Accessory DNA in the genomes of representatives of the Escherichia coli reference collection.
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Different strains of the Escherichia coli reference collection (ECOR) differ widely in chromosomal size. To analyze the nature of the differential gene pool carried by different strains, we have followed an approach in which random amplified polymorphic DNA (RAPD) was used to generate several PCR fragments. Those present in some but not all the strains were screened by hybridization to assess their distribution throughout the ECOR collection. Thirteen fragments with various degrees of occurrence were sequenced. Three of them corresponded to RAPD markers of widespread distribution. Of these, two were housekeeping genes shown by hybridization to be present in all the E. coli strains and in Salmonella enterica LT2; the third fragment contained a paralogous copy of dnaK with widespread, but not global, distribution. The other 10 RAPD markers were found in only a few strains. However, hybridization results demonstrated that four of them were actually present in a large selection of the ECOR collection (between 42 and 97% of the strains); three of these fragments contained open reading frames associated with phages or plasmids known in E. coli K-12. The remaining six fragments were present in only between one and four strains; of these, four fragments showed no similarity to any sequence in the databases, and the other two had low but significant similarity to a protein involved in the Klebsiella capsule synthesis and to RNA helicases of archaeal genomes, respectively. Their percent GC, dinucleotide content, and codon adaptation index suggested an exogenous origin by horizontal transfer. These results can be interpreted as reflecting the presence of a large pool of strain-specific genes, whose origin could be outside the species boundaries. (+info)
Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli.
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The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some correlations with the multilocus enzyme electrophoresis clusters were detected. The mode of variation of the rrl gene seems to be quite similar to that of the rrs gene. Homogenization of the gene families and transfer of sequences from different clonal lines could explain this pattern of variation detected; perhaps these factors are more relevant to evolution than single mutation. The spacer region between the 23S and 5S rRNA genes exhibited a highly polymorphic region, particularly at the 3' end. (+info)
A yellow head virus gene probe: nucleotide sequence and application for in situ hybridization.
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A portion of the genome of yellow head virus (YHV) of penaeid shrimp was cloned and the cDNA fragment (1161 bp) was designated clone 3-27. The fragment was labeled with digoxigenin and hybridized in situ to tissue sections of YHV-infected Penaeus vannamei. Positively reacting tissues included those of the lymphoid organ, cuticular epithelium, and gills. In addition, connective tissue of hepatopancreas, heart, antennal gland, hematopoietic organ, nerve tract, midgut cecum and muscle reacted to the probe. The probe was highly specific since it hybridized only to tissues from YHV-infected shrimp. It did not react to those of uninfected shrimp or shrimp infected with WSSV (white spot syndrome virus), IHHNV (infectious hypodermal and hematopoietic necrosis virus), or TSV (Taura syndrome virus). The clone was sequenced, and primers were synthesized for rapid detection of YHV in hemolymph using RT-PCR (reverse transcription-polymerase chain reaction). The strand that constituted the viral sequence in the cDNA was also determined via RT-PCR and in situ hybridization with a single-stranded RNA (ssRNA) probe. (+info)
Fluorescence In situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures.
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TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination. Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction. TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand. The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies. The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum, which is not identified by the probe. In total, 63 (87%) of the cultures were correctly identified as MTC (n = 46) or NTM (n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive. None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%. (+info)
Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler.
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BACKGROUND: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations. METHODS: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented. RESULTS: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; S(y|x) = 0.96; n = 24). CONCLUSIONS: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases. (+info)
A Na(+)-dependent nucleoside transporter in microglia.
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In the central nervous system, HIV-1 has a defined tropism for brain macrophages and microglia. Nucleoside analog drugs such as zidovudine improve the clinical and neuropsychological functions in HIV-demented patients. Multiple carrier-mediated transport systems can play an important role in the membrane permeation of nucleosides and nucleoside analog drugs in a number of cells. The purpose of this project was to characterize the uptake properties of the pyrimidine nucleoside probe thymidine by a continuous rat microglia cell line (MLS-9) grown as a monolayer on an impermeable substratum. Approximately 50% of thymidine (10 microM) uptake by the monolayer cells was found to be Na(+) dependent. Kinetics of specific thymidine uptake showed a single saturation system (K(m) = 44 microM at 37 degrees C) and a Na(+)/thymidine stoichiometry of 2:1. Pyrimidine and purine nucleoside probes (50 microM) exerted a competitive inhibitory effect on specific thymidine uptake with K(i) values of 40, 38, 45, and 39 microM for adenosine, uridine, guanosine, and cytidine, respectively. In addition, nucleoside analog drugs significantly decreased specific thymidine uptake, with IC(50) values of 135.1 microM for abacavir and 0.6 microM for zidovudine, which inhibited in a noncompetitive manner. These results suggest that a Na(+)-dependent nucleoside transport system is present in rat microglia and that long-range interactions between antiretroviral nucleoside analog drugs and the nucleoside substrates may occur at the transporter sites. (+info)
Comparison of Abbott LCx Chlamydia trachomatis assay with Gen-Probe PACE2 and culture.
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In this study the LCx assay (a nucleic acid amplification assay) for Chlamydia trachomatis in endocervical samples was compared with the Gen-Probe PACE2 assay (a nucleic acid probe assay) for endocervical samples, and with endocervical culture. In addition, the efficacy of the LCx assay was determined for midstream clean-catch urine samples because it is often necessary to obtain such a sample for routine urine culture and it is simpler to collect only a single sample without also collecting a first-void urine for LCx. Endocervical specimens from 205 patients were tested for C. trachomatis via LCx and PACE2. Of these patients, 203 were tested by culture. Midstream clean-catch urine samples from 75 of these patients were tested by LCx. The sensitivities and specificities for these assays, after discrepant analysis, were 100 and 98.9% for LCx of endocervical samples, 52.4 and 100% for PACE2; and 71.4 and 100% for culture. The sensitivity/specificity of LCx for midstream clean-catch urines was 66.7/98.5%. The apparent prevalence of C. trachomatis in our population was 10.2%. These data indicate that among the methods tested, LCx of endocervical samples had the highest sensitivity for C. trachomatis in this population. The sensitivity of the urine LCx assay using midstream clean-catch collected urines was considerably less than that reported in other studies that used first-void urines but was higher than that of PACE2. (+info)
Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future.
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Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA-DNA-PNA triplex with a looped-out DNA strand. Since its discovery, PNA has attracted major attention at the interface of chemistry and biology because of its interesting chemical, physical, and biological properties and its potential to act as an active component for diagnostic as well as pharmaceutical applications. In vitro studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. However, as with other high molecular mass drugs, the delivery of PNA, involving passage through the cell membrane, appears to be a general problem. (+info)