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Results for “replicative form”

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PubMed1991

The isolation and culture of DHBV-infected embryo and duckling hepatocytes and the effect of aflatoxin B1 or irradiation on these cells.

The preparation of primary cultures of control and DHBV-infected duck hepatocytes from embryos and young ducklings is described. Cultures of both embryo and duckling hepatocytes secreted duck serum proteins. Cultures of hepatocytes established from ducklings maintained initial morphology for up to 3 weeks in culture and also exhibited high levels of metabolism of aflatoxin B1. Embryonic cell cultures rapidly lost ability to metabolise AFB1 and became overgrown by spindle-shaped cells. Both embryo and duckling cell cultures secreted infective DHBV, and had intracellular replicative forms of the virus. No integration of the virus into the duck genome was observed, and attempts to induce viral integration in the duckling hepatocytes using irradiation and aflatoxin B1 toxicity were unsuccessful. The results of the study lend further support to the suggestion that the rarity of liver cancer in DHBV-infected experimental ducks is related to an innate resistance of the hepatocytes to develop DHBV-DNA integration. Another possibility may be related to the lower oncogenic potential of the DHBV strain used for the study. However DHBV infected duckling hepatocytes would appear to offer a suitable material for studying viral replication and mechanisms of aflatoxin B1 toxicity during prolonged cell culture.

Aflatoxin B1↗

PubMed2007

Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia.

The relationship between the occurrence of cryoglobulins and hepatitis C virus (HCV) productive infection in peripheral blood and bone marrow-derived lymphocytes was explored. HCV minus strand RNA, the viral replicative intermediate, was searched for by a polyA(+) tract strand-specific Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR) in lymphoid cells of 46 patients with acute and chronic infection. The HCV minus strand was demonstrated in RNA extracted from six (13%) and five (11%) peripheral blood and bone marrow-derived lymphocytes, respectively. The HCV replicating form in lymphoid cells was associated strictly with mixed cryoglobulinaemia (MCG), in that it was found in six of 13 (46%) MCG patients, including two with B cell non-Hodgkin's lymphoma (NHL). No traces of HCV-negative strand RNA were found in four patients with acute hepatitis C, in 15 with chronic active hepatitis without extrahepatic disorders, in seven with monoclonal gammopathy of undetermined significance, and in seven with B-NHL without MCG. These results emphasize the direct role of the virus in the pathogenesis of MCG and support the contention that HCV is not specifically lymphotropic, its entry and replication in lymphoid cells being determined largely by selective interactions.

Acute Disease↗

PubMed1985

Displacement synthesis of globin complementary DNA: evidence for sequence amplification.

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Animals↗

PubMed1996

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA.

An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

Bacteriophages↗

PubMed1972

Specific endonuclease R fragments of bacteriophage phiX174 deoxyribonucleic acid.

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.

Autoradiography↗

PubMed1980

Specificity of the spermidine requirement for the replication of phi X174 DNA be cell-free extracts of Escherichia coli.

A new experimental approach for assessing the biological significance of spermidine interactions in isolated systems is applied to the stimulation by spermidine of the conversion of phi X174 virion DNA to its replicative form by cell-free extracts of Escherichia coli. At 2.5 mM Mg2+, spermidine activated the reaction 20-fold. Varying the spermidine concentration affected both the rate and extent of this DNA synthetic reaction without altering the nature of the reaction products. We evaluated the biological significance of the spermidine requirement by measuring reaction rates in the presence of a homologous series of spermidine analogs of known activity in vivo. There was a lack of specificity, in that all of these analogs were capable of efficiently substituting for spermidine in stimulating the reaction rate. The relevance of this in vitro spermidine stimulation to Escherichia coli chromosome replication in vivo is discussed in light of the results obtained with the spermidine analogs.

Bacteriophage phi X 174↗

PubMed1981

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.

Bacteriophage phi X 174↗

PubMed1982

Effect of UV-irradiation on DNA replication of the parvovirus minute-virus-of-mice in mouse fibroblasts.

