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Polymerase Gene (polymerase + gene)
Selected AbstractsGenome sequences of two novel phages infecting marine roseobactersENVIRONMENTAL MICROBIOLOGY, Issue 8 2009Yanlin Zhao Summary Two bacteriophages, DSS3,2 and EE36,1, which infect marine roseobacters Silicibacter pomeroyi DSS-3 and Sulfitobacter sp. EE-36, respectively, were isolated from Baltimore Inner Harbor water. These two roseophages resemble bacteriophage N4, a large, short-tailed phage infecting Escherichia coli K12, in terms of their morphology and genomic structure. The full genome sequences of DSS3,2 and EE36,1 reveal that their genome sizes are 74.6 and 73.3 kb, respectively, and they both contain a highly conserved N4-like DNA replication and transcription system. Both roseophages contain a large virion-encapsidated RNA polymerase gene (> 10 kb), which was first discovered in N4. DSS3,2 and EE36,1 also possess several genes (i.e. ribonucleotide reductase and thioredoxin) that are most similar to the genes in roseobacters. Overall, the two roseophages are highly closely related, and share 80,94% nucleotide sequence identity over 85% of their ORFs. This is the first report of N4-like phages infecting marine bacteria and the second report of N4-like phage since the discovery of phage N4 40 years ago. The finding of these two N4-like roseophages will allow us to further explore the specific phage,host interaction and evolution for this unique group of bacteriophages. [source] Effects of interferon alpha therapy on the catalytic domains of the polymerase gene and basal core promoter, precore and core regions of hepatitis B virusJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2003ROBERT YUNG MING CHEN Aims: The aim of the present study was to examine the catalytic domains of the polymerase gene, the basal core promoter and the precore and core regions of the hepatitis B virus (HBV) genome for specific mutations. These may account for the response to interferon alpha (IFN-,) treatment, which may have prognostic value. Methods: Multiple serum samples were collected prospectively from 30 patients with chronic active hepatitis B who were treated with IFN-,. Patients were assigned to one of three groups: group A (n = 11) and group B (n = 10) individuals were hepatitis B e antigen (HBeAg)-positive prior to treatment. Group A patients underwent HBeAg seroconversion after treatment while group B patients did not. Group C (n = 9) patients were HBeAg-negative prior to treatment. The HBV DNA was extracted from the sera collected before, during and after treatment and the various genomic regions were amplified, sequenced and examined for mutations. Results: During IFN-, therapy, multiple changes were found in the catalytic domains of the HBV polymerase gene in all groups. The frequency of mutations and associated amino acid changes were highest in virus from group C patients and lowest in group A patients. The interdomain regions of the viral polymerase were the most affected. Multiple mutations were also found in the precore, core and core promoter regions. However, no specific mutations were associated with clinical response or outcome. Conclusions: During IFN-, treatment, multiple mutations occurred in the HBV genome, including the catalytic domains of the polymerase gene. Changes that did occur could not be correlated to the clinical response or treatment outcome. However, no mutations were found that have been linked to lamivudine escape, indicating that lamivudine therapy would be effective in IFN-, non-responder patients. [source] Increased detection of HBV DNA in HBsAg-positive and HBsAg-negative South African HIV/AIDS patients enrolling for highly active antiretroviral therapy at a Tertiary HospitalJOURNAL OF MEDICAL VIROLOGY, Issue 3 2009Azwidowi Lukhwareni Abstract This retrospective study investigated the prevalence of hepatitis B virus (HBV) in 192 stored sera from human immunodeficiency virus (HIV) positive South African patients initiating antiretroviral therapy (ART), and explored the implications of HBV,HIV co-infection on laboratory diagnosis of HBV. HBV serology (HBsAg, anti-HBs and anti-HBc) and nested HBV PCR assays targeting the HBV polymerase gene were performed, with HBV DNA positive samples being quantified with Cobas Taqman HBV test 48 assay (Roche Diagnostics). The study found that 63% (121/192) of patients had past or present HBV infection, and 40.6% (78/192) had detectable HBV DNA. Also, 22.9% (44/192) of patients were HBsAg positive and HBV DNA positive, while 23% (34/148) of HBsAG negatives had occult HBV infections. Of the 78 HBV DNA positive samples, 