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Results for “replicative form”

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At least 595 records · Page 33Linked to original sources

PubMed1984

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

The specific interaction between left-handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti-Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno-electron microscopy. DNA-antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti-Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti-Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti-Z IgG binding sites which were expressed with varying frequencies (53-3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non-cooperative due to relaxation of torsional stress by the B----Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.

Antigen-Antibody Complex↗

PubMed1991

Persistent rat parvovirus infection in individually housed rats.

The duration of infection with rat virus (RV), an autonomous rodent parvovirus, was examined at multiple intervals over 6 months in rats inoculated by the oronasal route at 2 days of age or 4 weeks of age and individually housed after weaning to prevent cross-infection. Infectious virus was recovered by explant culture from 32 of 80 rats inoculated as pups and was detected as late as 6 months after inoculation. Rats inoculated as juveniles developed acute infection, but virus was not detected beyond 7 weeks after inoculation. Tissues from rats in both age groups were surveyed for RV DNA by Southern blotting using a double-stranded DNA probe made from a 1700 bp cloned fragment of RV spanning map units 0.19-0.52. Band patterns representative of acute infection (juvenile rats) were consistent with the replicating form of RV DNA, whereas patterns representative of persistent infection (rats inoculated as pups) were suggestive of defective or non-productive viral replication.

Aging↗

PubMed1997

How is Helicobacter pylori transmitted?

Helicobacter pylori is one of the world's most common pathogens. It colonizes about 60% of the world's population, causes gastritis and peptic ulcer, and is strongly associated with gastric adenocarcinoma and lymphoma. However, most individuals never develop clinical disease. Thirteen years after the culture of H. pylori by Marshall and Warren, we still do not know its major mode of transmission. Childhood represents the major period of acquisition of infection in the third world, but infection is rare in children in the developed world. Possible routes of infection include either oral-oral or fecal-oral, iatrogenic spread with inadvertent use of unsterile pH probes and endoscopes, and vectorial spread by flies. Evidence to support each route of transmission is provided, but there is no predominant route. The only significant reservoir of infection appears to be humans themselves. The organism has been found in some domestic cats and in nonhuman primates, but the opportunities for human interaction with the latter are rare, making infection from this source an unlikely possibility. The organism has the propensity to become a coccoid form. This may represent a persistent form in which H. pylori can exist in the environment, but it has yet to be shown that it can revert to the replicative form.

Animals↗

PubMed1976

Multiplication of parvovirus LuIII in a synchronized culture system. III. Replication of viral DNA.

The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper function(s) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, double-stranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIII DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967).

Cell Division↗

PubMed1993

Characterization of chimeric full-length molecular clones of Aleutian mink disease parvovirus (ADV): identification of a determinant governing replication of ADV in cell culture.

The ADV-G strain of Aleutian mink disease parvovirus (ADV) is nonpathogenic for mink but replicates permissively in cell culture, whereas the ADV-Utah 1 strain is highly pathogenic for mink but replicates poorly in cell culture. In order to relate these phenotypic differences to primary genomic features, we constructed a series of chimeric plasmids between a full-length replication-competent molecular clone of ADV-G and subgenomic clones of ADV-Utah 1 representing map units (MU) 15 to 88. After transfection of the plasmids into cell culture and serial passage of cell lysates, we determined that substitution of several segments of the ADV-Utah 1 genome (MU 15 to 54 and 65 to 73) within an infectious ADV-G plasmid did not impair the ability of these constructs to yield infectious virus in vitro. Like ADV-G, the viruses derived from these replication-competent clones caused neither detectable viremia 10 days after inoculation nor any evidence of Aleutian disease in adult mink. On the other hand, other chimeric plasmids were incapable of yielding infectious virus and were therefore replication defective in vitro. The MU 54 to 65 EcoRI-EcoRV fragment of ADV-Utah 1 was the minimal segment capable of rendering ADV-G replication defective. Substitution of the ADV-G EcoRI-EcoRV fragment into a replication-defective clone restored replication competence, indicating that this 0.53-kb portion of the genome, wholly located within shared coding sequences for the capsid proteins VP1 and VP2, contained a determinant that governs replication in cell culture. When cultures of cells were studied 5 days after transfection with replication-defective clones, rescue of dimeric replicative form DNA and single-stranded progeny DNA could not be demonstrated. This defect could not be complemented by cotransfection with a replication-competent construction.

