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At least 559 records · Page 31Linked to original sources
PubMed2003

Linear plasmid SLP2 of Streptomyces lividans is a composite replicon.

SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase-like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini-linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.

Bacterial Proteins↗
PubMed2002

Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs.

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of approximately 22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin AC and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs. These data support the principle of siRNA-based HCV antiviral therapy.

Base Sequence↗
PubMed2006

The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons.

We report the complete 6,530,228-bp genome sequence of the symbiotic nitrogen fixing bacterium Rhizobium etli. Six large plasmids comprise one-third of the total genome size. The chromosome encodes most functions necessary for cell growth, whereas few essential genes or complete metabolic pathways are located in plasmids. Chromosomal synteny is disrupted by genes related to insertion sequences, phages, plasmids, and cell-surface components. Plasmids do not show synteny, and their orthologs are mostly shared by accessory replicons of species with multipartite genomes. Some nodulation genes are predicted to be functionally related with chromosomal loci encoding for the external envelope of the bacterium. Several pieces of evidence suggest an exogenous origin for the symbiotic plasmid (p42d) and p42a. Additional putative horizontal gene transfer events might have contributed to expand the adaptive repertoire of R. etli, because they include genes involved in small molecule metabolism, transport, and transcriptional regulation. Twenty-three putative sigma factors, numerous isozymes, and paralogous families attest to the metabolic redundancy and the genomic plasticity necessary to sustain the lifestyle of R. etli in symbiosis and in the soil.

Gene Transfer, Horizontal↗
PubMed2000

Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola.

Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

Bacterial Proteins↗
PubMed1981

On the possibility of metabolic control of replicon "misfiring": relationship to emergence of malignant phenotypes in mammalian cell lineages.

Constraints of a multireplicon chromosomal organization and of the necessity to maintain constant gene dosages demand that each origin of replication in a eukaryotic cell "fire" (initiate replication) only once per cell cycle. The central idea of this work is that a low probability of an extra ("illegitimate") round of DNA replication (called below "replicon misfiring") within any given chromosomal domain could be increased by certain substances of either intra- or extracellular origin. The term " "firone" is proposed for such a substance. It is shown that existence of firones could greatly speed up evolution of cellular systems under selection pressure, a developing tumor being one example of such a system. Experimentally testable predictions of the firone hypothesis are discussed.

Animals↗
PubMed1983

Primary structure of the essential replicon of the plasmid pSC101.

The replicon of the low copy number plasmid pSC101 has an obligatory requirement for the dnaA initiator protein of Escherichia coli as well as a plasmid-encoded initiator protein. We have identified the cistron of the plasmid-encoded initiator by DNA sequence analysis. Fusion of the initiator cistron with the lacZ gene of E. coli yielded a fusion protein of approximately equal to 150 kilodaltons, thus confirming that the open reading frame detected by DNA sequence analysis actually encoded a 37.5-kilodalton protein. Deletion of 26 amino acid residues from the COOH terminus of the plasmid initiator abolished autonomous replication from pSC101 origin. By in vitro deletion analysis we have shown that, although sequences downstream from the initiator cistron are dispensable, a maximum of 400 base pairs immediately upstream from the NH2-terminal region of the initiator is necessary for plasmid replication. These upstream sequences contain an A + T-rich region and three tandem repeats of a 21-base pair sequence; these features are characteristics of other replication origins.

Bacterial Proteins↗
PubMed1986

Host participation in plasmid maintenance: dependence upon dnaA of replicons derived from P1 and F.

Nonparticipation of the bacterial dnaA gene in plasmid replication has been assumed to be the general rule. In conditional dnaA mutants of Escherichia coli, only plasmid pSC101 has been shown to have a dnaA requirement. Experiments with dnaA null mutants of E. coli, presented here, show that dnaA plays a critical and direct role in the replication of miniplasmids derived from P1 and F as it does in the initiation of bacterial replication. Evidence is also presented for the existence of a dnaA-independent secondary replicon of P1 that is able to drive bacterial chromosome replication but is inadequate to support the maintenance of P1 as a plasmid in E. coli.

