Nucleotide Sequence Analysis (nucleotide + sequence_analysis)

Distribution by Scientific Domains
Distribution within Life Sciences

Selected Abstracts

Rep helicase suppresses short-homology-dependent illegitimate recombination in Escherichia coli

GENES TO CELLS, Issue 11 2005
Kouya Shiraishi
To study roles of Rep helicase in short-homology-dependent illegitimate recombination, we examined the effect of a rep mutation on illegitimate recombination and found that the frequency of spontaneous illegitimate recombination is enhanced by the rep mutation. In addition, illegitimate recombination was synergistically enhanced by the rep mutation and UV irradiation, showing that Rep helicase plays a role in suppression of spontaneous as well as UV-induced illegitimate recombination. The defect in RecQ helicase also has a synergistic effect on the increased illegitimate recombination in the rep mutant. It was also found that the illegitimate recombination induced by the rep mutation is independent of the RecA function with or without UV irradiation. Nucleotide sequence analyses of the recombination junctions showed that the illegitimate recombination induced by the rep mutation mostly takes place between short homologous sequences. Based on the fact that the defect of Rep helicase induces replication arrest during replication, resulting in the formation of DNA double-strand breaks, we propose a model for illegitimate recombination, in which double-strand breaks caused by defect of Rep helicase promotes illegitimate recombination via short-homology-dependent-end-joining. In addition, the mechanism of synergistic action between the rep mutation and UV irradiation on illegitimate recombination is discussed. [source]

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples

Objective To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. Design A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. Results The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per µl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. Conclusion The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis. [source]

Molecular characterization of a human scavenger receptor, human MARCO

FEBS JOURNAL, Issue 3 2000
Nabil A. Elshourbagy
Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria-binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full-length hMARCO clone encoded a 519-amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand-binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low-density lipoprotein (LDL), despite its similarity to the elongated collagen-like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis. [source]

Richter syndrome in B-cell chronic lymphocytic leukemia

Naoya Nakamura
Richter syndrome (RS) is well known as a secondary high-grade lymphoma, mostly diffuse large B-cell lymphoma (DLBCL) developed in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this review, we describe clinicopathological, histological, immunophenotypical and genetic findings of RS. The patients with RS, regardless of transformation of pre-existing clone or de novo malignant clone, were resistant to conventional combined chemotherapy and died within months of diagnosis. Molecular techniques can provide convincing results for the clonal relationship of RS to pre-existing B-CLL. When RS carries a same rearrangement band or a same sequence as B-CLL by Southern blotting or nucleotide sequence analyses of immunoglobulin heavy and/or light chain genes, it is suggested to that RS transforms from original B-CLL. These analyses have showed that approximately two-thirds of RS cases evolved from a B-CLL clone. How and where does the B-CLL clone evolve to RS? The genetic alteration of transforming B-CLL clone into RS has been addressed. Abnormalities of chromosomes 11 and 14 were most frequently involved in RS, but non-specific. In addition, RS does not include chromosomal translocation between Ig locus and oncogenes or rearrangements of bcl-6 gene, both of which were found in some de novo DLBCL. Several candidates, such as mutation of p53 gene and abnormalities of cyclin dependent kinase inhibitor, have been proposed to play an important role in the transformation of a part of B-CLL. However, there is still uncertainly as to how B-CLL progresses or develops into RS. [source]

A novel neisserial shuttle plasmid: A useful new tool for meningococcal research

Clíona A. O' Dwyer
Abstract We report the identification and nucleotide sequence analysis of a cryptic plasmid pMIDG2830 from the Gram-negative bacterium Neisseria flavescens. The largest open reading frame encodes a protein similar to the replication protein, RepA, found in pAB49 from Acinetobacter baumannii and pNI10 from Pseudomonas. Modified by the incorporation of a kanamycin resistance cassette, the plasmid can be stably maintained in Escherichia coli and Neisseria meningitidis, and can be used as a shuttle plasmid in meningococcal research. [source]

