SEARCH · Search PubMed

Results for “replicon”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,081 records · Page 60Linked to original sources

PubMed2006

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp.

A plasmid, pGB112, has recently been developed to transfer DNA from Escherichia coli to Streptomyces spp via conjugation. This technique made use of (A) E. coli replicon, (B) ampicillin (amp) resistance gene for selection in E. coli and thiostrepton (tsr) resistance gene for selection in Streptomyces, (C) a fragment of SCP2* replicon, (D) a 2.6 kb fragment of tra-cassette which consists of pIJ101 transfer gene (tra) and two ermE promoters, (E) a 0.8 kb fragment of oriT of (IncP) RK2. The results showed that this plasmid was able to transfer plasmid DNA from E. coli to Streptomyces coelicolor via conjugation, and that it could also transfer DNA between Streptomyces strains. Since this plasmid has both pBR322 and SCP2* replicons, it may provide a novel and useful method for genetic operation in E. coli and Streptomyces.

Conjugation, Genetic↗

PubMed2004

Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication.

Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3'/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n = 53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection.

5' Untranslated Regions↗

PubMed2006

Reactivation of an integrated disabled viral vector using a Cre-loxP recombination system in Arabidopsis thaliana.

We developed an inactivated DNA replicon of Turnip Mosaic Virus (TuMV), which was reactivated by a recombination event based on the Cre-loxP system. Viral replication was prevented by the insertion of a translation terminator sequence flanked by two loxP sites at the junction of the P1-HCPro-coding genes. In vitro recombination was tested with purified Cre, which excised the floxed sequence from the TuMV DNA, leaving a single loxP site in the reactivated viral genome, and restored the open reading frame of the replicon. Arabidopsis thaliana plants were made transgenic for the inactivated TuMV replicon. Removal of the translation terminator sequence was achieved by the controlled expression of Cre. Delivery of the Cre recombinase to the transgenic plants was obtained by three methods: agroinfiltration, PVX-based production, or transgenic chemical-inducible expression. In each case, reactivation of TuMV replication was observed.

Arabidopsis↗

PubMed1985

Transcription of the replication control region of the IncFII R-plasmid NR1 in vitro and in vivo.

The minimal replicon of the 90,000 base-pair IncFII R plasmid NR1 consists of a 2700 base-pair region of the DNA. Minireplicator plasmids consisting of the 2700 base-pair minimal replicon plus a 2200 base-pair region coding for chloramphenicol acetyltransferase (cat) were used as templates for in vitro transcription. Six RNA transcripts were synthesized from these templates in vitro. We have determined the directions of transcription and the approximate sites of initiation and termination of each of the in vitro RNA transcripts. One RNA transcript was synthesized from the cat gene, while the other five were transcribed from the minimal replicon. Four of the RNA transcripts also were identified by quantitative hybridization of RNA synthesized in vivo from these minireplicator plasmids. The strengths of the promoters for the RNA transcripts were estimated by the relative rates of transcription both in vitro and in vivo. Transcription from convergent promoters reduced the rate of RNA synthesis in vivo in both directions. In vivo, a significant fraction of the cat mRNA was extended past its in vitro termination point. Transcription of mutants that have altered plasmid copy number and/or incompatibility properties also were examined. The possible roles of each of the transcripts as mRNA and their involvement in regulation of DNA replication are discussed.

Base Sequence↗

PubMed1992

Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector.

A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of R1 and has a lambda PR promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon. The vector contains a unique Bg/II site between PR and the replicon which is suitable for insertion of genomic DNA.

Escherichia coli↗

PubMed1985

DNA repair and replication in human fibroblasts treated with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene .

DNA repair and replication were examined in diploid human fibroblasts after treatment with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 microM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4-6 h after treatment with 0.7 microM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 microM) were found to inhibit replicon initiation by up to 50% within 30-60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1 X 10(7) Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 microM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 microM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.

Benzopyrenes↗

PubMed1989

Histogenesis of malignant melanoma of the skin: the role of lymphocytes in the transformation of tissue developmental units.

