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At least 1,153 records · Page 64Linked to original sources
PubMed1989

Identification and characterization of a locus inhibiting extrachromosomal maintenance of the Streptomyces plasmid SLP1.

We report here the existence and initial characterization of a genetic locus (imp) that inhibits maintenance of SLP1-derived plasmids as extrachromosomal replicons in a manner distinct from normal incompatibility between autonomous SLP1 replicons. The trans-acting imp function has been localized to a 1.8 kb Eco47III restriction fragment present on integrated SLP1 elements. At least part of this DNA segment is absent from SLP1-derived plasmids. DNA sequence analysis of the imp region indicates that it contains three overlapping open reading frames (ORFs) that may constitute a polycistronic operon. The effects of insertions within the imp region indicate that uninterrupted transcription through all three ORFs is necessary for imp activity.

Amino Acid Sequence↗
PubMed2000

Cloning and analysis of the replication origin and the telomeres of the large linear plasmid pSLA2-L in Streptomyces rochei.

The replication origin and both terminal segments were cloned from the large linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. The basic replicon consists of a 1.9-kb DNA fragment, which contains the genetic information required for autonomous replication in circular form. Sequence analysis revealed two ORFs, RepL1 and RepL2, with no similarity to any of the replication initiator proteins in the database. Deletion and mutational analysis showed that RepL1 is essential for replication and RepL2 has a subsidiary function. The origin of replication may be located 800 bp upstream of repL1. Sequencing of the left and right terminal segments revealed the presence of 12 palindromes. The sequence of the first 90 bp, including palindromes I-IV, shows great similarity to that of other Streptomyces linear chromosomes and plasmids. These results suggest that the internal replication origins of the linear replicons vary widely, in contrast to the high degree of conservation of their telomeres.

Amino Acid Sequence↗
PubMed2006

Polypyrimidine-tract-binding protein is a component of the HCV RNA replication complex and necessary for RNA synthesis.

The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane protein-associated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

Cell Line, Tumor↗
PubMed1983

Fiber DNA studies of premeiotic mouse spermatogenesis.

The technique of fiber DNA measurement was used to study the possibility that the lengthening of the DNA "S" phase previously reported for mouse premeiotic spermatogonia was due to a reduced number of initiation sites. The mean replicon size of neonatal mouse preleptotene cells was similar to sizes reported for adult mouse somatic cells. A slow rate of DNA chain growth was observed in all cells from day 1 through days 10-12 of age. It was felt that the meiotic entry in male mouse germ cells may involve a slower replication fork rate and other factors which increased the time between activation and replication of replicon families.

Animals↗
PubMed1984

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells.

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.

Animals↗
PubMed1985

Does capacity of DNA replication change during in vitro ageing?

We described elsewhere how a lack of change in the rate of DNA chain elongation occurred during in vitro ageing of human diploid fibroblasts. Here we further examined the rate of actual incorporation of tritiated thymidine, the center-to-center distance of replicons and the length of each phase of the cell cycle in order to extend our previous results to the other aspects of DNA replication. The results obtained showed that the rate of net DNA synthesis, the replicon size and the duration of S phase did not change during in vitro ageing. Our findings indicated that the reason why the greater part of the cell population at high population doubling levels becomes incapable of proliferating might not be the gradual decline in the ability of DNA replication. The regulation system(s) of DNA replication may alter during the period of culturing without any change in the capacities of the DNA replication machinery and, consequently, the non-cycling cells increase.

Autoradiography↗
PubMed1980

Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance.

We have identified and characterized a genetic function (designated par, for partition) that is required for stable maintenance of plasmids within exponentially growing cell populations. This function, which accomplishes the active distribution of plasmid DNA molecules to daughter cells, has been localized within the pSC101 plasmid to a 270 bp segment adjacent to the replication origin. The par locus, which appears to be functionally equivalent to the centromere of eucaryotic cells, is able to rescue unstable pSC101-derived replicons or an unrelated par- P15A-derived multicopy replicon in the cis, but not the trans, configuration. It is independent of copy number control and dose not specify plasmid incompatibility. Furthermore, it is not associated directly with plasmid replication functions.

Cell Division↗
PubMed1986

Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids.

To identify DNA sequences that function in the control of DNA replication, we designed a hybrid replicon consisting of linked SV40 and BPV DNA sequences. In the composite SV40-BPV plasmid negative control encoded by BPV is dominant over the uncontrolled replication encoded by the positive factor, SV40 T antigen. Using a transient replication assay, we show that replication control requires three BPV elements. Two cis-acting sequences are closely linked to BPV replication origins. A third trans-acting element is encoded within the 5' part of the BPV E1 open reading frame (ORF) and is separable from the positive replication factor encoded within the 3' part of the same ORF. The controlled replication of SV-BPV composite replicons has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes.

