula and three patterns (types A, B, and C) ofA. jezoensis. One pattern (type B) of the former species and two patterns (types B and C) of the latter species were each different from those of the other species.Armillaria sinapina gave only oneAlu I digestion pattern, which was identical to that ofA. jezoensis (type A) andA. singula (type A). However, by digestion withDde I,A. singula (type A) could be distinguished fromA. jezoensis (type A) andA. sinapina.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

AIMS: To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. METHODS AND RESULTS: The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. CONCLUSIONS: There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.     

Analysis of intergenic spacer region length polymorphisms to investigate the halophilic archaeal diversity of stromatolites and microbial mats

Hamelin Pool in Western Australia is one of the two major sites in the world with active marine stromatolite formation. Surrounded by living smooth and pustular mats, these ancient laminated structures are associated with cyanobacterial communities. Recent studies have identified a wide diversity of bacteria and archaea in this habitat. By understanding and evaluating the microbial diversity of this environment we can obtain insights into the formation of early life on Earth, as stromatolites have been dated in the geological record as far back as 3.5 billion years. Automated ribosomal intergenic spacer analysis (ARISA) patterns were shown to be a useful method to genetically discriminate halophilic archaea within this environment. Patterns of known halophilic archaea are consistent, by replicate analysis, and the halophilic strains isolated from stromatolites have novel intergenic spacer profiles. ARISA–PCR, performed directly on extracted DNA from different sample sites, provided significant insights into the extent of previous unknown diversity of halophilic archaea within this environment. Cloning and sequence analysis of the spacer regions obtained from stromatolites confirmed the novel and broad diversity of halophilic archaea in this environment.     

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

Molecular approach to the phylogeny and systematics of Cytisus (Leguminosae) and related genera based on nucleotide sequences of nrDNA (ITS region) and cpDNA (trnL-trnF intergenic spacer)

 Phylogenetic relationships of Cytisus and allied genera (Argyrocytisus, Calicotome, Chamaecytisus, Cytisophyllum, and Spartocytisus) were assessed by analysis of sequences of the nrDNA internal transcribed spacer (ITS) and the cpDNA trnL-trnF intergenic spacer. Genera of the Genista-group (Chamaespartium, Echinospartum, Genista, Pterospartum, Spartium, Teline and Ulex) were included to check the position of Cytisus species transferred to Teline. The tree obtained by combining both sets of data indicates that the Genista and Cytisus groups form two separate clades. Cytisus heterochrous and C. tribracteolatus are more closely related to the Cytisus-group, thus their transfer to Teline is not supported by molecular data. Cytisus fontanesii (syn. Chronanthos biflorus) groups with Cytisophyllum sessilifolium and Cytisus heterochrous within the Cytisus-group. Similarly, Argyrocytisus battandieri falls within the Cytisus-group as a well differentiated taxon. All these taxa seem to have early diverged from the Cytisus-group. Their taxonomic rank should be reconsidered to better reflect their phylogenetic separation from Cytisus. On the contrary, Chamaecytisus proliferus and Spartocytisus supranubius enter in the main core of Cytisus, and they should better be included in sections of Cytisus (sect. Tubocytisus and Oreosparton, respectively). Sect. Spartopsis is not monophyletic and the position of several species, currently included in this section, deserves reevaluation: C. arboreus aggregate is closely related to C. villosus (sect. Cytisus) and to Calicotome; C. striatus is closely related to Cytisus sect. Alburnoides; and the position of C. commutatus (incl. C. ingramii) remains unclear. The relationships and positioning of several minor taxa (C. transiens, C. megalanthus, and C. maurus) are also discussed. Received November 22, 2001; accepted March 16, 2002 Published online: October 14, 2002 Addresses of the authors: Paloma Cubas (e-mail: cubas@farm.ucm.es) and Cristina Pardo (e-mail: cpardo@farm.ucm.es), Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense, E-28040 Madrid, Spain. Hikmat Tahiri Faculté des Sciences, Université Mohammed V, BP 1014 Rabat, Morocco (e-mail: tahiri@ fsr.ac.ma).     

Molecular structure of the Frankia spp. nifD—K intergenic spacer and design of Frankia genus compatible primer

The nifD—K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN₇). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807-85 amplified the nifD—K IGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 and AgB1-9) and yet failed to amplify DNA of all other nitrogen–fixing bacteria. Conversely, the failure of primer FGPK700′-92 to amplify Alnus-infective strains could be explained by point mutations in the 3′ part of the primer.     

Conservation and diversity among the three-dimensional folds of the Dicistroviridae intergenic region IRESes

Internal ribosome entry site (IRES) RNAs are necessary for successful infection of many pathogenic viruses, but the details of the RNA structure-based mechanism used to bind and manipulate the ribosome remain poorly understood. The IRES RNAs from the Dicistroviridae intergenic region (IGR) are an excellent model system to understand the fundamental tenets of IRES function, requiring no protein factors to manipulate the ribosome and initiate translation. Here, we explore the architecture of four members of the IGR IRESes, representative of the two divergent classes of these IRES RNAs. Using biochemical and structural probing methods, we show that despite sequence variability they contain a common three-dimensional fold. The three-dimensional architecture of the ribosome binding domain from these IRESes is organized around a core helical scaffold, around which the rest of the RNA molecule folds. However, subtle variation in the folds of these IRESes and the presence of an additional secondary structure element suggest differences in the details of their manipulation of the large ribosomal subunit. Overall, the results demonstrate how a conserved three-dimensional RNA fold governs ribosome binding and manipulation.