DNA Replication↗
PubMed1988

Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone.

A methodology was developed for stable gene transfer into cloned nontransformed human T lymphocytes. Stable high-level gene expression was achieved in cloned human T cells by using a self-replicating Epstein-Barr virus (EBV) episomal replicon. A comparison of five eukaryotic promoters established that the Rous sarcoma virus 3' long terminal repeat (RSV 3' LTR) and the lymphopapilloma virus (LPV) 5' LTR are optimal for episome-based expression in T cells. Effective (greater than 95%), selective, and reversible anti-sense RNA-mediated gene inhibition of a model T-cell-associated molecule (CD8) was achieved in a cytotoxic human T-cell clone by using an EBV episome-based, RSV 3' LTR-driven expression system. The linking of anti-sense RNA mutagenesis and T-cell cloning technologies should contribute significantly to studies of human T-cell function.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons.

The genome of the porcine transmissible gastroenteritis coronavirus is a plus-strand, polyadenylylated, infectious RNA molecule of approximately 20 kilobases. During virus replication, seven subgenomic mRNAs are generated by what is thought to be a leader-priming mechanism to form a 3'-coterminal nested set. By using radiolabeled, strand-specific, synthetic oligodeoxynucleotide probes in RNA blot hybridization analyses, we have found a minus-strand counterpart for the genome and for each subgenomic mRNA species in the cytoplasm of infected cells. Subgenomic minus strands were found to be components of double-stranded replicative forms and in numbers that surpass full-length antigenome. We propose that subgenomic mRNA replication, in addition to leader-primed transcription, is a significant mechanism of mRNA synthesis and that it functions to amplify mRNAs. It is a mechanism of amplification that has not been described for any other group of RNA viruses. Subgenomic replicons may also function in a manner similar to genomes of defective interfering viruses to lead to the establishment of persistent infections, a universal property of coronaviruses.

Animals↗
PubMed1990

Wheat dwarf virus Ac/Ds vectors: expression and excision of transposable elements introduced into various cereals by a viral replicon.

The maize transposon Activator (Ac) and in vitro-generated nonautonomous derivatives thereof [Ac delta or Dissociation (Ds) elements] were inserted into the genome of a geminivirus of graminaceous plants, wheat dwarf virus, at a site that does not interfere with viral replication. These recombinant viral genomes were introduced into wheat, maize, and rice protoplasts, where rapid and efficient excision of Ac was observed. Excision was detected only in vectors in which, after transfection, the virus could replicate. This result is not restricted to the autonomous Ac; excision of Ds elements was also induced by transposase activity provided in trans by plasmids expressing the cDNA of Ac. The potential of this combination of a transposon with a viral replicon for plant molecular genetic engineering is discussed.

Base Sequence↗
PubMed1994

Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number.

The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the tau initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called tau 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal tau, thus supporting the handcuffing model. A second mutant called tau 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type tau protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of tau 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of tau or interaction of tau with the primary binding sites at gamma. Taken together, the results are consistent with the notion that tau-mediated handcuffing is a mechanism, but not the only mechanism, of copy control in R6K. Interaction of tau with host proteins is likely to provide additional facets of the copy control mechanism.

Amino Acid Sequence↗
PubMed1998

Mechanism of DNA segregation in prokaryotes: replicon pairing by parC of plasmid R1.

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

Bacterial Proteins↗
PubMed1995

The unusual structure of a novel RNA replicon in rice.

