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At least 523 records · Page 29Linked to original sources
PubMed1987

Isolation and structure of the replicon of the promiscuous plasmid pCU1.

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.

Base Composition↗
PubMed1989

New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria.

Three types of new variants of the broad-host-range transposon Tn5 are described. (i) Tn5-mob derivatives with the new selective resistance (R) markers GmR, SpR and TcR facilitate the efficient mobilization of replicons within a wide range of Gram-negative bacteria. (ii) Promoter probe transposons carry the promoterless reporter genes lacZ, nptII, or luc, and NmR, GmR or TcR as selective markers. These transposons can be used to generate transcriptional fusions upon insertion, thus facilitating accurate determinations of gene expression. (iii) Tn5-P-out derivatives carry the npt- or tac-promoter reading out from the transposon, and TcR, NmR or GmR genes. These variants allow the constitutive expression of downstream genes. The new Tn5 variants are available on mobilizable Escherichia coli vectors suitable as suicidal carriers for transposon mutagenesis of non-E. coli recipients and some on a phage lambda mutant to be used for transposon mutagenesis in E. coli.

Base Sequence↗
PubMed1990

Identification of a low-copy-number mutation within the pUB110 replicon and its effect on plasmid stability in Bacillus subtilis.

A mutation (cop1) within the minimal replicon of plasmid pUB110, reducing its copy number from 48 +/- 2 to 11 +/- 2 in a Bacillus subtilis host, was isolated. This mutation is a G----T transversion within the translation control region of the replication initiation gene (repU). The cop1 mutation, when present in the recombinant plasmid, increases both its structural and segregational stability.

Bacillus subtilis↗
PubMed1995

Analysis of the halobacterial plasmid pHK2 minimal replicon.

The pMDS series of cloning vectors developed for use in halophilic archaea have utilized a 10.5-kb plasmid, pHK2, from Haloferax sp. Aa2.2. The minimal replicon of pHK2 has now been determined (3359 bp) and completely sequenced. No significant sequence similarity was found between the pHK2 subfragment and plasmid pHV2 from the closely related H. volcanii. However, a long open reading frame (ORF), named rep, was identified which encodes a putative protein with approx. 30% sequence identity to ORFs within plasmids pGRB1, pHGN1 and pHSB1 from Halobacterium sp. All these putative Rep proteins contain sequence motifs conserved in bacterial plasmids and phage genomes known to replicate via a rolling-circle mechanism.

Amino Acid Sequence↗
PubMed1994

Vectors with the fd replicon for in vivo cloning and analysis of genes.

We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.

Cloning, Molecular↗
PubMed1988

Genetic relatedness of the basic replicon of the virulence plasmid in shigellae and enteroinvasive Escherichia coli.

The regions required for replication (Rep) and for stable maintenance (Stb) of the 230 kilobase virulence plasmid, pMYSH6000, in Shigella flexneri 2a YSH6000 were identified on the restriction enzyme Sa/l fragments, C and O, respectively. The stable plasmid, pMYSH6610, consisting of adjacent fragments C and O was found to belong to the incompatibility group Fl. In all strains of shigellae and enteroinvasive Escherichia coli (EIEC) tested, virulence plasmids were found to be highly homologous with the Rep region of pMYSH6000. The restriction map of this probe was very similar to that of the inc region of RepFIIA replicon. The virulence plasmid of S. flexneri 6, S. boydii, S. dysenteriae and EIEC all coexisted stably with pMYSH6610, whereas that of S. flexneri other than serotype 6 and S. sonnei did not. These findings indicate that all the virulence plasmids of shigellae and EIEC are highly related but can be divided into at least 2 groups.

DNA, Bacterial↗
PubMed1989

Effects of UV light on DNA chain growth and replicon initiation in human cells.

Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. For rodent cells the extent and duration of this blockage is both cell line- and fluence-dependent. Using DNA fiber autoradiography we report here similar findings for human cells. Wild-type human cells did not exhibit significant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. Such activation would enable cells to replicate areas of DNA which do not contain a 'normal' site of initiation, yet contain blocking lesions both upstream and downstream. We have previously shown (Griffiths and Ling, 1987) that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. We report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation.

Cell Division↗
PubMed1991

Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal.

Three models have been proposed for the nature of the SOS-inducing signal in E. coli. One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.

Bacteriophage lambda↗
PubMed1991

Incompatibility between pSC101 and lambda dv replicons.

Escherichia coli cells are unable to co-maintain a lambda dv and a minimal pSC101 replicon. Selection for both plasmids results in a much reduced growth rate of the cells, whereas selection for one plasmid results in a rapid loss of the other. A chromosomal mutant has been isolated, which restores the normal growth rate of cells harbouring both plasmids, but does not alleviate the incompatibility. The nature of this new case of incompatibility is discussed.

Bacteriophage lambda↗
PubMed2006

Replication-associated strand asymmetries in vertebrate genomes and implications for replicon size, DNA replication origin, and termination.

Strand compositional asymmetry has been observed in prokaryotes and used in predicting prokaryotic DNA replication origins and termini. However, it was not found in eukaryotic genomes by the same methods. We propose that transcription-associated strand asymmetries mask the replication-associated ones. By analyzing the nucleotide composition of intergenic sequences larger than 50 kb by cumulative skew diagrams (CSD), we found replication-associated strand asymmetry in vertebrate genomes. Furthermore, we found that the most common replicon sizes in vertebrates are 50-100 kb, and show evidence that the replication origin and termination regions of vertebrate genomes range from a discrete site to a broad zone.

