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PubMed1979

The rep mutation. VI. Purification and properties of the Escherichia coli rep protein, DNA helicase III.

The protein product of the rep gene of Escherichia coli is required for the replication of certain bacteriophage genomes (phi X174, fd, P2) and for the normal replication of E. coli DNA. We have used a specialized transducing phage, lambda p rep+, which complements the defect of rep mutants, to identify the rep protein. The rep protein has been purified from cells infected with lambda p rep+ phage; it has a molecular weight of about 70 000 and appears similar to the protein found in normal cells. Stimulation of phi X174 replicative form DNA synthesis in vitro was observed when highly purified rep protein was supplied to a cell extract derived from phi X-infected E. coli rep cells and supplemented with replicative form DNA. The purified protein has a single-stranded DNA-dependent ATPase activity and is capable of sensitizing duplex DNA to nucleases specific for single-stranded DNA. For this reason we propose the enzyme be called DNA helicase III. We infer that the rep protein uses the energy of hydrolysis of ATP to separate the strands of duplex DNA; the E. coli DNA binding protein need not be present. The rep3 mutant appeared to make a limited amount of active rep protein.

Bacterial Proteins↗
PubMed1975

[RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda].

Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides). RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E. coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease. They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174. These RNA fragments are also required for in vitro replication of lambda phage DNA.

Adenosine Monophosphate↗
PubMed1977

Replication process of the parvovirus H-1. IX. Physical mapping studies of the H-1 genome.

The cleavage map of H-1 replicative-form DNA to the bacterial restriction endonuclease EcoRI, HaeII, HaeIII, HindII, HindIII, and HpaII has been determined. The 5'-phosphoryl end of the viral strand is on the right end of the molecule at or near the replication origin. Evidence is presented for the presence of inverted self-complementary sequences at the right end that differ from those at the left end. These sequences allow a foldback of the DNA after denaturation, and a minority of the native replicative-form DNA has the foldback configuration. The possible role of these structures in H-1 DNA synthesis is discussed.

Base Sequence↗
PubMed1976

Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA.

Two virus-specific species of newly synthesized DNA were isolated from rat fibroblast cell cultures infected with the Kilham rat virus (RV). These two DNA species were purified; their behavior on hydroxyapatite chromatography and their sedimentation coefficients in sucrose gradients were determined. One of the two species corresponds to the linear double-stranded form of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a fraction of both species showed an intramolecular renaturation; these molecules are composed of viral strand covalently linked to complementary strand. Models for the structure of both species are posposed. Both species may be considered as double-strand replicative intermediates of the single-stranded RV DNA.

Cell Line↗
PubMed1991

[IgM anti-HBc in the differential diagnosis of co- and HDV superinfection].

EIA was used to study the titers of IgM anti-HBc in 38 patients with chronic hepatitis B, 10 with acute viral hepatitis B and 18 patients with D-infection (6 with coinfection and 12 with D-superinfection) depending on the presence of HBe/anti-HBe. Chronic active hepatitis (CAH) was diagnosed in 18 patients, chronic persistent hepatitis (CPH) in 15, and the "healthy" HBsAg carriership in 5. The titers of IgM anti-HBc 1:100 were detectable in 14 out of the 18 patients with CAH and in 3 out of the 15 patients with CPH and correlated with the clinico-biochemical activity of the process and the presence of HBeAg in serum. In coinfection, the antibody titers were determined in the range of the antibody titers were determined in the range of 1:3200 to 1:25600 for the first two weeks of the disease and did not depend on the presence of HBe/anti-HBe. Anti-D were identified only on the 3d week since the first disease manifestations. In the presence of the replicative form of CAH, HDV-superinfection was diagnosed in 3 patients with HBeAg in serum and the titers of IgM anti-HBc 1:100 and higher. The follow-up studies demonstrated inhibition of HBV replication shown up by HBeAg elimination from serum and a decrease in the titers of IgM anti-HBc. Therefore, the titers 1:100 cannot serve as a diagnostic criterion for coinfection, for in the replicative form of CAH, their concentration may be higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers↗
PubMed1983

The use of bacteriophage M13 carrying defined fragments of the Escherichia coli gltA gene to determine the location and structure of the citrate synthase promoter region.

The gltA gene from Escherichia coli, which encodes citrate synthase, has been located on a 3.24 Kb HindIII/EcoRl restriction fragment. This region contains one restriction site for BamHl and two for BglII. Defined restriction fragments from this region were cloned into suitably cleaved replicative form M13mp8 and M13mp9. The recombinants (M13gtlA1 leads to 10) were isolated as single stranded DNA and characterised on the basis of molecular weight and DNA sequence. The single stranded DNA was converted to the double stranded replicative form and used to transform E. coli strain JM103 from which bacteriophage were isolated. Infection of JM103 with different bacteriophage followed by measurement of expressed citrate synthase activity showed that the complete gltA gene must span the BamHl restriction site, that the control region was on the 5'-terminal side of this restriction site and that the coding region for citrate synthase protein commenced on the 3'-terminal side. Analysis of the DNA sequence of this region allowed us to confirm this model, to identify the start sequence for translation of the structural gene and a number of sequences controlling the initiation of transcription. Of special interest is the fact that there must be an extensive leader sequence (305 nucleotides) separating the predicted sites for initiation of transcription and translation.

