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At least 631 records · Page 35Linked to original sources
PubMed1999

Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid.

The nucleotide sequence of a cryptic plasmid (pRMG1) isolated from a type 1 Streptococcus pneumoniae has been determined and two recombinant plasmids, pRMGE1 and pRMGE2, bearing the pRMG1 replicon have been constructed. pRMGE2 is a shuttle vector for Escherichia coli and S. pneumoniae. The important characteristics of this cloning vector are: a size of 5.5 kb including a 1.4 kb fragment of pRMG1 (containing a double-stranded replication origin and an open reading frame encoding a putative replication initiation protein), a multicloning site, two antibiotic resistance markers for selection of plasmid containing cells, and blue-white colony screening in E. coli for identification of insert-containing plasmids.

Base Sequence↗
PubMed2000

Alphavirus DNA and particle replicons for vaccines and gene therapy.

Alphaviruses have several features that make them attractive as gene delivery platforms, and vectors derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are currently being developed as prophylactic and therapeutic vaccines for infectious diseases and cancer. Alphavirus vectors, termed "replicons", retain the nonstructural protein genes encoding the viral replicase, that in turn programme high level cytoplasmic amplification of the vector RNA. We have developed plasmid DNA and recombinant vector particle delivery systems derived from the prototype alphavirus, SIN. Each system uses RNA polymerase II-based expression of alphavirus genome components and both vector formats are highly efficacious towards inducing robust antigen-specific immune responses in vaccinated animals. To increase the potency of SIN vector particles, which are not known to be lymphotropic, the tropism was re-directed for efficient infection of dendritic cells, both in vitro and in vivo.

Alphavirus↗
PubMed2001

Genetic variability of potato spindle tuber viroid RNA replicon.

The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome.

Genetic Variation↗
PubMed2002

Expression of angiostatin using DNA-based semliki forest virus replicon.

Angiogenesis is recognized as a critical factor in the growth of tumor cells and plays a key role in the tumor metastasis. Recent studies for antiangiogenic substances are getting popular. The angiostatin, one of the antiangiogenic substances, leads to the increased apoptosis of the tumor cells by inhibiting the neovascularization of the tumor. The angiostatin was identified as the internal fragments of the plasminogen which has no antiangiogenic activity. By hydrolysis of the plasminogen, the angiostatin can be produced. In this study, we constructed the SFV-derived DNA vector by employing the cytomegalovirus immediate early enhancer/ promoter (CMV). This vector makes it possible to transfect the cells with DNA without the in vitro transcription process. The C-myc epitope and polyhistidine residue sequences were placed in downstream of the angiostatin gene to make it eligible to detect the expressed protein. The murine Ig k-chain V-J2-C signal sequence was placed in upstream to secrete the expressed protein from the cells. We confirmed the expression of angiostatin in the BHK-21 cells using DNA-based SFV replicon.

Angiostatins↗
PubMed2004

High cell surface expression of Newcastle disease virus proteins via replicon vectors demonstrates syncytia forming activity of F and fusion promotion activity of HN molecules.

For functional studies, the hemagglutinin-neuraminidase (HN) and the fusion protein (F) of Newcastle disease virus (NDV) were expressed in BHK cells using two vectors which are based on the Semliki Forest virus (SFV) replicon. The first system of high protein expression works by transfection of RNA which before has been in vitro transcribed from a vector containing the gene for the SFV self-amplifying replicase (REP) and a foreign gene using the SP6 promoter. A high level of protein (HN or F) expression was detected 18-20 h after transfection. To study the host range of this expression system, a panel of different cell lines were compared for transfections with SFV RNA. A wide range of expression efficiency was observed, the highest being BHK cells. The second system is based on a DNA plasmid in which the SFV-REP and a foreign gene are expressed in cells under the transcriptional and translational control of the cytomegalovirus immediate-early enhancer T7 promoter. DNA-electroporated BHK cells expressed also high levels of the recombinant proteins but at a delayed time point (40-48 h) as compared with the corresponding RNA. Co-expression of the two NDV proteins, HN and F, via this DNA vector in the same cells led to syncytium formation in the cell monolayer, showing that both proteins expressed in this way, were functionally active. F alone, expressed via this vector, displayed residual fusion activity suggesting its proteolytic cleavage and its functional independence from HN.

Animals↗
PubMed1991

Yeast 20 S RNA replicon. Replication intermediates and encoded putative RNA polymerase.