Aleutian Mink Disease Virus↗

PubMed2001

Forced recombination between distinct strains of Maize streak virus.

Recombination between divergent virus genomes is believed to be a major mechanism for generation of novel virus genotypes. We have examined the recombination process in geminiviruses by forcing recombination between two distinct isolates of Maize streak virus (MSV), MSV-Kom and MSV-Set. Heterodimeric agroinfectious constructs containing tandemly cloned mixtures of complete or partial MSV-Set and MSV-Kom genomes were used to simulate a circular dimeric form similar to that which would be expected to occur following a single intermolecular crossing-over event between MSV-Set and MSV-Kom replicative form DNAs at the long intergenic region (LIR)-movement protein gene (MP) interface. We isolated, analysed and biologically characterized many of the recombinant MSV genomes that were generated from the constructs in planta. Apart from having the same simulated breakpoint at the LIR-MP interface, all the genomes examined had a second breakpoint that had been generated through either intramolecular homologous recombination or a replicational release mechanism. The pathogenicities of six predominantly MSV-Kom-like recombinants were tested in maize. While all were capable of producing a symptomatic infection in this host, none was more virulent than MSV-Kom and only two were more virulent than MSV-Set. The two most virulent recombinants were leafhopper transmitted to a range of differentially MSV-resistant maize, wheat and barley genotypes and both were found to have unique biological properties.

Base Sequence↗

PubMed1990

Polyoma DNA replication dependent upon growth condition of SEWA sarcoma cells.

Extrachromosomal replication of viral DNA sequences has been observed in transformed as well as in normal cells following "stress"-inducing treatments. To explore the effect of growth conditions on the ability to support such replication, we analyzed SEWA sarcoma cells that grew subcutaneously or as ascites tumors in vivo as well as cell lines that were established from each of these tumors. The replicative form of polyoma DNA sequences was observed in SEWA tumors grown in ascites fluids but not in cells maintained as solid tumors. Polyoma DNA replication was found in ascites-derived cells that were adapted to grow in culture, only when the cultured cells are stimulated with UV irradiation. Immunoprecipitation of T antigens enabled detection of large T antigen only in the ascites-derived cells. The mechanisms that may regulate this phenomenon and the possible role large T may play in different growth conditions of SEWA cells are discussed.

Animals↗

PubMed1996

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

Biological Evolution↗

PubMed1997

Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.

Natural resistance to flaviviruses in mice is controlled by a single genetic locus, FIv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flv(s)) and -resistant congenic C3H/RV (Flv(r)) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0.6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flv(r) interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.

Animals↗

PubMed1985

Mutational mechanisms by which an inactive replication origin of bacteriophage M13 is turned on are similar to mechanisms of activation of ras proto-oncogenes.

M13 viral strand synthesis is initiated by nicking of the viral strand of the duplex replicative form by the M13 gene II initiator protein at a specific site within a sequence of about 40 base pairs having dyad symmetry. Efficient replication of the M13 viral strand also requires the presence of an adjacent sequence of ca. 100 base pairs. Together these sequences constitute the minimal origin for M13 viral strand synthesis. A pBR322 derivative having a 182-base-pair insert of M13 DNA contains a functional M13 viral strand origin and, when provided with M13 gene functions in trans, replicates under conditions nonpermissive for the parent plasmid. Chimeric plasmids containing deletions within the sequence flanking the viral strand origin are unable to replicate under these conditions. We isolated spontaneous mutants of M13 based on their ability to activate replication of such plasmids. The mutations found in these strains, as well as several produced by oligonucleotide-directed mutagenesis, all result in the substitution of any of at least four different amino acids for a specific glycine residue near the amino-terminal end of the initiator protein. Other studies have shown that overproduction of the wild-type initiator protein also restores replication. These alternate mechanisms are discussed in terms of their striking similarity to the mechanisms of activation of the ras proto-oncogenes which can be activated either by increased expression of the wild-type protein or by substitution of any of several amino acids for a glycine residue near the amino terminus.

Bacteriophages↗

PubMed1975

DNA replication in vitro starting with an intact phiX174 phage.

