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PubMed1998

Development of specific nested oligonucleotide PCR primers for the Streptococcus iniae 16S-23S ribosomal DNA intergenic spacer.

Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer.

Animals↗
PubMed2003

16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains.

The diversity of 16S-23S rDNA intergenic spacer regions (ISR) among cellulolytic myxobacterial strains was assayed. Agarose gel electrophoresis of PCR amplification products from ten strains shows that there are at least four copies of rRNA operons in the genus Sorangium, based on their size and restriction enzymatic digest maps. There are two sequence organization patterns: tRNA(Ile)-tRNA(Ala)-containing ISR and tRNA-lacking ISR. The tRNA-containing ISRs are highly similar among strains and within a strain (more than 98% similarity) and contain the essential functional regions, such as a ribonuclease III recognition site and an antiterminator recognition site boxA. The tRNA-lacking ISR has no such functional sites that are important for yielding mature rRNA, which suggests that this type of rRNA operons might be degenerate. The tRNA-lacking ISR is divided into two types based on their sizes and sequences, which exhibits about 90% similarity within each type. Thus, the tRNA-lacking ISR polymorphisms can be used to discriminate among different strains of sorangial species.

Bacterial Typing Techniques↗
PubMed2000

New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island.

The population of the gastric pathogen Helicobacter pylori shows a high degree of genetic diversity. It is well established that heterogeneity at the isolate level is caused by nucleotide transitions within genes, differences in the gene order, and by genetic instability of single genes as well as of a large virulence-associated genomic DNA region, the cag pathogenicity island (PAI). Analysis of intergenic regions with specific PCR-assays developed in this study, revealed that DNA polymorphisms in the noncoding DNA localized in front of the genes ribA and vacA and at the insertion site of the cag PAI contribute to the genetic diversity of H. pylori and are useful for differentiation of individual isolates. Thirteen individual genotypes were identified by PCR analysis of these polymorphic loci in 487, 241, and 182 clinical H. pylori isolates. Sequence analysis revealed that genetic variability in front of genes ribA and vacA, and in the intergenic region at the PAI insertion site is caused by insertion and deletions of so-far-unknown DNA sequences as well as by parts of the H. pylori IS elements IS605 and IS606, respectively. The new genotypes identified could be used to differentiate antrum and corpus isolates from the same patients. Their combination with vacA allele subtypes and with the cagA status allowed to differentiate 140 isolates in 51 subtypes. In 36 cases the corresponding genotype patterns were isolate specific. In summary, the results confirm that DNA polymorphisms in intergenic regions contribute to the genetic diversity of H. pylori. Although individual H. pylori genotypes were not associated with peptic ulcer disease, the PCR-based approaches for their detection developed here should be of use for further investigation of genetic diversity in H. pylori and for epidemiological purposes.

Adult↗
PubMed2005

Plant viral intergenic DNA sequence repeats with transcription enhancing activity.

BACKGROUND: The geminivirus and nanovirus families of DNA plant viruses have proved to be a fertile source of viral genomic sequences, clearly demonstrated by the large number of sequence entries within public DNA sequence databases. Due to considerable conservation in genome organization, these viruses contain easily identifiable intergenic regions that have been found to contain multiple DNA sequence elements important to viral replication and gene regulation. As a first step in a broad screen of geminivirus and nanovirus intergenic sequences for DNA segments important in controlling viral gene expression, we have 'mined' a large set of viral intergenic regions for transcriptional enhancers. Viral sequences that are found to act as enhancers of transcription in plants are likely to contribute to viral gene activity during infection. RESULTS: DNA sequences from the intergenic regions of 29 geminiviruses or nanoviruses were scanned for repeated sequence elements to be tested for transcription enhancing activity. 105 elements were identified and placed immediately upstream from a minimal plant-functional promoter fused to an intron-containing luciferase reporter gene. Transient luciferase activity was measured within Agrobacteria-infused Nicotiana tobacum leaf tissue. Of the 105 elements tested, 14 were found to reproducibly elevate reporter gene activity (>25% increase over that from the minimal promoter-reporter construct, p < 0.05), while 91 elements failed to increase luciferase activity. A previously described "conserved late element" (CLE) was identified within tested repeats from 5 different viral species was found to have intrinsic enhancer activity in the absence of viral gene products. The remaining 9 active elements have not been previously demonstrated to act as functional promoter components. CONCLUSION: Biological significance for the active DNA elements identified is supported by repeated isolation of a previously defined viral element (CLE), and the finding that two of three viral enhancer elements examined were markedly enriched within both geminivirus sequences and within Arabidopsis promoter regions. These data provide a useful starting point for virologists interested in undertaking more detailed analysis of geminiviral promoter function.