Importance of Cryphonectria parasitica stromata production and intermediate-pigmented isolates in spread of Cryphonectria hypovirus 1 on grafted American chestnut trees

E. P. Hogan
Summary Large, surviving American chestnut trees, grafted in 1980, were inoculated in 1982 and 1983 with four ,white' European (French and Italian) hypovirulent strains of Cryphonectria parasitica infected with Cryphonectria hypovirus 1 (CHV1). Spread of Italian CHV1-Euro7, indicated by nucleotide sequence analysis of CHV1 isolates, and a high level of blight control, occurred on these trees for over 20 years. However, the means by which CHV1 spreads and the possible role of stromata production in that spread are unknown. In this study, 249 C. parasitica isolates were recovered from stromata excised from natural cankers on the grafted trees and plated on an agar medium; 5.2% of the stromata yielded white phenotype isolates, 9.2% yielded intermediate-pigmented isolates (30,70% pigmentation) and the remainder were normal-pigmented isolates. For comparison, cankers artificially established on blight-free, forest-clear-cut American chestnut trees, following inoculation with three Italian white hypovirulent strains, were evaluated in a similar manner. Of 241 C. parasitica isolates recovered from stromata, 66.4% had a white colony phenotype, 19.1% had an intermediate-pigmented phenotype and the remainder were normal-pigmented isolates. For single conidia collected from stromata and plated, nearly equal frequencies of only white and intermediate-pigmented colony phenotypes were obtained. Following dsRNA extraction and electrophoresis, 21 of 33 intermediate-pigmented isolates were positive for CHV1. Some normal pigmented isolates also were positive for CHV1. Single-sporing a CHV1-positve, normal-pigmented, natural-canker, stroma isolate (Str 1) from the grafts resulted in several deeply red-orange pigmented (JR) isolates as well as some white isolates. The dsRNA in the JR isolate was extracted and cDNAs made by reverse transcriptase-polymerase chain reaction (RT-PCR) for part of a region (p29) in ORF A. Nucleotide sequencing indicated 100% identity to CHV1 present in the inoculated Italian white strain, Ep 47. The results indicate that stromata production on the grafted trees may contribute to CHV1 spread, and the presence of CHV1 in intermediate-pigmented isolates and some normal pigmented isolates indicates these isolates, often overlooked, may be important in CHV1 spread and the high level of blight control on the grafted trees. [source]

Hepatitis B virus genotypes and serotypes in western India: Lack of clinical significance

Swati S. Gandhe
Abstract To determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection. J. Med. Virol. 69:324,330, 2003. © 2003 Wiley-Liss, Inc. [source]

Identification and Molecular Characterization of ,Candidatus Phytoplasma mali' Isolates in North-western Italy

Paola Casati
Abstract Apple proliferation (AP) is an important disease and is prevalent in several European countries. The causal agent of AP is ,Candidatus Phytoplasma mali' (,Ca. Phytoplasma mali'). In this work, isolates of ,Ca. Phytoplasma mali' were detected and characterized through polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses of 16S rRNA gene and non-ribosomal DNA fragment. The presence of three AP subtypes (AT-1, AT-2 and AP-15) was identified in 31 symptomatic apple trees and two samples each constituted by a pool of five insects, collected in north-western Italy, where AT-1 is a dominant subtype. Subsequent nucleotide sequence analysis of the PCR-amplified 1.8 kb (P1/P7) fragment, containing the 16S rDNA, the 16S,23S intergenic ribosomal region and the 5,-end of the 23S rDNA, revealed the presence of at least two phytoplasmal genetic lineages within the AT-1 subtype, designed AT-1a and AT-1b. Moreover, in silico single nucleotide polymorphism (SNP) analysis based on 16S rDNA sequence can differentiate AT-1 subtype from AT-2 and AP-15 subtypes. Our data showed a high degree of genetic diversity among ,Ca. Phytoplasma mali' population in north-western Italy and underlined the possible use of the 16S rDNA analysis for the identification and the geographical origin assignation of isolates of AP phytoplasma. Molecular markers on 16S rDNA, here identified, could be useful for studying the epidemiology of AP disease. [source]

Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection

M. S. Choi
Summary., We investigated the overall and site-specific prevalence of pre-S mutations and its clinical significance in patients with genotype C hepatitis B virus (HBV) infection. Three hundred subjects were included: 50 asymptomatic carriers (AC), 87 chronic hepatitis (CH), 91 liver cirrhosis (LC) and 72 hepatocellular carcinoma (HCC). Pre-S mutations were determined by nucleotide sequence analysis. Possible correlations between pre-S mutations and clinical/virological parameters were examined. Pre-S mutations were detected in 82 cases (27.3%); it was more frequently found in HCC (43.1%) and LC (35.2%) group than in the CH (20.7%) and AC (2.0%) group. Pre-S2 deletion was the most commonly found mutation (10.7%), followed by pre-S2 start codon mutation (9.7%), pre-S1,S2 deletion (3.0%) and both pre-S2 deletion and start codon mutation (2.7%). Pre-S2 deletion and pre-S2 start codon mutation were more frequently detected in advanced diseases (LC and HCC). Pre-S mutations were associated with older age and higher rates of positive HBV DNA (,0.5 pg/mL). Advanced disease and positive HBV DNA were shown to be independent predictors of pre-S mutations by logistic regression analysis. These findings suggest that pre-S mutations, especially pre-S2 deletions and pre-S2 start codon mutations, are common in patients with genotype C HBV infection and are associated with advanced liver disease and active viral replication. [source]

A rapid real-time PCR assay for determination of hepatitis C virus genotypes 1, 2 and 3a

A. Moghaddam
Summary., The genotypes of hepatitis C virus (HCV) in serum of patients have been described as independent predictors of success of antiviral therapy. Therefore, different antiviral regimens have been proposed depending on the infecting HCV genotype. HCV strain is usually determined by polymerase chain reaction (PCR) amplification of genome followed by sequencing or by line-probe assays. We report a new one step real-time PCR assay for genotyping of HCV strains that are prevalent in patients in Norway. HCV types 1, 2 and 3a were genotyped unambiguously in 37 patient serum samples with 100% correlation to genotyping by nucleotide sequence analysis and line-probe assays. Genotyping could also be confirmed against an HCV genotype panel from the National Institute for Biological Standards and Control. This assay does not require manipulation of amplified PCR products, it involves very little hands on and analysis time. This assay can be used for rapid genotyping of HCV-RNA in infected patients to aid physicians decide suitability of patients for treatment and subsequent length of treatment. [source]

Characterization of Potato rough dwarf virus and Potato virus P: distinct strains of the same viral species in the genus Carlavirus

C. Nisbet
In an attempt to resolve whether two putative carlaviruses, potato rough dwarf virus (PRDV) and potato virus P (PVP), reported infecting potato in Argentina and Brazil, respectively, are the same virus in the genus Carlavirus, they were characterized using nucleotide sequence analysis, serology and bioassay. For PRDV and PVP, sequences of 2016 and 1492 bp, respectively, were obtained. Similarity searches of coat-protein amino acid sequences and the presence of the nucleic-acid-binding protein exhibiting the characteristic motif C-X2 -C-X11 -C-X4 -C, highly conserved in carlaviruses, showed PRDV and PVP to be members of the genus Carlavirus. Further analysis showed that they were the same virus species since the full coat-protein sequence, translated from the nucleotide sequence, exceeded the 84% minimum identity required for members of the same species within the genus Carlavirus, at 90·8% identity in an ungapped alignment of 304 amino acids. Moreover, the viruses could not be distinguished using polyclonal antibodies raised to PRDV and PVP in ELISA. They could, however, be distinguished using an anti-PVP monoclonal antibody, which did not react to PRDV, and by differences in the susceptibility of plant species and potato cultivars to infection and/or symptom expression following mechanical inoculation. Host plants Datura metel, Nicandra physalodes and Nicotiana edwardsonii were reliably infected systemically with PVP, but not with PRDV. Although all potato cultivars tested were infected, there were marked differences between cultivars in the symptoms produced. The cultivars Ditta and King Edward showed symptoms (including stunting and mottle) when infected with PRDV, but not when infected with PVP. Based on the results, it is proposed that PRDV is a strain of PVP within the genus Carlavirus. For detection of the viruses in postentry quarantine, it is recommended that PRDV or PVP polyclonal antibodies are used, together with inoculation to Nicotiana occidentalis N. megalosiphon -P1. For differentiation of PRDV and PVP, the PVP monoclonal antibody may be used together with inoculation to D. metel and N. physalodes (systemic infection with PVP but not PRDV, determined using ELISA). [source]

