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Results for “Nested Genes”

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At least 757 records · Page 42Linked to original sources

PubMed1992

Frequency and DNA sequence of tal-1 rearrangement in children with T-cell acute lymphoblastic leukemia.

Using nested polymerase chain reaction (PCR) a gene rearrangement named tal-1 deletion was found in five of 56 leukemic bone marrow samples from children with T-cell acute lymphoblastic leukemia (ALL). The DNA sequences of the PCR fragments consisted of the known conserved germline sequences in addition to short DNA insertions at the breakpoint region, which were different in each patient. Moreover, one patient was examined at diagnosis and at relapse 11 months later, revealing identical DNA sequences at the rearrangement site. The recombination site of the tal rearrangement therefore may be used as a genetic marker for detecting minimal residual disease in about 10% of T-cell ALL in childhood.

Base Sequence↗

PubMed1996

Dual function of the intron of the rat insulin I gene in regulation of gene expression.

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Animals↗

PubMed2002

Fcgamma receptor IIIA polymorphism in Korean patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the presence of various autoantibodies and the deposition of immune complex, which is cleared by Fcgamma receptors. Genotype analysis was done to investigate whether the FcgammaRIIIA-176F/V polymorphism is a risk factor for SLE in Koreans. We genotyped 145 Korean SLE patients and 75 control subjects for FcgammaRIIIA-176F/ V. After amplifying a 1.7-kb fragment containing the Fcgamma/RIIIA-176F/V polymorphic site using two FcgammaRIIIA gene-specific primers, we performed a nested polymerase chain reaction (PCR) for allele-specific genotyping at position 559 in FcgammaRIIIA. FcgammaRIIIA genotype or allele distribution was not significantly different between lupus patients and controls, and also between lupus nephritis patients and healthy controls. Neither creatinine clearance, 24 h urine proteinuria, number of American College of Rheumatology (ACR) criteria, nor the Systemic Lupus International Collaborating Clinics (SLICC)/ACR damage index was different according to the genotype. In conclusion, FcgammaRIIIA-176F/V polymorphism is not associated with SLE in Koreans.

Asian People↗

PubMed2004

Pneumocystis jiroveci isolates with dihydropteroate synthase mutations in patients with chronic bronchitis.

Since mutations in the dihydropteroate synthase (DHPS) gene possibly associated with sulfonamide resistance have been reported in patients with Pneumocystis jiroveci (previously carinii) pneumonia, and since P. jiroveci colonization has been recently demonstrated in patients with chronic pulmonary diseases, the present study aimed to investigate the possible occurrence of P. jiroveci DHPS mutations in patients with chronic bronchitis. P. jiroveci colonization was detected in 15 of 37 non-selected patients with chronic bronchitis by amplifying the large subunit of the mitochondrial gene of the ribosomal RNA using nested PCR. DHPS mutations were demonstrated using touchdown PCR and restriction enzyme analysis in two of eight patients with chronic bronchitis and in two of six patients from the same region who had AIDS-associated Pneumocystis pneumonia. In all cases, mutations were observed in subjects with no prior exposure to sulfonamides. These data could have important implications for public health, since (i) P. jiroveci colonization could speed the progression of chronic bronchitis, and (ii) these patients, who are customary sputum producers, could represent a reservoir for sulfonamide-resistant strains with the potential ability to transmit them to immunocompromised hosts susceptible to Pneumocystis pneumonia.

Age Distribution↗

PubMed2005

Oligonucleotide microarray for identification of Enterococcus species.

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.

DNA, Bacterial↗

PubMed2004

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis.

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.

Amino Acid Sequence↗

PubMed2006

Prevalence of selected bacterial named species and uncultivated phylotypes in endodontic abscesses from two geographic locations.