62.8% had viral loads ranging from 102 to ,108 IU/ml, and 37.2% had HBV viral loads <200 IU/ml. There was a statistically significant positive association between HBsAg-positivity and high viral loads, with 27% (12/44) of HBsAg positives having HBV viral loads between 104 and ,108 IU/ml, compared to only 5.9% (2/34) of HBsAg negatives (relative risk: 4.64; 95% confidence interval: 1.11, 19.35; chi-square P -value,=,0.015). The study shows that the majority of HIV/AIDS patients initiating ART have either acute or chronic HBV infections, and further confirms that HIV remains a risk factor for occult HBV infections in South African patients as previously shown. The findings strongly support HBV screening in all HIV-positive patients initiating ART in South Africa, considering that current ART regimens include drugs with anti-HBV activity (e.g., lamivudine). J. Med. Virol. 81:406,412, 2009. © 2009 Wiley-Liss, Inc. [source] Susceptibility of hepatitis B virus to lamivudine restored by resistance to adefovirJOURNAL OF MEDICAL VIROLOGY, Issue 3 2009H.L. Zaaijer Abstract Serial monotherapy and add-on regimes for treatment of chronic hepatitis B virus (HBV) infection may induce the accumulation of viral resistance mutations in patients, reducing the options for ongoing viral suppression. The induction of antiviral resistance by serial application of polymerase inhibitors does not necessarily imply that the subsequent combined use of the drugs will fail. Some HIV strains resistant to one polymerase inhibitor show increased susceptibility to another polymerase inhibitor. After failure of sequential lamivudine and adefovir monotherapy, two patients with hepatitis B changed to treatment with lamivudine plus adefovir and had renewed suppression of HBV. To study the mutational history of resistant HBV subpopulations in the two patients, a part of the HBV polymerase gene was amplified, cloned, sequenced, and analyzed for the presence of mutations, in sequential plasma samples. In both patients serial monotherapy caused the replacement in all HBV clones of wild-type virus by classical lamivudine resistant mutants (L180M and M204V/I), which were replaced subsequently by adefovir resistant mutants (A181V and N236T). When finally lamivudine was added to adefovir, the A181V adefovir mutation persisted in all clones and lamivudine-related mutations did not reappear. During 18 months of combination therapy, HBV-DNA levels decreased 10,000, respectively, 1,000-fold, despite the earlier resistance to lamivudine and adefovir. Although clinically insufficient, this effect indicates that HBV polymerase resistance mutations may be antagonistic, which is relevant if chronic HBV infection is to be treated by a combination of polymerase inhibitors. J. Med. Virol. 81:413,416, 2009. © 2009 Wiley-Liss, Inc. [source] Epidemiology of enterovirus types causing neurological disease in Austria 1999,2007: Detection of clusters of echovirus 30 and enterovirus 71 and analysis of prevalent genotypesJOURNAL OF MEDICAL VIROLOGY, Issue 2 2009Birgit Ortner Abstract Between 1999 and 2007 1,388 stool specimens from patients with acute flaccid paralysis or aseptic meningitis were submitted to the Austrian reference laboratory for poliomyelitis. Samples (201) yielded non-poliovirus enterovirus in culture. One hundred eighty-one viruses were available for typing and 78 isolates which remained serologically untyped were further analyzed by CODEHOP-PCR and sequencing of the VP1 gene and the 5,-untranslated region (5,-UTR). Typing revealed an Echovirus 30 outbreak in northwestern Austria in 2000, which was in accordance with the situation in Europe, and no dramatic seasonal changes of Coxsackie viruses were observed. In 2002/2003 a small outbreak of enterovirus 71 (EV71), affected 12 patients in the province of Styria. This virus was identified as genotype C1 and appeared to be genetically distinct from the isolates observed in 2001/2002 in Vienna. In 2004 two unrelated cases occurred in Lower Austria, which were identified as genotype C4, which has been described associated with high mortality most recently in China. In contrast to the situation in Asia the detected EV71 cases were not associated with hand,foot,mouth disease, but with serous meningitis only. This was surprising as a recent publication suggested a reduced neurovirulence of C1 genotype in children in Norway, presumably due to alterations in 5,-UTR and polymerase gene. However, comparing the 5,-UTR of the Austrian isolates and established virulent reference strains to the Norwegian isolate and an attenuated EV71 laboratory strain we did not find an indication that the genotype C1 possesses a RNA structure in its 5,-UTR leading to reduced neurovirulence. J. Med. Virol. 