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Results for “replicative form”

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At least 307 records · Page 17Linked to original sources

PubMed1982

Role of SSB protein in RecA promoted branch migration reactions.

The RecA protein of Escherichia coli is essential for genetic recombination and postreplicational repair of DNA. In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of phi X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981 a0. In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle. Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA. The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein. In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA. These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.

Adenosine Triphosphatases↗

PubMed1984

Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions.

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.

Autoradiography↗

PubMed1991

Nonstructural protein NS2 of parvovirus H-1 is required for efficient viral protein synthesis and virus production in rat cells in vivo and in vitro.

We generated a mutation in the gene for the nonstructural protein NS2 of parvovirus H-1 in which the highly conserved dinucleotide AG at the 3' splice acceptor site of NS2 intron 1 was mutated to CG. The mutation does not change the amino acid sequence for NS1. The splice acceptor (SA) mutant gene was introduced into the H-1 virus (H-1SA) and an infectious clone of LuIII (pLuH1SA). The R2 transcripts encoding NS2 were absent by both Northern blot and primer extension analysis in the LuH1SA or H-1SA virus-infected cells and the NS2 protein was undetectable in the infected cell lysate by immunoprecipitation. These NS2 null mutant viruses were capable of lytic growth in cell lines that were derived from human, hamster, and dog, but they produced lower virus titers than wild-type H-1. The H-1SA virus nonproductively infected Rat2 rat fibroblasts and transformed Rat2 cell lines. Analysis of synchronized infections of rat fibroblasts demonstrated that H-1SA viral duplex replicative form DNA replication was reduced and that single-stranded progeny DNA was deficient compared to wild-type H-1. In addition, H-1SA viral protein synthesis was about 10% of wild-type virus and virions were not detectable in rat fibroblasts. However, H-1SA mRNAs R1 and R3 accumulated to wild-type levels. NS2 was also required for productive infection in newborn rats but not in newborn hamsters. These results indicate that NS2 plays an important role in the regulation of viral protein synthesis in rat cells in vivo and in vitro.

Animals↗

PubMed1983

The fine structure of Cymbidium ringspot virus infections in host tissues. III. Role of peroxisomes in the genesis of multivesicular bodies.

Ultrastructural studies of Nicotiana clevelandii plants systemically infected with Cymbidium ringspot virus, a member of the tombusvirus group, have shown that a clear-cut relationship exists between perioxisomes and multivesicular bodies (MVB). In infected cells, peroxisomes undergo a progressive vesiculation of the bounding membrane through the possible addition of membranous material by the endoplasmic reticulum and become very plastic. Portions of the ground cytoplasm are engulfed either through the invagination of the limiting membrane or the production of membranous appendages that fold back on the main body. Cytochemical tests have shown MVB to possess catalase and glycolate oxidase activity in the matrix. The vesicles contain RNA, a substantial amount of which is double stranded, as indicated by differential RNase digestion tests in high- and low-salt media. The double-stranded RNA may consist of replicative forms or replicative intermediates of the viral nucleic acid. If so, MVB (i.e., modified peroxisomes) may be directly involved in the replication of Cymbidium ringspot virus.

Catalase↗

PubMed1979

Origin and direction of DNA replication of plasmid RSF1030.

An in vitro replication system has been used to study the origin and direction of replication of the covalently closed, circular DNA of plasmid RSF1030, a nonconjugative R factor. We have enriched for replicative intermediates in these studies either by isolating them on the basis of their unique structure or by limiting the extent of synthesis in the in vitro system. Circular molecules that have replicated to various extents migrate to characteristic positions in agarose gels, thus providing a rapid and efficient method for isolating partially replicated forms. Alternatively, replicative intermediates can be isolated directly from reaction mixtures that contain dideoxyTTP (ddTTP), a compound that limits the average extent of synthesis in vitro. Electron microscopic analysis of such intermediates linearized with either Hpa I or BamHI indicates that RSF1030 replicates in vitro from a unique origin located 70% from one end of Hpa I-cleaved molecules and 47% from the BamHI site. The unidirectional mode of replication has been confirmed by the order in which the six HincII fragments of RSF1030 DNA are labeled in vitro when synthesis is limited to various extents with ddTTP. Finally, a physical map of RSF1030 has been constructed using the restriction endonucleases BamHI, Hpa I, and HincII, and the origin and direction of replication have been defined relative to the map.