Evidence is mounting that the prevalence of some putative endodontic pathogens can significantly vary according to the geographic location in which samples were taken. This study aimed to provide additional knowledge on this subject by comparing the frequencies of 10 selected candidate endodontic pathogens in samples of acute apical abscesses obtained from two distinct geographic locations; Portland, OR, and Rio de Janeiro, Brazil. DNA was extracted from aspirates and used as template in nested PCR assays using 16S rRNA gene taxon-specific oligonucleotide primers. Of the target species/phylotypes, Treponema denticola (73% of the cases), Porphyromonas endodontalis (70%), and Tannerella forsythia (57%) were the most prevalent taxa found in Brazilian samples. Dialister invisus (70% of the cases), P. endodontalis (63%) and Dialister pneumosintes (55%) were the most frequent taxa in the Portland, OR samples. Data analysis revealed that T. denticola and T. forsythia were significantly more detected in Brazilian samples than in the Portland, OR samples. Although D. invisus, Filifactor alocis, and Synergistes oral clone W090 were detected in many more samples from the Portland, OR patients, differences were not found to be statistically significant. These findings confirmed that some bacterial taxa can markedly differ in the frequencies they occur in samples from different locations. It remains to be clarified whether this observation translates into relevant therapeutic implications.

Bacterial Infections↗

PubMed2006

In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia.

Neospora caninum was isolated and established in vitro from the skin lesion of a naturally infected dog. The identity of the parasite was evaluated by immunofluorescent antibody test (IFAT), microscopy, Western blotting and polymerase chain reaction (PCR). N. caninum DNA was detected in the whole blood, serum, skin lesion, rectal scrapings and faeces of the infected dog utilising a nested PCR targeting the Nc-5 gene of N. caninum. Antigenic and genetic characterisation of the isolate, designated WA-K9, at a number of loci including the Nc-5 gene, heat shock protein 70 (HSP-70) gene, alpha-tubulin and beta-tubulin genes revealed no variation between this isolate and two N. caninum isolates from different geographic areas. Clinical aspects of this case, which included cutaneous and neurological disease, are also discussed.

Animals↗

PubMed2003

Detection of influenza virus from throat and pharyngeal swabs with a nested duplex light cycler RT-PCR.

A rapid duplex RT-PCR method was developed using the Roche LightCycler technology for detection of influenza type A and influenza type B viruses. Ninety-seven clinical specimens were analyzed using the Lightcycler method compared with conventional viral culture. Thirty-seven specimens (38.1%) were positive by RT-PCR using matrix protein (MP) primers for influenza A or B virus, compared to thirteen culture positive specimens (13.4%). All culture positive specimens were also positive by RT-PCR. A nested PCR reaction using hemagglutination (HA) gene primers confirmed all of the earlier positive PCR reactions. This LightCycler PCR technique was found to be more sensitive than viral culture for influenza virus detection.

Cohort Studies↗

PubMed1995

Viremia induced by influenza virus.

A mouse model of influenza A/PR/8 virus infection was adopted to investigate the blood and various tissues of intranasally infected mice for the presence of viral RNA by using the nested polymerase chain reaction. The nucleoprotein gene was detected in the red blood cell fraction from 1 to 5 days post-inoculation, while it was found in the lung and brain up to 14 days and in the liver, spleen, kidney, heart, and skeletal muscle up to 7 days. The virus-specific messenger RNA was transiently found in these organs. When mice received the uv-inactivated virus, viremia did not occur. The prior transfer of the hyperimmune serum prevented pneumonia but not bronchitis, and viremia was totally abolished. These results suggest: (1) viremia occurs during the acute phase of infection, (2) the virus is present in various organs and there the virus gene is transiently expressed, and (3) the virus enters the blood stream possibly through capillaries of the infected alveolar wall. Viremia may influence the pathogenesis of influenza.

Animals↗

PubMed2001

Extreme morphological and ecological homoplasy in tropical salamanders.

Fossorial salamanders typically have elongate and attenuated heads and bodies, diminutive limbs, hands and feet, and extremely elongate tails. Batrachoseps from California, Lineatriton from eastern México, and Oedipina from southern México to Ecuador, all members of the family Plethodontidae, tribe Bolitoglossini, resemble one another in external morphology, which has evolved independently. Whereas Oedipina and Batrachoseps are elongate because there are more trunk vertebrae, a widespread homoplasy (parallelism) in salamanders, the genus Lineatriton is unique in having evolved convergently by an alternate "giraffe-neck" developmental program. Lineatriton has the same number of trunk vertebrae as related, nonelongated taxa, but individual trunk vertebrae are elongated. A robust phylogenetic hypothesis, based on sequences of three mtDNA genes, finds Lineatriton to be deeply nested within a clade characterized by generalized ecology and morphology. Lineatriton lineolus, the only currently recognized taxon in the genus, shows unanticipated genetic diversity. Surprisingly, geographically separated populations of L. lineolus are not monophyletic, but are sister taxa of different species of the morphologically generalized genus Pseudoeurycea. Lineatriton, long thought to be a unique monospecific lineage, is polyphyletic. Accordingly, the specialized morphology of Lineatriton displays homoplasy at two hierarchical levels: (i) with respect to other elongate lineages in the family (convergence), and (ii) within what is currently recognized as a single taxon (parallelism). These evolutionary events are of adaptive significance because to invade the lowland tropics salamanders must be either arboreal or fossorial; the repeated evolution of elongation and attenuation has led to multiple lowland invasions.