Prunus necrotic ringspot virus isolates in stone fruit germplasm accessions and cultivars in Israel

Summary Prunus necrotic ringspot virus (PNRSV) was detected in almonds, plum and apricot germplasm accessions and local almond cultivars in Israel. PNRSV was widespread both in wild and cultivated almond trees and uncommon in wild apricots and plums. The possible variation among the PNRSV isolates was initially evaluated by restriction analysis of PCR products representing the CP gene with the endonuclease RsaI and followed by nucleotide sequence analysis of selected isolates. It was concluded that all 13 isolates belong to group PV96, the largest cluster of PNRSV isolates, described previously. Two PNRSV isolates, one from a plum accession and one from an almond cultivar, were found to be distinct members of group PV96 with unique nucleotide modifications not found in other documented isolates of this virus. However, no PNRSV isolate typical to a specific host and/or to the Middle East region could be identified. This study expands the body of data on variability of PNRSV isolates and highlights the importance of assessing the virus status of germplasm collections by applying reliable diagnostic and differentiating methods. [source]

Phase II randomized study of daily gefitinib treatment alone or with vinorelbine every 2 weeks in patients with adenocarcinoma of the lung who failed at least 2 regimens of chemotherapy

CANCER, Issue 9 2007
Yuh-Min Chen MD
Abstract BACKGROUND. The objective of this study was to assess the efficacy of adding chronic, intermittent, low-dose vinorelbine to gefitinib treatment for patients who had adenocarcinoma of the lung who failed ,2 regimens of chemotherapy. METHODS. Patients were randomized into 2 arms: Oral gefitinib 250 mg daily (the G arm) or vinorelbine 15 mg/m2 as an intravenous infusion on Day 1 and oral gefitinib 250 mg daily on Days 2 through 14 every 2 weeks (the GV arm). From August 2004 to October 2005, 48 patients were enrolled. Epidermal growth factor receptor (EGFR) exon 18 through 21 nucleotide sequence analysis and fluorescence in situ hybridization were performed in patients who had tumor tissue specimens available for analysis. RESULTS. After randomization, each arm had 24 patients. However, 3 patients refused vinorelbine treatment and were given gefitinib treatment only. Thus, 27 patients received G treatment, and 21 patients received GV treatment. Objective response rates were 55.6% in the G arm and 52.4% in the GV arm. All toxicities in both arms were mild. The 1-year progression-free survival rate was 57.1% in the GV arm and 21.2% in the G arm (P = .008). The median survival was 13.3 months in the G arm and 23.4 months in the GV arm (P = .1231). Three of 6 patients (50%) had an exon 19 in-frame deletion, and 2 of 10 patients had EGFR gene high polysomy or amplification (20%). CONCLUSIONS. Gefitinib was highly effective in ethnic Chinese patients with adenocarcinoma of the lung who failed previous platinum and taxane treatment. The addition of low-dose vinorelbine every 2 weeks produced a significantly better 1-year progression-free survival rate. Cancer 2007. © 2007 American Cancer Society. [source]