Conversion of the single-stranded DNA in the intact phiX174 phage particle to the duplex replicative form (RF) has been demonstrated in lysates form phage-sensitive cells. The conversion is resistant to rifampicin and requires participation of both a "membrane" fraction of the lysate and a multienzyme replicative system. The lipopolysaccharide phage receptor, while essential, does not replace the membrane fraction. Clear, nonsedimentable extract fractions prepared with a certain nonionic detergent can replace the membrane fraction. Purification of the activity in these extracts by adsorption to polypropylene film yields a fraction with a 5-fold increase in activity relative to lipopolysaccharide and 50-fold increase relative to protein. The low buoyant density (1.03 g/cm3) suggests a high phospholipid or detergent content in this fraction.

Centrifugation, Density Gradient↗

PubMed1989

Mechanisms of mutagenesis by chloroacetaldehyde.

A number of bifunctional chemical mutagens induce exocyclic DNA lesions. For example, 2-chloroacetaldehyde (CAA), a metabolite of vinyl chloride, readily reacts with single-stranded DNA to predominantly form etheno lesions. Here, we report on in vivo mutagenesis caused by CAA treatment of DNA in vitro. These experiments used partially duplex phage M13AB28 replicative form DNA in which a part of the lacZ gene sequence was held in single-stranded form to direct reaction with CAA. CAA-treated partial duplex DNA was transfected into Escherichia coli, and the induced base changes were defined by DNA sequencing. These experiments suggested that CAA treatment induced mutations at cytosines, much less efficiently at adenines, but not at guanines or thymines. Among mutations targeted to cytosine, 80% were C-to-T transitions and 20% were C-to-A transversions. Application of a post-labeling method detected dose-dependent formation of ethenoadenine and ethenocytosine in CAA treated DNA. These data indicate that ethenocytosine is a highly efficient mutagen with properties suggestive of a non-instructional DNA lesion in vivo. Paradoxically, ethenoadenines are efficiently bypassed by a mechanism which appears to be largely nonmutagenic.

Acetaldehyde↗

PubMed1983

Reduced synthesis of Sindbis virus negative strand RNA in cultures treated with host transcription inhibitors.

Host cell involvement in Sindbis virus (SB) RNA synthesis was examined in cells which had been treated before infection with actinomycin D or alpha-amanitin (alpha-A). Overall synthesis of SB RNA was reduced significantly in CHO cells treated for 18 h before infection with alpha-A. However, SB RNA was produced at near normal levels in CHOama-1 cells, a line which contains an alpha-A-resistant RNA polymerase II. In BHK or CHO cells infected with SBamr, a mutant which replicates normally in cells pretreated with either actinomycin D or alpha-A, viral RNA synthesis was not decreased. The levels of negative strand RNA and of replicative forms I, II, and III in SB-infected cells were progressively reduced with increasing times of pretreatment with host transcription inhibitors, indicating fewer functional replicative intermediates in treated cells. Replicative events after replicative intermediate formation also were inhibited but only to the extent predicted by the reduction in replicative intermediates. Similarly, events preceding negative strand synthesis, adsorption, penetration, uncoating, and translation of nonstructural proteins, apparently were not impeded in treated cells. Therefore, our results are consistent with the involvement of a host component after translation of the nonstructural proteins but before or during the synthesis of SB negative strand RNA.

Amanitins↗

PubMed1991

Yeast 20 S RNA replicon. Replication intermediates and encoded putative RNA polymerase.

The 20 S RNA genome is a circular single-stranded replicon, present in most laboratory yeast strains, whose copy number is induced 10,000-fold by transfer of cells to acetate medium without a carbon source. We have sequenced most of the 20 S RNA genome, and the (+) strand has a long open reading frame with the potential to encode a protein with homology to viral RNA-dependent RNA polymerases. The presence of a typical cAMP-dependent phosphorylation site in the putative RNA polymerase suggests that the acetate amplification of the 20 S RNA genome might be mediated by cAMP, a signal known to transmit the same nutritional status information to the sporulation-control system. Our inability to clone across the gap in the sequence suggests either autocatalytic cleavage of the RNA in the reverse transcriptase reaction, an unusual linkage of 5' and 3' ends of a fundamentally linear molecule, or a structure unusually resistant to reverse transcription. The identity of our sequence with that of the accompanying paper (Rodriguez-Cousino, N., Esteban, L.M., and Esteban, R. (1991) J. Biol. Chem. 266, 12772-12778) for W double-stranded RNA (dsRNA) suggests that W is the replicative form of 20 S RNA. The presence of single-stranded (+) and (-) strands and greater than unit length molecules suggests a rolling circle mode of replication as has been suggested for viroids.