Animals↗
PubMed2006

Hsp90 inhibitors suppress HCV replication in replicon cells and humanized liver mice.

Persistent infection with hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Here we report that inhibition of heat shock protein 90 (Hsp90) is highly effective in suppressing HCV genome replication. In HCV replicon cells, HCV replication was reduced by Hsp90 inhibitors and by knockdown of endogenous Hsp90 expression mediated by small-interfering RNA (siRNA). The suppression of HCV replication by an Hsp90 inhibitor was prevented by transfection with Hsp90 expression vector. We also tested the anti-HCV effect of Hsp90 inhibition in HCV-infected chimeric mice with humanized liver. Combined administration of an Hsp90 inhibitor and polyethylene glycol-conjugated interferon (PEG-IFN) was more effective in reducing HCV genome RNA levels in serum than was PEG-IFN monotherapy. These results suggest that inhibition of Hsp90 could provide a new therapeutic approach to HCV infection.

Animals↗
PubMed2006

Characterization of the ColE2-like replicon of plasmid pTT8 from Thermus thermophilus.

We identified the 1.6-kb region of Thermus thermophilus plasmid pTT8 capable of autonomous replication, which shows a significant sequence similarity to the replicon regions of the ColE2-related plasmids. We showed the requirement of DNA polymerase I for pTT8 replication. The putative rep gene coding for the replication initiator protein, Rep, similar to those of the ColE2-related plasmids was cloned into an expression vector. The 6xHis-Rep protein expressed in Escherichia coli was successfully purified by stepwise denaturing with urea and refolding in the presence of glycerol on Ni-resin. We identified the nucleotide sequence recognized by the pTT8 Rep protein by the SELEX experiment using the purified protein, and proposed the existence of the third origin of pTT8 replication different from those predicted previously.

Bacterial Proteins↗
PubMed2005

Synthesis and anti-viral activity of a series of sesquiterpene lactones and analogues in the subgenomic HCV replicon system.

Hepatitis C virus (HCV) infection is a severe liver disease that often leads to liver cirrhosis and hepatocellular carcinoma (HCC). Current therapy is inadequate to conquer this viral disease. In this study, we identified parthenolide (1), an active component in feverfew, a popular remedy for fever and migraine, as a lead compound with an EC50 value of 2.21 microM against HCV replication in a subgenomic RNA replicon assay system. Parthenolide is able to potentiate the interferon alpha-exerted anti-HCV effect. Several commercially available sesquiterpene lactones (2-5) structurally analogous to parthenolide and a series of synthesized Michael-type adducts of parthenolide (12-18) also exhibit micromolar concentrations for anti-HCV activities. Structure-activity relationship was elucidated to reveal that the spatial arrangement of the terpenoid skeleton fused with an alpha-methylene-gamma-lactone moiety produces maximal anti-HCV activity. In addition, a strong anti-HCV potency indicates a possibility of secondary amino adducts (12-18) converting back to parthenolide or being replaced by the nucleophilic residues of proteins inside cells. This work shows that screening of natural products is a viable and fast way for identifying novel molecular diversity as potential drug leads.

Blotting, Northern↗
PubMed2006

Improved replicon cellular activity of non-nucleoside allosteric inhibitors of HCV NS5B polymerase: from benzimidazole to indole scaffolds.

Benzimidazole-based allosteric inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified to a variety of topologically related scaffolds. Replacement of the polar benzimidazole core by lipophilic indoles led to inhibitors with improved potency in the cell-based subgenomic HCV replicon system. Transposing the indole scaffold into a previously described series of benzimidazole-tryptophan amides generated the most potent inhibitors of HCV RNA replication in cell culture reported to date in this series (EC(50) approximately 50 nM).

Allosteric Regulation↗
PubMed2006

Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories.

Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.

Chromosomes, Fungal↗
PubMed2006

Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli.

Antibody molecules bind to antigen with six complementary determining region (CDR) loops, three of which are located on each variable heavy (V(H)) and light (V(L)) chains. Discovery and optimization of antibodies that bind antigen using in vitro techniques require diversification of one or more of these CDRs. Since antibodies are dimeric, simultaneous diversification of heavy and light chains on separate genetic elements would allow "chain shuffling" to occur simply and efficiently. Efficient expression of antibody V(H) and V(L) requires that the two separate replicons be compatible with one another, but also have similar properties, such as copy number in E. coli. Standard plasmids that are compatible with one another in E. coli exist at widely variable copy numbers. Recently we described the isolation of ColE1 mutants that have similar copy numbers but different incompatibility characteristics. Thus, new compatibility groups in the ColE1 family were established. Herein we describe the E. coli expression of V(H) and V(L) genes to form a functional Fab. The ability to express antibody heavy and light chains from separate but compatible high copy plasmids should allow new opportunities in antibody engineering, such as rapid chain shuffling and generation of more complex antibody libraries.

Biological Evolution↗
PubMed2004

A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenomic replicon cells.

Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EGdelta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(delta4AB)SEAP. EG(delta4AB)SEAP is a fusion protein in which Enhanced Green Fluorescent Protein (EGFP) was fused to SEcreted Alkaline Phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, delta4AB, which spans the NS4A and NS4B junction region. The secretion of SEAP into culture medium has been shown to depend on the cleavage of delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(delta4AB)SEAP cells. In conclusion, Ava5-EG(delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs.

Alkaline Phosphatase↗