The 20 S RNA genome is a circular single-stranded replicon, present in most laboratory yeast strains, whose copy number is induced 10,000-fold by transfer of cells to acetate medium without a carbon source. We have sequenced most of the 20 S RNA genome, and the (+) strand has a long open reading frame with the potential to encode a protein with homology to viral RNA-dependent RNA polymerases. The presence of a typical cAMP-dependent phosphorylation site in the putative RNA polymerase suggests that the acetate amplification of the 20 S RNA genome might be mediated by cAMP, a signal known to transmit the same nutritional status information to the sporulation-control system. Our inability to clone across the gap in the sequence suggests either autocatalytic cleavage of the RNA in the reverse transcriptase reaction, an unusual linkage of 5' and 3' ends of a fundamentally linear molecule, or a structure unusually resistant to reverse transcription. The identity of our sequence with that of the accompanying paper (Rodriguez-Cousino, N., Esteban, L.M., and Esteban, R. (1991) J. Biol. Chem. 266, 12772-12778) for W double-stranded RNA (dsRNA) suggests that W is the replicative form of 20 S RNA. The presence of single-stranded (+) and (-) strands and greater than unit length molecules suggests a rolling circle mode of replication as has been suggested for viroids.

Amino Acid Sequence↗
PubMedDate not supplied

[The modern system for selection of cryptic plasmids based on replicon pMBI].

Cytotoxicity of hybrid plasmide pGalI was associated with the genome site measuring for 1.1.mD and flanked by the recognizing sites of Eco RI restrictase. The cytotoxic effect of pGalI might be limited not only by plasmide pMB9 but also by other plasmides constructed on the base of pMBI replicone. Limitation of the cytotoxicity occurred due to a decrease in amount of the pGalI copies. Capacity of the plasmides studied to limit the pGalI cytotoxicity enabled to clone them as cryptic plasmides under conditions of both simultaneous with pGalI transformation of E. coli and the subsequent selection of clones with the Gal+ phenotype.

Cloning, Molecular↗
PubMed1993

Persistent inhibition of DNA synthesis in irradiated rat embryo fibroblasts expressing the oncogenes H-ras plus v-myc derives from inhibition of replicon initiation and is mitigated by staurosporine.

We have previously shown that rat embryo fibroblasts expressing the oncogenes H-ras plus v-myc experience a prolonged inhibition of DNA replication after exposure to ionizing radiation as compared to normal rat embryo fibroblasts, or rat embryo fibroblasts expressing H-ras or v-myc alone. Here we show that this enhanced inhibition of DNA replication in cells expressing H-ras plus v-myc is due to inhibition of the main controlling event of DNA replication, i.e., replicon initiation, that this inhibition is reversible, and that the expression of this phenotype is reverted by staurosporine, a protein kinase inhibitor. These findings implicate genetic influences in the processes that control DNA replication in irradiated cells and identify events in the regulation of DNA replication that become apparent several hours after irradiation. The products of the oncogenes H-ras and v-myc appear to be members of, or exert influence on, this controlling pathway.

Alkaloids↗
PubMed1995

[The retardation of DNA synthesis in adjacent replicon clusters in the lymphocytes of patients with Down's syndrome as a model of premature aging].

In peripheral blood lymphocytes taken from 7 patients with Down syndrome (DS) and 2 normal donors, using DNA fiber autoradiography, we estimated the rate of DNA-chain growth, which depends oil the number of simultaneously replicating adjacent replicon clusters, but not on the rate of fork movement. No difference was found in the rate of fork movement in these cells. 6 of 7 DS patients showed a significant reduction in the rate of DNA-chain growth as compared to that in normal control. Thus, a new molecular defect in DS lymphocytes has ben first revealed. Besides, new arguments have been provided in favour of genetic heterogeneity of this genetic disorder belonging to premature ageing diseases. Relation of molecular DNA replication defects with ageing is discussed.

Aging↗
PubMed1997

Identification by replicon-typing of indigenous plasmids of Burkholderia (Pseudomonas) solanacearum.

Fifteen strains of Burkholderia (Pseudomonas) solanacearum were isolated, identified from different parts of India and screened for plasmid profile. They harboured 1-3 plasmids, but most of the isolates contained a single plasmid. Plasmids were classified on the basis of replicon typing by Southern hybridization using incompatible specific rep probes. Plasmids of 9 strains hybridized with repP probe, plasmids of 5 strains hybridized with repW probe and one plasmid hybridized with repQ probe. Fingerprint and cluster analyses of plasmids from 10 strains of B. solanacearum digested with Eco RI revealed that plasmids of race 3 strains clustered in one group and the plasmids of race 1 strains clustered in another group.