Amino Acid Sequence↗

PubMed1989

Identification of single-strand initiation signals in the terC region of the Escherichia coli chromosome.

On the basis of clear-plaque formation, we detected initiation signals in the terC region of the Escherichia coli chromosome. At least two single-strand initiation signals were identified from the terC region. The nucleotide sequences of these two signals were determined. Sequence homologies, variations of the consensus of n' protein recognition sites, 5'-GAAGCGG-3', were found within these signals. A novel conserved sequence was also found within these signals. Their initiation activities were measured both by the infection growth assay and by the ability to convert the single-stranded DNA to the duplex replicative form DNA in vivo.

Bacteriophage phi X 174↗

PubMed1989

Biologic significance of the detection of HBsAg and HBcAg in liver and tumor from 204 HBsAg-positive patients with primary hepatocellular carcinoma.

Hepatitis B virus surface and core antigens (HBsAg, HBcAg) were examined in the resected primary hepatocellular carcinoma from 204 patients who had HBsAg in serum. Ninety patients had small (less than 5 cm) and 114 had large hepatocellular carcinoma (greater than 5 cm). HBsAg was detected in hepatocellular carcinoma in 65 cases (32%) and HBcAg in 30 cases (14.7%); hepatitis B virus antigens were more frequently detected in small (HBsAg in 42.2% and HBcAg in 20%) than in large hepatocellular carcinoma (HBsAg 23.7% and HBcAg 10.5%). These results suggest that replicative forms of hepatitis B virus DNA may exist in hepatocellular carcinoma more frequently than previously believed and that the malignant hepatocytes can support hepatitis B virus replication. A lymphocytic infiltration in hepatocellular carcinoma was more often observed in hepatocellular carcinoma expressing HBsAg (71%) or HBcAg (63%) than in hepatocellular carcinoma with no detectable HBsAg (26%) or HBcAg (37%), p less than 0.01. The reaction was mild in the majority (85%) of the cases. These findings suggest that hepatitis B virus antigen expression in hepatocellular carcinoma can provoke a local immune response. The most striking finding was that patients with hepatitis B virus antigens in small hepatocellular carcinoma had a 5-year survival rate (13%) lower than that (50%) of the antigen-negative patients (p less than 0.05). In contrast, patients with a marked local immune response in hepatocellular carcinoma, regardless of the viral antigen status, had significantly better 5-year survival rates (43%) than those with no or a mild lymphocytic reaction (18%). These findings indicate that a marked immune response in hepatocellular carcinoma is a favorable prognostic sign.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗

PubMed2001

Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments.

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.

DNA Helicases↗

PubMed1980

Studies on the functions of DNA helicase I and DNA helicase II of Escherichia coli.

Inactivating antibodies raised against DNA helicase I and DNA helicase II were applied to Escherichia coli DNA-replicating systems. Antibody against DNA helicase II was found to inhibit the replication of E. coli DNA, lambda phage DNA (during early and late phases), and ColE1 plasmid DNA during the elongation step. The antibody did not inhibit the replication of fd replicative form (RF) DNA, the unwinding of which is known to depend on the rep protein. Antibody against DNA helicase I failed to inhibit any of the replication processes. The replication of E. coli DNA, lambda-DNA, and ColE1 DNA is known to be initiated in a closed circle, in contrast to fd RF which is known to be initiated in a nicked circle. In conjunction with data given in the literature, our results suggest that replicative unwinding is carried out by DNA helicase II or rep protein, depending on the mechanism by which DNA replication is initiated. The concentration of DNA helicase II in E. coli, as determined by immunological methods, is 5000 to 8000 copies/cell; that of DNA helicase I is 500 to 700.

Adenosine Triphosphatases↗