Base Sequence↗
PubMed1983

Characterization of replicative intermediate RNA of mouse hepatitis virus: presence of leader RNA sequences on nascent chains.

Mouse hepatitis virus A59 codes for seven mRNAs in infected cells. These mRNAs are transcribed from a minus (-) strand template of genome length and contain a leader RNA at their 5' ends. To further elucidate the mechanism of coronavirus transcription, we examined the structure of mouse hepatitis virus replicative intermediates (RIs) isolated by 2 M NaCl precipitation and Sepharose 2-B column chromatography. Purified RIs migrated as a single species on agarose gels and sedimented between 12 and 38S on 10 to 25% sucrose gradients. The complexes were readily heat denatured into a heterogeneous population of smaller RNA molecules which probably represent nascent plus (+) strands. RNase A digestion of RIs produced a single replicative form which sedimented between 30 and 32S. These data suggest that the RI is composed of a single genome-sized (-) strand hydrogen bonded to an average of 4 to 6.5 nascent (+) strands. In contrast, a column-purified replicative form was extremely resistant to RNase A digestion and heat denaturation and migrated as a single RNA species on agarose gels and sucrose gradients. Oligonucleotide fingerprinting of an RI revealed the presence of the 5' leader RNA on the nascent (+) strands. In addition, an average of 6.2 cap structures were present in each RI, which agrees with the average number of nascent (+) strands per RI. These data suggest that the leader RNA is utilized as a primer for mouse hepatitis virus RNA transcription and is not added to mRNA post-transcriptionally.

Animals↗
PubMed1987

[Restriction analysis of the genome of swine abortion parvovirus and cloning of its PstI-EcoRI fragment].

The cultured pig kidney cells infected by the porcine parvovirus (PPV) produced the virions and viral DNA. The latter was used as a matrix to synthesize the double stranded DNA. The obtained preparation is more homogenic than the natural replicative form and was used for restriction analysis of porcine parvovirus genome and for molecular cloning of its fragment. The isolated recombinant plasmids contained the PstI-EcoRI fragment of PPV DNA, containing 70% of the viral genome. The restriction analysis of replicative PPV DNA isolated from the infected cells has resulted in finding of the replicative form containing a 300 bp deletion in the 5'-region of PPV genome.

Abortion, Veterinary↗
PubMedDate not supplied

HCV and lymphoproliferative diseases.

METHODS: Genomic and replicative forms of HCV-RNA in B lymphocytes were detected by RT-PCR, and HCV genotyping was performed using universal and type-specific primers for the core region. Immunoglobulin gene rearrangement was detected by RT-PCR. RESULTS: The presence of genomic and replicative forms of HCV-RNA in the 5'NC region was investigated on total RNA extracted from subpopulations of PBMC. The frequency of HCV-RNA was higher in the B lymphocytes than in other PBMC. In two patients a larger sized band was present in the B lymphocytes and PMN; this band could represent either another form of HCV-RNA or a cross-reaction between cellular RNA and HCV primers. HCV-RNA detected using primers for the core region was negative in the patients examined. Immunoglobulin monoclonal gene rearrangement was present on the cDNA in all of the HCV and type II cryoglobulinemia positive samples except two; in contrast, it was absent in the HCV positive and cryoglobulinemia negative samples. The analysis of immunoglobulin monoclonal gene rearrangement on DNA showed the presence of new positive samples among the HCV positive, type II cryoglobulinemia negative patients, who had been negative when PCR was performed on cDNA. Denaturing sequencing gel showed clearer results than agarose gel. CONCLUSIONS: The early detection of immunoglobulin monoclonal gene rearrangement and expression is very important because it could provide evidence of the possible lymphoproliferative evolution of HCV infection. In addition, these investigations together with PCR product sequencing could show us the steps in the clonal selection of B lymphocytes towards malignant transformation, in which HCV plays a direct and/or indirect role.

Base Sequence↗
PubMed2006

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-gamma.

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-gamma, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-gamma at 0.1 to 5 microg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5 microg/L, IFN-gamma also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-gamma showed no additive effect, sequential treatment first with lamivudine and then IFN-gamma was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 micromol/L lamivudine for two days, followed by 1 microg/L IFN-gamma for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 micromol/L lamivudine for two days, followed by 5 microg/L IFN-gamma for six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-gamma and lamivudine, especially in IFN-alpha non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Antiviral Agents↗
PubMed1983

In vitro synthesis of bacteriophage phi X174 by purified components.

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.