81:317,324, 2009. © 2008 Wiley-Liss, Inc. [source] Identification of Three Strains of a Virus Associated with Cassava Plants Affected by Frogskin DiseaseJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2008L. A. Calvert Abstract Cassava Frogskin Disease (CFSD) can cause severe damage to cassava roots and is one of the most important diseases of cassava in Latin America. The principal objective of this study was to identify the causal agent of CFSD. Electron microscopy, viral purifications, double-stranded RNA (dsRNA) analysis, cloning, sequencing, rtPCR and hybridizations were carried out to characterize and associate a novel virus with the disease. Virus-like particles of 70 and 45 nm in diameter were found in affected cassava plants and partially purified preparations respectively. Nine species of dsRNA were associated with this disease and cDNA clones to six genomic segments were synthesized from the purified dsRNAs. The putative proteins predicted from the sequence of the cassava virus cDNA clones have similarity with the P1, P2, P3, P4, P5 and P10 proteins of Rice ragged stunt virus (RRSV). Phylogenic analysis confirmed that this virus is a member of the family Reoviridae and is most closed related to RRSV. Hybridization analyses of dsRNA identified S1, S2, S3, S4, S5 and S10 genomic segments in the CFSD-affected plants, but not in healthy controls. Additionally, 26 isolates were compared using a portion of the putative polymerase gene. The virus was detected in all 26 isolates, and they were classified into three distinct races. The association of this virus with CFSD was strengthened by the detection of this virus in diseased plants collected from different locations throughout Colombia. [source] Evolution of hepatitis B virus precore/basal core promoter gene in HBeAg-positive chronic hepatitis B patients receiving lamivudine therapyLIVER INTERNATIONAL, Issue 1 2004Chih-Lin Lin Abstract: Aim: Lamivudine is effective in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, but the relapse rate after cessation of treatment is high. The evolution of viral genome may contribute to the viral replication under antiviral pressure of lamivudine. We therefore determined the evolution of hepatitis B virus (HBV) precore/basal core promoter and polymerase genes in HBeAg-positive chronic hepatitis B patient during lamivudine therapy. Method: Thirteen patients with HBeAg-positive chronic hepatitis who had received short-term lamivudine therapy (mean, 30 weeks) during 1999,2001 were enrolled. The precore/basal core promoter region and polymerase gene were amplified and directly sequenced before, during and post lamivudine treatment. Result: HBeAg loss or seroconversion occurred in 11, but eight relapsed after stopping therapy and five had reversion of HBeAg. Before treatment, basal core promoter mutation was found in 1. In the first 3 months of therapy, a rapid decline of serum HBV DNA level accompanied with basal core promoter mutation appeared in 11 of 13 patients (vs. before therapy; P=0.003). However, this mutant was replaced by wild-type virus in four of eight patients who relapsed after treatment. There was no significant change of precore sequences before and during therapy. Conclusions: Lamivudine therapy may result in the rapid development of basal core promoter mutation of HBV, but this mutation may revert to wild type gradually after cessation of therapy. [source] SVISS , a novel transient gene silencing system for gene function discovery and validation in tobacco plantsTHE PLANT JOURNAL, Issue 5 2002Véronique Gosselé Summary We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family. [source] The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 proteinCELLULAR MICROBIOLOGY, Issue 5 2008Igor Mazur Summary The 11th influenza A virus protein PB1-F2 was previously shown to enhance apoptosis in response to cytotoxic stimuli. The 87 amino acid protein that is encoded by an alternative reading frame of the PB1 polymerase gene was described to localize to mitochondria consistent with its proapoptotic function. However, PB1-F2 is also found diffusely distributed in the cytoplasm and in the nucleus suggesting additional functions of the protein. Here we show that PB1-F2 colocalizes and directly interacts with the viral PB1 polymerase protein. Lack of PB1-F2 during infection resulted in an altered localization of PB1 and decreased viral polymerase activity. Consequently, mutant viruses devoid of a functional PB1-F2 reading frame exhibited a small plaque phenotype. Thus, we have identified a novel function