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At least 1,063 records · Page 59Linked to original sources
PubMed2005

E2 of hepatitis C virus inhibits apoptosis.

Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and can be involved in very long chronic infections up to 30 years or more. Therefore, it has been speculated that HCV possesses mechanisms capable of modulating host defense systems such as innate and adaptive immunity. To investigate this virus-host interaction, we generated HCV replicons containing various HCV structural proteins and then analyzed the sensitivity of replicon-containing cells to the apoptosis-inducing agent, TRAIL. TRAIL-induced apoptosis was monitored by cleavage of procaspase-3 and procaspase-9 as well as that of their substrate poly(ADP-ribose) polymerase. TRAIL-induced apoptosis was inhibited in cells expressing HCV E2. Moreover, expression of HCV E2 enhanced the colony forming efficiency of replicon-containing cells by 25-fold. Blockage of apoptosis by E2 seems to be related to inhibition of TRAIL-induced cytochrome c release from the mitochondria. Based on these results, we propose that E2 augments persistent HCV infection by blocking host-induced apoptosis of infected cells.

Apoptosis↗
PubMed2005

ATM and ATR check in on origins: a dynamic model for origin selection and activation.

Initiation of DNA replication occurs at origins of replication, traditionally defined by specific sequence elements. Sequence-dependent initiation of replication is the rule in prokaryotes and in the yeast Saccharomyces cereviseae. However, sequence-dependent initiation does not appear to be absolutely required in metazoan eukaryotes. Origin firing is instead likely dependent on stochastic initiation from chromatin-defined loci, despite the demonstration of some specific origins. Based on some recent observations in Xenopus laevis egg extracts and in mammalian cell culture, we propose that timing of origin firing is dependent on feedback from active replicons. This dynamic regulation of replication is mediated by sensing of ongoing replication by the DNA-damage checkpoint kinases ATM and ATR, which in turn downregulate neighboring and distal origins and replicons by inhibition of the S-phase kinases Cdk2 and Cdc7 and by inhibition of the replicative Mcm helicase. Origin selection, activation, and replicon progression are therefore constrained in both space and time via feedback from the cell cycle and ongoing replication.

Animals↗
PubMed1991

[Biochemical kinetics of plasmid replication processes].

Two groups of plasmid replication reactions occurring during the growth of cell populations were kinetically characterized: 1) short-term intermediate processes coupled with changes in plasmid concentrations in host cells and occurring in the first few generations; 2) long-term processes resulting in the loss of plasmids and occurring during prolonged cultivation of cells. It was shown that first group processes are determined by the ratios of plasmid replication rates relative to cell growth. Using growing populations of E. coli as examples, it was shown that the shape of kinetic curves reflecting the plasmid copy number depends on the nature of their replicon. In this case plasmids with ColE1 replicon exhibit kinetic behaviour of the first type (temporal loss of plasmid copy number in the cells), whereas plasmids with p15 replicon display the second type behaviour consisting in the decrease of the plasmid copy number followed by its short-term drastic increase. For both types of kinetic behaviour a kinetic description adequately reflecting these processes is given. It is supposed that the loss of plasmids during long-term cultivation of cell populations may be associated with both segregation (lower growth rates of plasmid-containing cells in comparison with cells containing no plasmids) and mutation mechanisms. Kinetic descriptions of both mechanisms are proposed and principles of their discrimination are formulated. Practical recommendations for the prevention of the plasmid loss by the cells that are based on theoretical data are given.

Kinetics↗
PubMed1986

[Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties].

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.

Chromosome Mapping↗
PubMed1983

Aphidicolin arrest irreversibly impairs replicating simian virus 40 chromosomes.

The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this drug inhibits DNA polymerase alpha reversibly. Yet, its in vivo effect on SV40 DNA replication, which depends on DNA polymerase alpha, was found to be irreversible. Thus, exposure of infected cells to aphidicolin led to a progressive loss in their ability to incorporate [3H]dT into viral DNA in a subsequent pulse without drug. This loss was time-dependent (t1/2 at 37 degrees C at 2 microgram/ml of drug was approximately 20 min) and increased with drug concentration. Likewise, replicating SV40 DNA, pulse-labeled prior to exposure, lost the ability to mature into form I DNA upon removal of the drug. No degradation of replicating SV40 DNA molecules was detected by neutral sucrose gradient analysis during or up to 1 h after aphidicolin exposure. However, longer incubations resulted in breakdown of the arrested replicative intermediate, concomitant with the resumption of viral DNA synthesis. Origin-synchronized SV40 replicons were less affected by exposure to aphidicolin than were ongoing replicons, as judged from comparing recoveries of tsA replicons from 40 degrees C restriction, with or without the drug. The data indicate that replicating SV40 chromosomes become selectively impaired during aphidicolin arrest and prevent thereby the initiation of new replication rounds, perhaps by occupying fixed nuclear replication sites.

Animals↗
PubMed1984

The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression.

For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity. The trfA gene is transcribed as part of a polycistronic operon which also includes kilD. Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci. Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself. Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter. This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans. When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2. Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis. These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.

Bacterial Proteins↗
PubMed1993

A new and rapid method for visualising DNA replication in spread DNA by immunofluorescence detection of incorporated 5-iododeoxyuridine.