Cell-Free System↗

PubMed1991

Plum pox potyvirus RNA replication in a crude membrane fraction from infected Nicotiana clevelandii leaves.

In vitro synthesis of plum pox potyvirus (PPV)-specific nucleic acid has been measured in a crude fraction prepared from leaves of PPV-infected Nicotiana clevelandii plants. Using alkali and DNase treatments, the synthesized nucleic acid was shown to be RNA. The electrophoretic mobility and the differing sensitivity to RNase at high and low salt concentrations allowed the identification of in vitro products probably corresponding to replicative form and replicative intermediate RNA, as well as to single-stranded RNA. Most of the PPV-specific RNA synthesized was shown to be of positive polarity. The in vitro RNA synthesis, performed in the presence of actinomycin D, required all four ribonucleoside triphosphates and Mg2+ ions. This enzyme extract contained about 6% of the leaf protein and most of the identified virus-encoded proteins. Altogether, the results presented in this paper suggest that in vitro RNA synthesis was carried out by the PPV replicase complex.

Blotting, Northern↗

PubMed1966

Allomorphic forms of bacteriophage phiX-174 replicative DNA.

The bacteriophage øX-174 replicative form of DNA exists in two configurations, as judged by electron microscopy and sucrose density gradient sedimentation, and on methylated albumin-coated kieselguhr column chromatography. The predominant 21S form of the DNA is a tightly twisted circle. Treatment with deoxyribonuclease causes a single scission in one of the two strands, resulting in an open circle which sediments at 16S.

Chemical Phenomena↗

PubMed1976

Replication process of the parvovirus H-1 V. Isolation and characterization of temperature-sensitive H-1 mutants.

Two temperature-sensitive mutants of the parvovirus H-1 have been isolated and characterized. These mutants are distinguishable by the immunofluorescent staining of cells infected by them and by the thermolability of their hemagglutinins. Under restrictive conditions both mutants synthesize a missense capsid protein defective in hemmaglutination. Synthesis of the viral DNA strand was shown to be diminished for both wild-type virus and these mutants at the restrictive temperature of 39.5 C, but the mutants were more defective than the wild type at the less restrictive temperature of 38 C. Replicative form DNA replication was not decreased in these mutants. It is proposed that H-1 requires one of the capsid proteins for the synthesis of the single-stranded progeny DNA.

Culture Techniques↗

PubMed1978

Defective interfering particles of parvovirus H-1.

Defective interfering particles of the parvovirus H-1 were produced by serial propagation at high multiplicities of infection. Such particles interfere with the synthesis of capsid proteins and infectious virus of standard H-1. The interference is sensitive to UV irradiation, dependent on the multiplicity of the challenge virus, and is active in heterotypic infections against parvovirus H-3 or LuIII. Defective interfering particle genomes have alterations characterized by integral numbers (1 to 10 or more) of a 60-base-pair addition in the neighborhood of the origin of replicative-form DNA replication and deletions that are located primarily within two regions, 32 to 44 or 80 to 90 on the genome map. Some of the implications of these findings are discussed.

Capsid↗

PubMed1979

Studies of two temperature-sensitive mutants of Mengo virus.

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA- ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 degrees C) to the nonpermissive (39 degrees C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA- phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 degrees C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant. Subviral (53S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 degrees C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

Animals↗

PubMed1975

Defective particles in alphavirus infections.

This article summarizes our studies with defective-interfering particles of Sindbis virus obtained by high multiplicity passaging of the virus in BHK cells. Cells infected with these defective passages accumulate a species of RNA (20S) at the expense of 26S RNA--the mRNA coding for the viral structural proteins. Although the structure of the RNA in defective particles remains undefined, our studies of replicative forms and replicative intermediates suggest that it is larger than the intracellular 20S RNA. The defective particles are unable to synthesize detectable amounts of viral structural proteins when they infect a cell in the absence of standard virus and they do not contribute to the stimulation of intracellular viral RNA synthesis. We have proposed a model for the mechanism of interference by these defective particles in which standard and defective RNAs compete for a limited amount of viral-specific replicase.