Animals↗

PubMed2006

Chlamydophila pneumonia: Specific mRNA in aorta ascendens in patients undergoing coronary artery by-pass grafting.

The objective of this prospective study was to investigate if Chlamydophila pneumoniae (Cp)-specific DNA and mRNA are present in tissue samples from the wall of aorta ascendens in patients undergoing by-pass surgery for coronary artery disease (CAD) that includes stable angina pectoris (SAP, 25 patients) and acute coronary syndrome (ACS, 19 patients). Viable Cp was detected in 8/44 (18%) patients using reversed transcriptase PCR (RT-PCR) against bacterial mRNA with detection of cDNA using real-time PCR against the MOMP gene. Cp DNA was detected by nested PCR in 22/44 (50%) patients and by real-time PCR in 13/44 (30%) patients. In total, 24/44 (55%) patients were positive for Cp nucleic acid in any PCR. Antibodies to Cp were detected in 13/24 (54%) Cp PCR-positive and in 15/20 (75%) Cp PCR-negative patients. Nested PCR was run on throat swabs from all patients. No significant differences were noted between SAP and ACS patients regarding PCR results or serology. It has been suggested that Cp may be a 'silent passenger' picked up by the atherosclerotic plaque. Our findings of viable and metabolically active bacteria in aortic tissue add further support to the hypothesis that Cp may have an active role in the pathogenesis of atherosclerosis.

Aged↗

PubMed2005

Differentiation of Culicoides obsoletus and Culicoides scoticus (Diptera: Ceratopogonidae) based on mitochondrial cytochrome oxidase subunit I.

Culicoides obsoletus (Meigen) and Culicoides scoticus Downes & Kettle (Diptera: Ceratopogonidae) are sibling species of the Obsoletus group. This group comprises species of biting midges that are suspect vectors of bluetongue virus (family Reoviridae, genus Orbivirus, BTV) and African horse sickness virus (family Reoviridae, genus Orbivirus, AHSV). BTV and AHSV have been isolated several times from females of this group, although it has not been possible to determine the particular species harboring the virus, because of the inability to clearly identify the females of each species based on morphology. Both sexes of C. obsoletus and C. scoticus midges from Catalonia and the Balearics (Spain) were sequenced for the mitochondrial gene cytochrome oxidase subunit I (COI), and these sequences were analyzed to determine intra- and interspecific genetic variability. Species-specific primers for C. obsoletus and C. scoticus were designed and a polymerase chain reaction (PCR)-based diagnostic assay based on the COI gene and using a hemi-nested PCR technique was developed for reliably distinguishing the females of both species. The species-specific PCR diagnostic was compared with morphological discrimination of C. obsoletus and C. scoticus females. The morphologic characters were not fully reliable.

African Horse Sickness↗

PubMed1989

The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes.

The Pseudomonas aeruginosa capsule, composed of polysaccharide alginate, is an important Pseudomonas virulence factor encountered primarily in cystic fibrosis. The regulatory algR gene positively controls transcription of a key alginate biosynthetic gene, algD. The algR gene was subcloned and sequenced by creating a set of nested deletions in M13 bacteriophage. DNA sequence analysis of algR revealed the homology of its gene product with a recently recognized class of environmentally responsive bacterial regulatory genes, including ompR, phoB, sfrA, ntrC, spoOA, dctD, and virG; these transcriptional activators control cellular reactions to osmotic pressure, phosphate limitations, or specific chemical compounds present in the medium or released from wounded host tissue. These findings indicate that novel conditions in lungs affected by cystic fibrosis may be participating in the control of mucoidy.