Arabidopsis↗
PubMed1995

Variation in ribosomal DNA intergenic spacers among populations of Anopheles albimanus in South and Central America.

Variation in the length and copy number of intergenic spacers (IGS) of nuclear ribosomal DNA were examined to test for genetic differentiation among Anopheles albimanus populations. Extensive collections were made in Guatemala but populations were also sampled over a large range of its distribution in Central and South America. Discriminant analysis of IGS patterns in individual mosquitoes indicated that populations generally had unique sets of IGS length variants. The IGS patterns from populations on the Pacific side of Central America were distinct from those on the Atlantic side or from South America. Cluster analysis indicated a similar trend. The IGS diversity in Central America was 50% greater than in South America. These results suggest that barriers to gene flow exist among Atlantic and Pacific coast populations of An. albimanus. No gene flow barriers were detected among populations from Colombia, Ecuador, Peru, and Venezuela.

Animals↗
PubMed2004

The regulatory content of intergenic DNA shapes genome architecture.

BACKGROUND: Factors affecting the organization and spacing of functionally unrelated genes in metazoan genomes are not well understood. Because of the vast size of a typical metazoan genome compared to known regulatory and protein-coding regions, functional DNA is generally considered to have a negligible impact on gene spacing and genome organization. In particular, it has been impossible to estimate the global impact, if any, of regulatory elements on genome architecture. RESULTS: To investigate this, we examined the relationship between regulatory complexity and gene spacing in Caenorhabditis elegans and Drosophila melanogaster. We found that gene density directly reflects local regulatory complexity, such that the amount of noncoding DNA between a gene and its nearest neighbors correlates positively with that gene's regulatory complexity. Genes with complex functions are flanked by significantly more noncoding DNA than genes with simple or housekeeping functions. Genes of low regulatory complexity are associated with approximately the same amount of noncoding DNA in D. melanogaster and C. elegans, while loci of high regulatory complexity are significantly larger in the more complex animal. Complex genes in C. elegans have larger 5' than 3' noncoding intervals, whereas those in D. melanogaster have roughly equivalent 5' and 3' noncoding intervals. CONCLUSIONS: Intergenic distance, and hence genome architecture, is highly nonrandom. Rather, it is shaped by regulatory information contained in noncoding DNA. Our findings suggest that in compact genomes, the species-specific loss of nonfunctional DNA reveals a landscape of regulatory information by leaving a profile of functional DNA in its wake.

Animals↗
PubMed2001

Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Bradyrhizobium↗
PubMed1999

Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions.

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

Arthrodermataceae↗
PubMed2002

Differentiation of closely related Carnobacterium food isolates based on 16S-23S ribosomal DNA intergenic spacer region polymorphism.

A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763(T) and C. mobile DSM 4849(T) generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766(T) and C. piscicola NCDO 2762(T) were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNA(Ile) and tRNA(Ala) genes. The M-ISR included one tRNA(Ala) gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.

Bacteria↗
PubMed1998

Analysis of a ribosomal DNA intergenic spacer region from the yellow fever mosquito, Aedes aegypti.

We have sequenced the 1.8 kb intergenic spacer (IGS) region from an Aedes aegypti ribosomal DNA repeat and have identified conserved functional motifs shared with the related mosquito, Aedes albopictus. Despite the shorter length and greater homogeneity of the Ae. aegypti IGS region, the sequences of two potential RNA polymerase I core promoters and closely associated terminator elements were highly conserved. Primer extension analysis indicated that the predominant transcription initiation site in the Ae. aegypti rDNA repeat unit region lay at or near the A residue at nucleotide position 1003 in the 'upstream' RNA polymerase I promoter. This observation was supported by the higher sequence identity between the upstream promoters in Ae. aegypti and Ae. albopictus, relative to the downstream promoters. In contrast to strong similarities among proximal regulatory elements, the Ae. aegypti IGS sequence upstream of the transcription initiation site lacked the ordered array of contiguous approximately 200 nucleotide subrepeats previously found in the IGS of Ae. albopictus. In Ae. aegypti, only 4 approximately 50 nucleotide R subrepeats separated by unique sequences, followed by 2 approximately 50 nucleotide E subrepeats, occurred upstream of the transcription initiation site. Despite their differences in size and sequence, however, the four Ae. aegypti R subrepeats shared an internal structural organization that included a conserved core with 'spacer' promoters and recombinogenic elements similar to those in the longer Ae. albopictus subrepeats. These observations provide an important basis for further characterization of transcription specificity among mosquito RNA polymerase I promoters and associated regulatory elements, and contribute towards the eventual use of these elements in transgenic applications.