Bacteriophage phi X 174↗
PubMed1990

Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.

Animals↗
PubMed1982

Identification of ColE1 DNA sequences that direct single strand-to-double strand conversion by a phi X174 type mechanism.

A DNA single-strand initiation sequence, named rriA (called rri-1 previously), was detected in the origin region (Hae II fragment E) of the ColE1 plasmid [Nomura, N. & Ray, D. S. (1980) Proc. Natl. Acad. Sci. USA 77, 6566-6570]. Another site, called rriB, has been found on the opposite strand of Hae II fragment C. Both rriA and rriB (i) direct conversion of chimeric M13 phage single-stranded DNA to parental replicative form DNA in vivo by a rifampicin-resistant mechanism that is dependent on the dnaG and dnaB gene products, (ii) provide effector sites of dATP hydrolysis by primosomal protein n', and (iii) require the same primosomal proteins as phi X174 DNA for directing the in vitro conversion that rriA is the DNA sequence that determines the mechanism of lagging strand synthesis of ColE1 DNA and that the mechanism of discontinuous synthesis involves the primosomal proteins utilized in the in vitro conversion of phi X174 single strands to the double-stranded replicative form.

Bacterial Proteins↗
PubMed1975

Studies on bacteriophage M13 DNA. 1. A cleavage map of the M13 genome.

A physical map of the bacteriophage M13 genome has been constructed on the basis of specific cleavage of M13 replicative form DNA by bacterial restriction endonucleases. The 13 fragments produced by the enzyme from Haemophilus aphirophilus (endonuclease R.Hap II) as well as the 10 fragments produced by the enzyme from Haemophilus aegyptius (endonuclease R.Hae III) have been ordered by analysis of partial digest products and by analysis of overlapping sets of fragments. In addition, the single site in M13 replicative form DNA cleaved by the restriction enzyme from Haemophilus influenzae Rd (endonuclease R.Hin dII) has been located more precisely. With this unique site as a reference point, the H. aphirophilus cleavage sites and the H. aegyptius cleavage sites have been localized on the map.

Chromosome Mapping↗
PubMed1993

Aleutian mink disease parvovirus infection of mink peritoneal macrophages and human macrophage cell lines.

Aleutian mink disease parvovirus (ADV) mRNAs are found in macrophages in lymph nodes and peritoneal exudate cells from ADV-infected mink. Therefore, we developed an in vitro infection system for ADV by using primary cultures of mink macrophages or macrophage cell lines. In peritoneal macrophage cultures from adult mink, virulent ADV-Utah I strain showed nuclear expression of viral antigens with fluorescein isothiocyanate-labeled ADV-infected mink serum, but delineation of specific viral proteins could not be confirmed by immunoblot analysis. Amplification of ADV DNA and production of replicative-form DNA were observed in mink macrophages by Southern blot analysis; however, virus could not be serially propagated. The human macrophage cell line U937 exhibited clear nuclear expression of viral antigens after infection with ADV-Utah I but not with tissue culture-adapted ADV-G. In U937 cells, ADV-Utah I produced mRNA, replicative-form DNA, virion DNA, and structural and nonstructural proteins; however, virus could not be serially passaged nor could [3H]thymidine-labeled virions be observed by density gradient analysis. These findings indicated that ADV-Utah I infection in U937 cells was not fully permissive and that there is another restricted step between gene amplification and/or viral protein expression and production of infectious virions. Treatment with the macrophage activator phorbol 12-myristate 13-acetate after adsorption of virus reduced the frequency of ADV-positive U937 cells but clearly increased that of human macrophage line THP-1 cells. These results suggested that ADV replication may depend on conditions influenced by the differentiation state of macrophages. U937 cells may be useful as an in vitro model system for the analysis of the immune disorder caused by ADV infection of macrophages.

Aleutian Mink Disease Virus↗
PubMed1988

Mapping of porcine parvovirus DNA and development of a diagnostic DNA probe.

Dimeric and monomeric replicative forms of DNA of porcine parvovirus (PPV) strain NADL-2 were isolated and examined by restriction enzyme analysis and reciprocal Southern blot hybridization during development of a DNA probe for PPV. Genomic single stranded PPV DNA was 5.0 kb long, and results substantiated the rolling-hairpin model of parvovirus DNA replication with the primer sequence located in the 3' terminal hairpin loop. An additional finding was the generation of a 4.7 kb species of viral DNA which was considered to be a 0.3 kb deletion variant of genomic PPV DNA. A 3.0 kb DNA fragment obtained by Pst I/Hind III digestion of monomer replicative form DNA was cloned into a plasmid vector, pUC 19. The cloned fragment, recovered from transformed Escherichia coli strain TB1 and labelled with [32P] dCTP, was evaluated by dot hybridization as a probe for PPV in infected cell cultures. The probe was specific for PPV infected cells, and was 100 times more sensitive than the standard hemagglutination test.

Animals↗