The effect of UV-irradiation on the conversion of the single-stranded DNA of the parvovirus Minute-Virus-of-Mice (MVM) to duplex Replicative Forms (RF) was studied after infection of mouse A9 fibroblasts. UV-irradiation of the virus prior to infection of unirradiated cells resulted in a dose-dependent, single-hit, inhibition of RF formation. Restriction fragment analysis indicated that this inhibition could be ascribed to the introduction of absolute blocks which prevent elongation of the newly synthesized complementary strand. Cell exposure to UV-light prior to infection with UV-irradiated MVM enhanced the fraction of input viral DNA which was converted to RF. This enhancement required de novo protein synthesis during the interval between cell irradiation and virus infection. These results suggest that DNA replication constitutes a target in the viral life cycle that leads to the UV-enhanced Reactivation of virus survival, however, they do not permit us to identify the step of RF formation which is enhanced in UV-pretreated cells.

Animals↗

PubMed1979

Endonucleolytic activity for gamma-irradiated DNA in normal and ataxia telangiectasia fibroblast cell extracts.

The increased sensitivity of ataxia telangiectasia cells towards ionizing radiation may be related to their inability to incise DNA near sites of radiation-induced base damages. When compared to 3 unaffected controls, crude extracts from 5 lines of fibroblast cells derived from ataxia telangiectasia patients were capable of incising gamma-irradiated DNA to the same extent as normal cells as determined in a nicking assay, using the circular replicative form of phiX174. However, the types of alterations introduced into DNA by gamma-irradiation could be distinguished from sites of base loss due to depurination or depyrimidination and from sites of base modification by OsO4. The specific endonuclease involved was demonstrated to be distinct from the apurinic endonuclease by its rate of temperature inactivation.

Ataxia Telangiectasia↗

PubMed1968

Host cell participation in small virus replication. I. Replication of M-13 in a strain of Escherichia coli with a temperature-sensitive lesion in deoxyribonucleic acid synthesis.

The replication of M-13 in a strain of Escherichia coli with a thermosensitive lesion in deoxyribonucleic acid synthesis was studied. M-13 failed to replicate at the restrictive temperature, even when the parental replicative form was allowed to form at the permissive temperature. When cells which were actively producing phage at the permissive temperature were shifted to the restrictive temperature, phage production continued. The incorporation of radioactive label into phage particles at 42 C indicated that continued single-strand synthesis was unaffected by the lesion in the host cell.

Chloramphenicol↗

PubMed1987

Escherichia coli mutT mutator effect during in vitro DNA synthesis. Enhanced A.G replicational errors.

The mechanism of the Escherichia coli mutT mutator effect was investigated using single-stranded phage as a mutational target. In vivo experiments showed that two M13mp2 lacZ alpha nonsense mutants reverted at a higher rate on a mutT1 host than on the wild-type host. The specificity of this mutator effect was identical to that observed for E. coli genes: A.T----C.G transversions. The mutT effect was subsequently demonstrated in vitro during DNA replication of M13mp2 DNA in cell-free extracts of E. coli. Replication (the single-stranded----replicative form conversion) in mutT1 extracts proceeded with a higher error rate than in wild-type extracts, and DNA sequence analysis of the in vitro revertants revealed the specific induction of A.T----C.G transversions. On the basis of the template specificity of the mutT effect in vitro, we conclude that the mutT effect involves the aberrant processing of A.G rather than T.C mispairs.

Bacterial Proteins↗

PubMed2002

Detection of hepatitis C virus sequences in brain tissue obtained in recurrent hepatitis C after liver transplantation.