A linear, plasmid-like, double-stranded RNA (dsRNA) was isolated from rice, and its entire sequence of 13,952 nucleotides (nt) was determined. The dsRNA encodes a single, unusually long, open reading frame (13,716 nt, 4,572 amino acid residues), which includes an RNA helicase-like domain and an RNA-dependent RNA polymerase-like domain. A series of Northern hybridization and primer extension experiments revealed that the coding (sense) strand of the dsRNA contains a discontinuity (nick) at a position 1,211 nt (or 1,256 nt) from the 5' end. This discontinuity divides not only the coding strand of dsRNA molecule into a 1,211-nt fragment and a 12,741-nt fragment (or a 1,256-nt fragment and a 12,696-nt fragment) but also divides the long open reading frame into a 5' part of 1,045 nt (348 amino acid residues) and a 3' part of 12,671 nt (4,224 amino acid residues) or a 5' part of 1,090 nt (363 amino acid residues) and a 3' part of 12,626 nt (4,209 amino acid residues). It seems likely that almost all dsRNA molecules in rice plants contain such a discontinuity. This rice dsRNA appears to be a novel and unique RNA replicon.

Base Sequence↗
PubMed1998

Yeast positive-stranded virus-like RNA replicons. 20 S and 23 S RNA terminal nucleotide sequences and 3' end secondary structures resemble those of RNA coliphages.

Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qbeta. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3' ends. Both 20 S and 23 S RNAs have GGGGC at the 5' ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3' ends. S1 and V1 secondary structure-mapping of the 3' ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3' end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3' ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast.

Cloning, Molecular↗
PubMed2001

Human immunodeficiency virus type 1 Gag-specific vaginal immunity and protection after local immunizations with sindbis virus-based replicon particles.

The majority of human immunodeficiency virus (HIV) infections occur through vaginal and rectal transmission. In seeking a safe, nonreplicating gene-delivery vector that can induce mucosal and systemic immune responses and protection, Sindbis virus-based replicon particles expressing HIV-1 Gag (SIN-Gag) were developed. In mice, after nasal or intramuscular immunization with SIN-Gag and vaginal challenge with vaccinia virus (VV) expressing HIV-1 Gag (VV-Gag), CD8(+) T cell-mediated responses were detected locally, in the vaginal mucosa and in the draining iliac lymph nodes (ILNs), and systemically, in the spleen. However, the mice were not protected against VV-Gag replication in the ovaries. In contrast, after vaginal or rectal immunization with SIN-Gag and vaginal challenge with VV-Gag, despite lower local CD8(+) T cell-mediated responses in the vaginal mucosa and ILNs, the mice were protected against VV-Gag replication in the ovaries. Therefore, local immunization with SIN-Gag induced both local mucosal cell-mediated responses and protection.

AIDS Vaccines↗
PubMed2003

Toll-like receptor and cytokine expression patterns of CD56+ T cells are similar to natural killer cells in response to infection with Venezuelan equine encephalitis virus replicons.

Using the natural killer (NK) cell-surface marker CD56 to study NK T cells in peripheral blood, we found that their frequency in mononuclear cells among healthy individuals was 1%-20% (average, 7.3%) and sporadically increased 4-5-fold within individuals over the course of 8 months. Infection of mononuclear cells in vitro with Venezuelan equine encephalitis virus replicon particles (VRPs) resulted in a significant increase in CD56(+) T cells and in the expression of interferon-alpha, tumor necrosis factor (TNF)-alpha, and interferon-gamma by CD56(+) but not CD56(-) T cells. NK and CD56(+) T cells expressed higher levels of Toll-like receptor (TLR)-3 and TLR4 after infection with VRPs, whereas only NK cells expressed inducible TNF-alpha and TLR2. Most of these effects were duplicated by activating mononuclear cells with double-stranded RNA. These expression patterns indicate that T cells coexpressing NK markers respond like NK cells to viral infection or double-stranded RNA, potentially fulfilling innate and adaptive immune functions.

Animals↗
PubMed1998

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.

Bromodeoxyuridine↗
PubMed1983

Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix.

We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.

Cell Cycle↗