Aedes↗
PubMed1994

Intergenic DNA and the sequence requirements for replication initiation in eukaryotes.

Replication in eukaryotes initiates at many origins per chromosome. The locations of most of these origins appear to be restricted to intergenic spacers. In this review, I propose that the sequence dependence of initiation seen in lower eukaryotes may be a by-product of the small size of intergenic sequences and may not reflect a general requirement of the mechanisms that control the initiation of replication.

Base Sequence↗
PubMed1997

Discrimination of Streptomyces albidoflavus strains based on the size and number of 16S-23S ribosomal DNA intergenic spacers.

In an attempt to develop a rapid and accurate method for discrimination of streptomycetes at the strain level, 21 strains identified by fatty acid analysis as Streptomyces albidoflavus and the type strains of nine subjective synonyms of S. albidoflavus were selected for a full or partial 16S ribosomal DNA (rDNA) sequence analysis and an investigation of the 16S-23S rDNA intergenic spacer region. 16S rDNA sequence analysis showed that 27 of the strains exhibited 100% sequence similarity; these strains included the type strain of S. albidoflavus and the type strains of the subjective synonyms Streptomyces canescens, Streptomyces coelicolor, Streptomyces felleus, Streptomyces limosus, Streptomyces odorifer, and Streptomyces sampsonii. The type strains of the other subjective synonyms of S. albidoflavus (i.e., Streptomyces gougerotii, Streptomyces intermedius, and Streptomyces rutgersensis) were found to have levels of 16S rDNA sequence difference of 1.0 to 1.1% when they were compared to the type strain of S. albidoflavus. In order to discriminate between the strains which had identical 16S rDNA sequences, the 16S-23S rDNA intergenic spacer regions were amplified and cloned, and the sequences of the spacer regions were determined for four S. albidoflavus strains, including the type strain. The 16S-23S rDNA intergenic spacer region was found to vary in length and sequence composition among the strains and within each strain. The sizes and numbers of 16S-23S rDNA intergenic spacer regions for the 27 strains with identical 16S rDNA sequences were determined by high-resolution electrophoresis of FAM-labeled PCR products and a subsequent size analysis with GeneScan 672 software. This was shown to be a useful method for discrimination of S. albidoflavus strains. Strains with the same 16S-23S rDNA intergenic spacer band pattern, as determined by high-resolution electrophoresis of FAM-labeled PCR products, could be further discriminated on the basis of the sequence composition of the spacer region.

Cloning, Molecular↗
PubMed1983

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.

Animals↗
PubMed2002

Molecular typing of papillomatous digital dermatitis-associated Treponema isolates based on analysis of 16S-23S ribosomal DNA intergenic spacer regions.

Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms.

Animals↗
PubMed2003

Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes.

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.

Animals↗
PubMed1996

Two 16S-23S ribosomal DNA intergenic regions in different Treponema pallidum subspecies contain tRNA genes.

The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum, Nichols strain, and Treponema pallidum subspecies pertenue, Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p.pallidum and T.p. pertenue, and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi.

Alleles↗
PubMed1991

Transcription of ribosomal DNA intergenic spacer sequences in normal and regenerating rat liver.

In regenerating rat liver both the transcriptional activity of the intergenic spacer rDNA promoter and the steady-state abundance of spacer transcripts are increased about 2-fold, as compared to normal liver. These changes are parallel to the observed 2.5-fold increase in regenerating liver of rRNA gene promotor activity and gene promotor transcripts abundance. These results suggest that both gene and spacer rDNA promotors are subject to common regulatory mechanisms. Our results indicate also that the stability of spacer transcripts in regenerating liver is not significantly altered.

Animals↗