Patients with chronic hepatitis C frequently report tiredness, easy fatigability, and depression. The aim of this study is to determine whether hepatitis C virus (HCV) replication could be found in brain tissue in patients with hepatitis C and depression. We report two patients with recurrent hepatitis C after liver transplantation who also developed severe depression. One patient died of multiorgan failure and the other, septicemia caused by Staphylococcus aureussis. Both patients had evidence of severe hepatitis C recurrence with features of cholestatic fibrosing hepatitis. We were able to study samples of their central nervous system obtained at autopsy for evidence of HCV replication. The presence of HCV RNA-negative strand, which is the viral replicative form, was determined by strand-specific Tth-based reverse-transcriptase polymerase chain reaction. Viral sequences were compared by means of single-strand conformation polymorphism and direct sequencing. HCV RNA-negative strands were found in subcortical white matter from one patient and cerebral cortex from the other patient. HCV RNA-negative strands amplified from brain tissue differed by several nucleotide substitutions from serum consensus sequences in the 5' untranslated region. These findings support the concept of HCV neuroinvasion, and we speculate that it may provide a biological substrate to neuropsychiatric disorders observed in patients with chronic hepatitis C. The exact lineage of cells permissive for HCV replication and the possible interaction between viral replication and cerebral function that may lead to depression remain to be elucidated.

Base Sequence↗

PubMed1978

Heterologous transfection with bacteriophage phiX174 DNA: and improved system.

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage phiX174 DNA was established. By mild washing of the speroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of phiX174 progeny phage production and not on those of phiX174 dougle-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage phiX174 DNA can be detected and the number of molecules can be determined with accuracy.

Bacteria↗

PubMed1994

Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds.

We have developed an assay using flavivirus RNA-dependent RNA polymerase to test the inhibitory activity of potential antiviral agents. The effects of antiviral agents on RNA synthesis were examined in this assay using extracts of Vero cells infected with dengue virus type 2 or Kunjin virus. Several different classes of known polymerase inhibitors were tested. The synthesis of double-stranded replicative form RNA was inhibited in a dose-dependent fashion in the presence of the polyoxometalate HPA-23 [(NH4)18(NaW21Sb9O86)17].14 H2O and several structurally related compounds.

Animals↗

PubMed1976

Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage phi X174.

A DNA protein complex that is a precursor of mature phi X174 phage was isolated. The complex sedimented with an S value of 50 in a sucrose gradient and contained phage DNA consisting of a replicative form molecule with an extended tail of single-stranded viral DNA. The viral-strand DNA ranged from one to two genomes in length. Proteins coded on the phi X174 genome as well as the host genome were associated with the viral DNA in the 50S precursor complex. Our results indicated that both viral DNA synthesis and cleavage of the growing viral-strand DNA occurred in the 50S complex.

Coliphages↗

PubMed1979

[Comparative antiviral and interferonogenic activity of natural and synthetic interferon inducers in vitro and in vivo].

Biological activities of the RNA replicative form of phage f2, a natural interferon inductor and poly-I -- poly-C, a synthetic polyribonucleotide complex were studied comparatively. Differences in the comparative interferonogenic and antiviral activity of the inductors were as dependent on the type of the cell system. It was shown that DEAE-dextran increased the interferon-inducing activity of RFf2 in the cell culture by 4 to 8 times. The dynamics of the interferonogenic and antiviral activity of RFf2 in the L-929 cell culture was studied. Interferon appeared in the culture fluid in 6--8 hours and reached its maximum titers (128 IU50/ml) by the 24th hour, the maximum protection of the cells being also developed by the 12th--24th hour, reaching on an average 51 g PFU/ml. It was shown in the experiments with green marmosets that administration of RFf2 in the form of aerosol in a dose of 2.3 mg/kg induced interferon production in the blood serum the titers of which amounted to 80--160 IU50/ml 24 hours after the administration.

Animals↗

PubMed1988

Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X.

Gene II protein is required for all phases of filamentous phage DNA synthesis other than the conversion of the infecting single strand to the parental double-stranded molecule. It introduces a specific nick into the double-stranded replicative form DNA, is required for the initiation of (+) strand synthesis and is responsible for termination and ring closure of the (+) strand product. Here we show that the gene II protein also promotes minus strand synthesis later in infection. Over-expression of gene II protein can induce the conversion of all nascent single-stranded phage DNA to the double-stranded form, even in the presence of the single-stranded DNA-binding gene V protein that would normally sequester the newly synthesized single strands. We also present evidence that the gene X protein (separately translated from an initiator codon within gene II, and identical to the C-terminal one-third of the gene II protein) is a powerful inhibitor of phage-specific DNA synthesis in vivo.

Base Sequence↗

PubMed1984

Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.

Bacteriophage phi X 174↗