Burkholderia↗
PubMed2004

Unexpected host range of hepatitis C virus replicons.

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known if extrahepatic tissues are also infected by the virus nor if nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and other species. We have tested this hypothesis and found that subgenomic HCV RNA can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.

Comment↗
PubMed1999

Initiation of DNA replication in a eukaryotic rolling-circle replicon: identification of multiple DNA-protein complexes at the geminivirus origin.

The mechanism of initiation of DNA replication in eukaryotic rolling- circle replicons is still poorly understood in molecular terms. Geminiviruses, a family of plant DNA viruses, which use this strategy during part of their replicative cycle, replicate in the nucleus and are amenable to molecular studies. Except for the virally encoded initiator protein (Rep), geminivirus DNA replication relies on cellular factors, likely interfering with cell cycle regulation of the infected cell. Here, we report the identification of three distinct DNA-protein complexes of the DNA replication initiator protein encoded by wheat dwarf geminivirus (WDV) within viral regulatory sequences controlling DNA replication and transcription. We have mapped the WDV Rep binding sites by combining gel-shift assays, electron microscopy and DNase I footprinting. Two of the Rep-DNA complexes (C and the V) are high-affinity complexes, located in the proximity of the two divergent TATA boxes, at 150 and 90 bp, respectively, from the DNA replication initiation site. The third one, the O-complex, is a low-affinity complex, which can assemble under conditions supporting the DNA cleavage reaction. This suggests that it might be responsible for initiation of rolling-circle DNA replication in WDV and other members of the Mastrevirus genus.

Base Sequence↗
PubMed2000

Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF.

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.

Amino Acid Sequence↗
PubMed1981

Double-stranded DNA with the size expected of replicons can be released from Chironomus polytene chromosomes.

The effect of the drug 5-fluorodeoxyuridine on DNA synthesis in Chironomus polytene chromosomes was investigated. The DNA was labelled by injection of radioactive precursor into living animals pre-treated with the drug, extracted with an neutral non-denaturing buffer at 25 degrees C and then characterized by gel electrophoresis. After a short pulse a heterogeneous double-stranded DNA population is released from the polytene chromosome. These fragments are later jointed together to produce a double-stranded DNA with a size ranging between 8-13 X 10(6) D. The release of both types of fragments from the polytene chromosome is prevented by lysing the cells at 0 degree C instead of at 25 degrees C. The larger double-stranded DNA has the size expected of replicons in Chironomus. The results are discussed in relation to the organization of replication in polytene chromosomes.

Animals↗
PubMed1978

The stable carriage of two TnA units on a single replicon.

Bacterial plasmids which contain a copy on TnA are refactory to the uptake of a second by transposition. However plasmids containing two such copies can be constructed by in vitro recombination techniques. Some plasmids containing two copies of TnA have been obtained by conventional transposition, but in all cases they arose by the virtually simultaneous insertion of both units into a replicon that carried no TnA. All stable plamids containing two copies of TnA carried the transposons in opposite orientation.

DNA Replication↗
PubMed1984

High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon.

A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.

Chromosomes, Bacterial↗
PubMed2002

Use of the pAL5000 replicon in PAH-degrading mycobacteria: application for strain labelling and promoter probing.

Three environmental Mycobacterium strains (LB501T, LB307T and VM552) able to degrade anthracene, phenanthrene or pyrene, respectively, were successfully electroporated with pAL5000-based plasmids containing the green fluorescent protein (gfp) gene of Aequoria victoria under the control of the hsp60 promoter of Mycobacterium bovis following a slightly modified standard procedure. Transformants showed irregular gfp expression profiles. Four plasmid derivatives were constructed that contained gene promoters isolated from, and adapted to, gene expression in polycyclic aromatic hydrocarbon (PAH)-degrading mycobacteria. One derivative directed strong and homogeneous expression of GFP, allowing dual analysis of both GFP- and PAH-derived fluorescence as assessed by confocal laser scanning microscopy. The results reported here demonstrate the suitability of the pAL5000 replicon for the development of recombinant DNA-based studies in PAH-degrading Mycobacterium spp.

DNA, Bacterial↗