Cell Line↗

PubMed1987

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication.

The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.

Bacteriophage phi X 174↗

PubMed2005

Possession, use, and transfer of select agents and toxins--reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments. Interim final rule.

We are adding reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments to the list of HHS select agents and toxins. We are taking this action for several reasons. First the pandemic influenza virus of 1918-19 killed up to 50 million people worldwide, including an estimated 675,000 deaths in the United States. Also, the complete coding sequence for the 1918 pandemic influenza A H1N1 virus was recently identified, which will make it possible for those with knowledge of reverse genetics to reconstruct this virus. In addition, the first published study on a reconstructed 1918 pandemic influenza virus demonstrated the high virulence of this virus in cell culture, embryonated eggs, and in mice relative to other human influenza viruses. Therefore, we have determined that the reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments have the potential to pose a severe threat to public health and safety.

Animals↗

PubMed1999

Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction.

Numerous environmental influences have been demonstrated to enhance recombinant adeno-associated virus (rAAV) transduction. Such findings are the foundation of developing new and innovative strategies to improve the efficiency of rAAV as a gene therapy vector. Several of these environmental factors included genotoxic stresses such as UV and y irradiation as well as certain adenoviral gene products such as E4orf6. The mechanisms by which these environmental stimuli increase rAAV transduction are only partially understood but have been suggested to involve both endocytosis and uptake of virus to the nucleus, as well as conversion of single-stranded DNA viral genomes to double-stranded expressible forms. Two molecular intermediates of rAAV genomes, which have been demonstrated to correlate with transgene expression and/or the persistence of rAAV, include both replication form (Rf) monomers and dimers as well as circular intermediates. In the present study, we demonstrate that augmentation of rAAV transduction by UV irradiation and the adenoviral protein E4orf6 correlates with distinct increases in either circular or replication form intermediates, respectively. UV irradiation of primary fibroblasts at 15 J/m2 resulted in a 15-fold induction of head-to-tail circular intermediates, with minimal induction of replication form rAAV genomes. In contrast, E4orf6-augmented rAAV transduction was correlated with the formation of replication form intermediates, with no alteration in the abundance of circular intermediates. These findings demonstrate that rAAV transduction can occur through two independent molecular pathways that convert single-stranded AAV genomes to expressible forms of DNA.

Adenovirus E4 Proteins↗

PubMed1986

Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment.

1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1978

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.

Coliphages↗

PubMed1991

Interferon alfa therapy in patients with chronic hepatitis B virus infection. Effects on hepatitis B virus DNA in the liver.

Pretrial and posttrial liver biopsy samples from 124 adult patients who participated in two randomized, controlled trials of interferon alfa therapy for chronic hepatitis B virus (HBV) infection were analyzed to determine the effects of interferon on the replication of HBV in the liver. Replicative forms of HBV DNA were detected in the pretrial biopsy samples from all and posttrial biopsy samples from 74% treated patients and 86% controls. Replicative forms of HBV DNA were detected in the posttrial biopsy samples from all patients who remained positive for hepatitis B e antigen and HBV DNA in the serum, in 77% treated patients and 80% controls who cleared HBV DNA in the serum but who remained positive for hepatitis B e antigen, but in only 19% treated patients and 40% controls who cleared HBV DNA as well as hepatitis B e antigen in the serum. Serum alanine aminotransferase levels were significantly lower in patients whose posttrial biopsies did not contain replicative forms of HBV DNA. In summary, we demonstrated that in most patients with chronic HBV infection treated with interferon alfa, serological response was associated with the disappearance of replicative forms of HBV DNA in the liver.

Adolescent↗

PubMed1997

The emergence of form by replication.

It is shown with a simple mathematical model that if a system exhibits a given form (a spatial structure) and is put in contact with another system of the same type but in a state of spatial disorder, then under certain conditions their mutual interaction as they evolve in time allows replication of form in the disordered system with a controllable degree of faithfulness.

Biological Evolution↗