We have developed a new and rapid immunofluorescent method for visualisation of replicated regions on fixed DNA fibers. Using this method we have found in 5-fluorodeoxyuridine (FrdU)-blocked human cells distinct replication units covering about 60 kb of DNA and corresponding in size to single replicons or chromatin loops. Our results also suggest that nonadjacent replicons within a replicon cluster may be activated after FrdU arrest and that the method may be adapted for localization on fibers of specific DNA sequences.

Chromosomes, Human↗
PubMed2002

Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.

Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.

5' Untranslated Regions↗
PubMed2001

Mapping sites where replication initiates in mammalian cells using DNA fibers.

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller ( approximately 46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.

Animals↗
PubMed1997

Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene.

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.

Amino Acid Sequence↗
PubMed1999

Replication of plasmids during bacterial response to amino acid starvation.

Amino acid starvation of bacterial cells leads to expression of the stringent (in wild-type strains) or relaxed (in relA mutants) response (also called the stringent or relaxed control, respectively). The stringent control is a pleiotropic response which changes drastically almost the entire cell physiology. Although starvation is a rule rather than an exception in natural environments of bacteria, and DNA replication is a fundamental cell process, until recently our knowledge about regulation of DNA replication in amino acid-starved cells has been unexpectedly poor. Within recent years the stringent control of DNA replication has been investigated mainly on plasmid models. Several plasmid replicons have been studied, including oriC plasmids, ColE1-like replicons, pSC101, F, R1, RK2, and R6K, and plasmids derived from bacteriophages lambda and P1. However, molecular models of replication regulation in amino acid-starved cells have been proposed to date only for lambda plasmids and ColE1-like replicons. Although further extensive studies are necessary in the understanding of molecular mechanisms of the stringent and relaxed control of replication of other plasmids, the results obtained to date (summarized and discussed in this review) show that studies on DNA replication in amino acid-starved cells may provide new insights into the regulatory mechanisms and lead to more general conclusions.

Amino Acids↗
PubMed1989

Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1.

Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 bp upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC mutation located at 62 min of the E. coli chromosome. The herC mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC mutation alone conferred cold-sensitive growth, suggesting that the herC gene product is essential for cell growth. The 1832 bp E. coli DNA fragment, containing the wild-type allele of the herC mutation, was cloned and an open reading frame for the HerC protein was determined.

Amino Acid Sequence↗
PubMed1979

Genetic analysis of the inter-relationship between plasmid replication and incompatibility.

The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1. The inability of the composite plasmid to replicate at 42 degrees in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nervertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.

DNA Replication↗
PubMed1977

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids.

Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1. Chloramphenicol immediately stops its replication. A stable relaxation complex is not formed. Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1. In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself. This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid. Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional. The composite plasmids are incompatible with both the parent replicons.

Chloramphenicol↗
PubMed1977

The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units.

The replication of chromosomal DNA in human and Chinese hamster cell populations has been studied by means of the DNA fiber autoradiography. It was found that the rate of DNA replication for one fork in human cells varies from 0.2 to 0.9 micron/min, the average being 0.6 micron/min. In the Chinese hamster cells the rate DNA replication is greater, varying from 0.3 to 1.2 micron/min, the average being 0.8 micron/min. There are no clusters containing a great number of replication units in human and Chinese hamster cells. Sequences consisting of two or three replicons which belong to single DNA molecule have been observed, but their frequency was relatively low. The distances between the initiation points in such sequences of replicons vary from 40 to 280 micron, the average value being 130 micron. This value represents the minimum size of the replication units which have completed the DNA synthesis within 3 h of the S-period. The DNA synthesis in most replication units fails to be accomplished within the three hours of labelling. The process can be completed only in the fragments of DNA molecules of 40 to 200 micron (the average value being 100 micron) in human cells, whereas in the Chinese hamster cells the fragments of 40 to 250 micron (the average being about 140 micron) are completely replicated. Provided that the replicaton is bidirectional the complete replicons are supposed to contain two such fragments. Consequently, the greater part of replication units in mammalian cells covers the pieces of a few hundred microns in DNA molecules. The relation between replication process at the DNA molecules level and that at the metaphase chromosome level is discussed.

Autoradiography↗
PubMed1983

Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2.

Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of RK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.

DNA Replication↗
PubMed1985

Maintenance of multicopy plasmid Clo DF13 III. Role of plasmid size and copy number in partitioning.

To elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell. From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cell population. The effect of plasmid copy number is dominant over the effect of plasmid size.

Cell Division↗
PubMed2002

A spontaneous runaway vector for production-scale expression of bovine somatotropin from Escherichia coi.

An Escherichia coli expression vector was constructed for the production-scale fermentation of recombinant bovine somatotropin (rBST). Gene expression is regulated by a spontaneous increase in copy number at a constant low temperature without the need for an external inducer. This vector, designated pURA-4, contains the ampicillin resistance gene, the replication origin from pBR322, the R1 temperature-inducible runaway replicon, and a gene encoding rBST. Optimized rBST expression levels of >35% total cell protein were achieved at a constant 28 degrees C. Shake-flask analysis of pURA-4 shows that the copy number spontaneously increases approximately 6-fold during rBST production. Investigation into the mechanism of pURA-4 spontaneous runaway shows that the increase in copy number is directed by the pBR322 ori and not by the R1 replicon. Although the R1 temperature-inducible replicon does not mediate spontaneous runaway, it does have a positive effect on rBST expression. Copy number analysis also confirmed the stability of pURA-4 spontaneous runaway from the shake-flask scale through the production scale.

Animals↗