It is proposed that the completion of both growth and differentiation, in highly specialized progressively evolving multicellular organisms, is achieved throughout two separated but sexually interacting populations of cells (bi-clonal model): 1) the germ line, seated in an obligatory axial position, required for the generation of cytoplasmic replicons, is the source of travelling vectors (spermatozoa, lymphocytes ...) endowed with these potentially transforming nucleoproteins. 2) The centrifugally growing somatic tissues subjected to mitotic senescence. The progressive loss of replicons probably triggers meiotic-like recombinations and differentiation in distal tissues. The shedding of axial replicons to distal meiotic targets seems to be instrumental in transformation, initiating both, embryogenesis and cancer. This theoretical approach is applied to the histogenetic model of malignant melanoma of the skin. In this setting, the axial population is identified as lymphocytes involved in a process of extrathymic maturation (lichenoid lesions), in the same field where melanoma would arise. Pagetoid nests of melanocytes are considered to be the expression of TDUs evolving in a distal population of senescent neurocristic cells. This situation establishes the likelihood of mixing between potentially transforming lymphocytes and the meiotic melanocytes resulting in a transformed hybrid, the earliest cellular expression of malignant melanoma.

Cell Differentiation↗

PubMed1987

High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection.

Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.

Base Sequence↗

PubMedDate not supplied

Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction.

The nucleotide sequence of the 1847-bp Bifidobacterium longum B2577 cryptic plasmid pMB1 was determined. The plasmid had a G+C content of 62.0%, and contained two open reading frames, orf1 and orf2, likely arranged in an operon. The proteins encoded by orf1 and orf2 show the highest degree of similarity with similarly arranged peptide sequences translated from Corynebacterium glutamicum pXZ10142 and Mycobacterium fortuitum pAL5000 plasmids. Recombinant plasmids containing the pMB1 replicon were able to replicate in Bifidobacterium animalis MB209. The successful transformation of this strain with pMB1-based plasmids facilitated characterization of this replicon, results of which showed that both orf1 and orf2 are necessary for plasmid replication. A family of new Escherichia coli-B. animalis shuttle plasmids, based on the pMB1 replicon and expressing a cat and an ery gene, was constructed.

Amino Acid Sequence↗

PubMed2006

Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro.

The combinations of Abbott Hepatitis C virus (HCV) polymerase A-782759 with either Boehringer Ingelheim HCV NS3 protease inhibitor BILN-2061 or interferon (IFN) displayed additive to synergistic relationships over a range of concentrations of two-drug combination. Treatment of HCV replicon with A-782759, IFN or BILN-2061 for about 16 days resulted in dramatic reductions in HCV RNA (5.1, 3.0 and 3.9 log10 RNA copies, respectively). However, none of the compounds tested alone lead to replicon RNA reduction to undetectable levels. Ongoing replication in the presence of A-782759 or BILN-2061 was associated with the appearance of resistant mutations M414T in NS5B and D168V in NS3, respectively. In contrast, a combination of A-782759 with BILN-2061 resulted in greater than 7 logs RNA reduction leading to undetectable replicon RNA after 16 days of treatment. Our findings suggest that a monotherapy with either drug alone is likely to result in development of resistant mutants. However, a combination therapy with polymerase inhibitor has the potential to improve the efficacy of IFN or a protease inhibitor alone in vivo, due to the lower likelihood of resistance development.

Antiviral Agents↗

PubMed2026

Remdesivir maintains antiviral potency against clinically relevant SARS-CoV-2 Nsp12 substitutions.

Remdesivir (RDV) is a nucleotide analog prodrug approved for COVID-19 treatment that inhibits the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; nsp12). Although RDV maintains activity against circulating variants of concern, ongoing evaluation of resistance-associated substitutions is critical for clinical care, particularly in settings of prolonged viral replication such as immunocompromised individuals. We assessed the phenotypic impact of nsp12 substitutions identified from in vitro resistance selection, RDV clinical reports, and global sequence surveillance. Using a recombinant infectious SARS-CoV-2 reporter virus, we compared susceptibility of these nsp12 substitutions to RDV and its parent nucleoside, GS-441524. After confirming concordant resistance profiles between RDV and GS-441524, we assessed RDV susceptibility in a complementary non-infectious replicon system. In both systems, single nsp12 substitutions remained fully susceptible to RDV within their respective assay variability limits. Of the double substitutions tested, S759A/V792I conferred the largest reduction in antiviral susceptibility (∼15-fold) but was associated with impaired replication kinetics. Given the strong concordance between the two assays, the replicon system also enabled phenotypic characterization of substitutions E802A, E802D, and P323L/E802D that could not be rescued as infectious virus. Analysis of >17 million SARS-CoV-2 genomes in GISAID showed that all tested nsp12 substitutions had low prevalence (≤0.1%), except P323L (98.8%). Collectively, these data reinforce the high genetic barrier to RDV resistance, as reduced susceptibility is typically accompanied by substantial reductions in replication. Our findings support the continued clinical utility of RDV and highlight the complementary value of SARS-CoV-2 infectious virus and replicon systems for antiviral resistance surveillance and phenotyping.