Animals↗
PubMed1984

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state.

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.

Chromosome Mapping↗
PubMed1991

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis.

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.

Bacillus subtilis↗
PubMed1991

Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2.

Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperature-sensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temperature sensitivity but the nonpermissive temperature pattern was different than that found in E. coli.

DNA Replication↗
PubMed1990

Inhibition of DNA synthesis and cytotoxic effects of some DNA topoisomerase II and gyrase inhibitors in Chinese hamster V79 cells.

In this study, some DNA topoisomerase II and gyrase inhibitors have been identified as inhibitors of polymerization of deoxyribonucleotides [novobiocin (NVB), nalidixic acid (NDA), oxolinic acid (OXA)], or inhibitors of replicon initiation and DNA-chain elongation [etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)methansulfon-m-anisidine (m-AMSA), ellipticine (ELT)]. The inhibitors of deoxyribonucleotide polymerization produced a significant (greater than 85%) suppression of [3H]thymidine incorporation into V79 cells within 20 min of treatment, followed by a rapid recovery of DNA synthesis, and reduced cell killing. In contrast, the inhibitors of replicon initiation and DNA-chain elongation needed about 60 min to induce a partial, but irreversible inhibition of DNA replication, associated with extensive cell killing.

Animals↗
PubMedDate not supplied

Cancer: re-evolution of eucaryotic to procaryotic replication units--a result of genetic transposition?

We have consistently tried to present our "procaryote-hypothesis" of oncogenesis in the light of evolution. It is suggested that the replication units are the essential starting points of oncogenesis, and cancer is the result of a short and compact re-evolution from eucaryotic to procaryotic replicons. In our view, the process of re-evolution or reactivation of procaryotic replication units and/or their precursors starts with the activity of a cancerogen and proceeds by genetic rearrangements, possibly by genetic transposition. Consequently, some remarks had to be made on: (a) the evolution of self-replicative species; (b) the evolvement of procaryotes to eucaryotes; and (c) the various mobile repeated genetic elements. Moreover, we have taken into consideration the important fact that most of the mammalian genome is reversibly repressed and "that there is in these modern genes still a memory of ancient sequences" ( Eigen et al., 1981). We can only speculate on the extent of the evolutionary conservation, particularly of certain ancient nucleotide sequences related to autonomous, indefinite self-replication. Nevertheless, no real argument can be made against the simple possibility that a re-evolution of eucaryotic to procaryotic replicons lead to cancer.

Animals↗
PubMed1990

Autonomy in tumor cell proliferation.

Autonomous replication of tumor cells seems to be an essential factor in the definition of the malignant tumor itself, although tumor cell proliferation is, in general, controlled by the host response including immunological reactions and microenvironment. The cause of the autonomy can hypothetically be classified into four categories as follow: (a) auto- and paracrine growth stimulation; (b) growth factor receptor abnormalities; (c) abnormal signal transduction; (d) self-incitement of 'initiator-replicon' system in DNA replication. These intracellular mechanisms may play important roles in the autonomy as shown in autocrine growth factors from the data obtained in protein-free cell culture. Hypothetically, negative regulation systems on the initiator-replicon may play roles of cell replication in multicellular organisms. Oncogene products and growth factors may affect this regulation system.

Animals↗
PubMed1986

Intramolecular transposition of IS102.

It has been postulated that deletions mediated by transposable elements are intramolecular transposition events. An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication. We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons. We show that when two origins (ori's) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions. On the contrary, when two ori's (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions. However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions.

Chromosome Deletion↗
PubMed1986

Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.

Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.

Cloning, Molecular↗
PubMed1988

Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae.

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.

Bacteriophage mu↗
PubMed1989

Analysis of IncF plasmids evolution: nucleotide sequence of an IncFIII replication region.

We determined the nucleotide sequence of RepFIII, the IncFIII replication region of the plasmid pSU316. Our data confirmed that RepFIII belongs to the RepFIIA family. The comparison of the nucleotide sequences from several RepFIIA-family plasmids revealed that pSU316 and R1 replicons are almost identical (96% similarity). Most of the differences between them are clustered in the incompatibility determinant. Analysis of the rest of the sequence suggested that the divergence between R1 and pSU316 replicons is very recent.

Base Sequence↗