of PB1-F2 as an indirect regulator of the influenza virus polymerase activity via its interaction with PB1. [source] The genome of Syntrophomonas wolfei: new insights into syntrophic metabolism and biohydrogen productionENVIRONMENTAL MICROBIOLOGY, Issue 8 2010Jessica R. Sieber Summary Syntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen- and/or formate-using microorganisms. This slow-growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains 10 RNA-directed, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate or unsaturated fatty acids to re-oxidize reduced cofactors. Syntrophomonas wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologues for ,-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from four to eight carbons in length.Syntrophomonas wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H2 from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron,sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions. [source] Genomic analysis of cancer tissue reveals that somatic mutations commonly occur in a specific motif,HUMAN MUTATION, Issue 1 2009Nick M. Makridakis Abstract Somatic mutations are hallmarks of cancer progression. We sequenced 26 matched human prostate tumor and constitutional DNA samples for somatic alterations in the SRD5A2, HPRT, and HSD3B2 genes, and identified 71 nucleotide substitutions. Of these substitutions, 79% (56/71) occur within a WKVnRRRnVWK sequence (a novel motif we call THEMIS [from the ancient Greek goddess of prophecy]: W=A/T, K=G/T, V=G/A/C, R=purine (A/G), and n=any nucleotide), with one mismatch allowed. Literature searches identified this motif with one mismatch allowed in 66% (37/56) of the somatic prostate cancer mutations and in 74% (90/122) of the somatic breast cancer mutations found in all human genes analyzed. We also found the THEMIS motif with one allowed mismatch in 88% (23/26) of the ras1 gene somatic mutations formed in the sensitive to skin carcinogenesis (SENCAR) mouse model, after induction of error-prone DNA repair following mutagenic treatment. The high prevalence of the motif in each of the above mentioned cases cannot be explained by chance (P<0.046). We further identified 27 somatic mutations in the error-prone DNA polymerase genes pol ,, pol ,, and pol , in these prostate cancer patients. The data suggest that most somatic nucleotide substitutions in human cancer may occur in sites that conform to the THEMIS motif. These mutations may be caused by "mutator" mutations in error-prone DNA polymerase genes. Hum Mutat 0, 1,10, 2008. © 2008 Wiley-Liss, Inc. [source] Evolution of hepatitis B virus precore/basal core promoter gene in HBeAg-positive chronic hepatitis B patients receiving lamivudine therapyLIVER INTERNATIONAL, Issue 1 2004Chih-Lin Lin Abstract: Aim: Lamivudine is effective in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, but the relapse rate after cessation of treatment is high. The evolution of viral genome may contribute to the viral replication under antiviral pressure of lamivudine. We therefore determined the evolution of hepatitis B virus (HBV) precore/basal core promoter and polymerase genes in HBeAg-positive chronic hepatitis B patient during lamivudine therapy. Method: Thirteen patients with HBeAg-positive chronic hepatitis who had received short-term lamivudine therapy (mean, 30 weeks) during 1999,2001 were enrolled. The precore/basal core promoter region and polymerase gene were amplified and directly sequenced before, during and post lamivudine treatment. Result: HBeAg loss or seroconversion occurred in 11, but eight relapsed after stopping therapy and five had reversion of HBeAg. Before treatment, basal core promoter mutation was found in 1. In the first 3 months of therapy, a rapid decline of serum HBV DNA level accompanied with basal core promoter mutation appeared in 11 of 13 patients (vs. before therapy; P=0.003). However, this mutant was replaced by wild-type virus in four of eight patients who relapsed after treatment. There was no significant change of precore sequences before and during therapy. Conclusions: Lamivudine therapy may result in the rapid development of basal core promoter mutation of HBV, but this mutation may revert to wild type gradually after cessation of therapy. [source] Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschiiMOLECULAR MICROBIOLOGY, Issue 5 2002Sophie Maisnier-Patin Summary We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea. [source] Six active phage-type RNA polymerase genes in Nicotiana tabacumTHE PLANT JOURNAL, Issue 6 2002Boris Hedtke Summary In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon. [source] | |||||||||||||