COVID-19↗

PubMed2005

Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors.

BACKGROUND/AIMS: Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR). METHODS: In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells. RESULTS: PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells. CONCLUSIONS: A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.

Antigens, Surface↗

PubMed2005

Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

Cells, Cultured↗

PubMed2001

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin.

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.

Animals↗

PubMed2003

Bovine viral diarrhea virus as a surrogate model of hepatitis C virus for the evaluation of antiviral agents.

The identification and development of new antiviral agents that can be used to combat hepatitis C virus (HCV) infection has been complicated by both technical and logistic issues. There are few, if any, robust methods by which HCV virions can be grown in vitro. The development of HCV RNA replicons has been a great breakthrough that has allowed for the undertaking of significant screening efforts to identify inhibitors of HCV intracellular replication. However, since replicons do not undergo a complete replication cycle, drug screening programs and mechanism of action studies based solely on these assays will not identify compounds targeting either early (virion attachment, entry, uncoating) or late (virion assembly, egress) stages of the viral replication cycle. Drugs that negatively affect the infectivity of new virions will also not be identified using HCV RNA replicons. Bovine viral diarrhea virus (BVDV) shares a similar structural organization with HCV, and both viruses generally cause chronic long-term infections in their respective hosts. The BVDV surrogate model is attractive, since it is a virus-based system. It is easy to culture the virus in vitro, molecular clones are available for genetic studies, and the virus undergoes a complete replication cycle. Like HCV, BVDV utilizes the LDL receptor to enter cells, uses a functionally similar internal ribosome entry site (IRES) for translation, uses an NS4A cofactor with its homologous NS3 protease, has a similar NS3 helicase/NTPase, a mechanistically similar NS5B RNA-dependent RNA polymerase, and a seemingly equivalent mechanism of virion maturation, assembly and egress. While the concordance between drugs active in either BVDV or HCV is largely unknown at this time, BVDV remains a popular model system with which drugs can be evaluated for potential antiviral activity against HCV and in studies of drug mechanism of action.

Antiviral Agents↗

PubMed1998

Application of the resident plasmid integration technique to construct a strain of Streptococcus godronii able to express the Bacillus circulans cycloisomaltooligosaccharide glucanotransferase gene, and secrete its active gene product.

A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H.K. Kuramitsu, Plasmid 34 (1995) 85-95. This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb Bg/II fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3'-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3'-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.

Bacillus↗

PubMed1998

Virulence plasmids of enterotoxigenic Escherichia coli isolates from piglets.

Virulence plasmids of 68 ETEC isolates from piglets belonging to different pathotypes and six ETEC isolates from calves with pathotypes typical of porcine ETEC were identified with seven virulence probes for the heat-stable (STa and STb) and heat-labile (LT) enterotoxins, for the F4, F5, F6, and F41 fimbrial adhesion subunit, and also with five Rep probes for the RepFIA and RepFIB basic replicons, and the RepFIC family of basic replicons. With the exception of the F41 probe, the other virulence probes hybridized with at least one plasmid band of a size range from 65 to more than 100 Mda. Common associations of virulence factor-encoding genes on plasmid bands were: STb/LT, STa/F5, STa/F6, STa/STb. Other associations, STa/F4, STa/F4/F6, and STa/STb/LT/F6, were rarer. On the other hand the F4 adhesin-encoding genes were isolated on one plasmid band in all but three F4+ isolates. All but one of the 92 virulence plasmids which were studied have Rep probe hybridization profiles and replicon types typical of the uni- or multireplicon plasmids belonging to the various incompatibility groups of the F incompatibility complex.

Animals↗

PubMed1989

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase.

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.

Animals↗