Alginates↗

PubMed2003

Detection of Borrelia lonestari, putative agent of southern tick-associated rash illness, in white-tailed deer (Odocoileus virginianus) from the southeastern United States.

To determine if white-tailed deer may serve as a reservoir host for Borrelia lonestari, we used a nested PCR for the Borrelia flagellin gene to evaluate blood samples collected from deer from eight southeastern states. Seven of 80 deer (8.7%) from 5 of 17 sites (29.4%) had sequence-confirmed evidence of a B. lonestari flagellin gene by PCR, indicating that deer are infected with B. lonestari or another closely related Borrelia species. Our findings expand the known geographic range of B. lonestari and provide the first evidence of this organism in a vertebrate other than humans.

Animal Diseases↗

PubMed2003

Evaluation of a PCR assay for detection of Streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, but it is undoubtedly underdiagnosed. We used a nested PCR assay (targeting the pneumolysin gene) to detect S. pneumoniae DNA in multiple sample types from 474 adults with community-acquired pneumonia and 183 control patients who did not have pneumonia. Plasma or buffy coat samples were PCR positive in only 6 of the 21 patients with positive blood cultures for S. pneumoniae and in 12 other patients (4 of whom had no other laboratory evidence of S. pneumoniae infection). Buffy coat samples from two control patients (neither having evidence of S. pneumoniae infection), but no control plasma samples, were PCR positive. Although pneumococcal antigen was detected in the urine from 120 of 420 (29%) patients, only 4 of 227 (2%) urine samples tested were PCR positive. Overall, 256 of 318 (81%) patients had PCR-positive sputum samples, including 58 of 59 samples from which S. pneumoniae was cultured. Throat swab samples from 229 of 417 (55%) patients were PCR positive and, in those who produced sputum, 96% also had positive PCR results from sputum. Throat swabs from 73 of 126 (58%) control patients were also PCR positive. We conclude that the pneumolysin PCR assay adds little to existing diagnostic tests for S. pneumoniae and is unable to distinguish colonization from infection when respiratory samples are tested.

Adult↗

PubMed1991

Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction.

Borrelia burgdorferi, the causative agent of Lyme disease, was detected in patients' serum by DNA amplification using the polymerase chain reaction (PCR). B burgdorferi was pelleted from serum samples by centrifugation (10,000 x g for 10 minutes) and lysed by treatment with ammonium hydroxide (100 degrees C for 15 minutes). Two pairs of "nested" PCR primers complementary to the gene encoding a major outer surface protein (OSP A) of B burgdorferi were used in DNA amplification under standard PCR conditions (Perkin-Elmer Cetus). Two out of five patients with erythema migrans, the characteristic primary skin lesion associated with early Lyme disease, were positive by the PCR. This method could form the basis of a useful routine laboratory test in those cases of early Lyme disease where conventional serological testing commonly yields equivocal or false negative results.

Adult↗

PubMed2001

Characterization of soybean bradyrhizobia for which serogroup affinities have not been identified.

The USDA, ARS National Rhizobium Germplasm Collection contains 143 accessions of slow-growing soybean strains among which there are 17 distinct serological groups. However, 11 strains appear to have no serological affinity with the 17 serogroups. Therefore, we determined whether these strains were diverse and examined their phylogenetic placement. Nine strains formed nitrogen-fixing symbioses with soybean indicating that these accessions were not contaminants. We concluded from results of amplified fragment length polymorphism (AFLP) analysis, using 3 selective primers with 8 strains, that they were genetically dissimilar. Nine strains were examined for their fatty acid composition using fatty acid methyl ester (FAME) derivatives. The FAME results with 5 strains and serotype strains of Bradyrhizobium elkanii were similar, while results with each of the remaining 2 pairs were either similar to the type strain of Bradyrhizobium japonicum (USDA 6) or to USDA 110. Evolutionary history of 9 strains was reconstructed from sequence divergence of a combination of the complete 16S rRNA gene, the internally transcribed spacer region, and about 400 bases of the 5' end of the 23S rRNA gene. Placement of 5 strains was nested within B. elkanii, 2 with USDA 110, and the other 2 with USDA 6. We concluded that soybean isolates that cannot be placed within one of the 17 established serogroups are phenotypically and genetically as diverse as the serotype strains.

Bacterial Typing Techniques↗