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Results for “replicative form”

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At least 649 records · Page 36Linked to original sources

PubMed1987

Maintenance of woodchuck hepatitis virus activity in woodchuck hepatocyte primary culture.

Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3.3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Animals↗

PubMed1981

Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA.

We have studied the relationship between adeno-associated virus (AAV) DNA replication and virus particle assembly. Formation of empty or full particles and accumulation of AAV capsid proteins was prevented in the presence of the arginine analogue, L-canavanine, or when a temperature-sensitive helper adenovirus was used at the nonpermissive temperature. In each case there was a concomitant inhibition of AAV single-stranded (progeny) DNA accumulation but little or no effect upon synthesis of AAV duplex, replicating form DNA. These results indicate that AAV protein, perhaps in the form of assembled capsids, is required for AAV single-stranded progeny DNA accumulation.

Canavanine↗

PubMed1986

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences.

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

Base Sequence↗

PubMed1993

O6-methylguanine in DNA inhibits replication in vitro by human cell extracts.

To study the effects of methylation damage on DNA replication in vitro, the plasmid pSVori containing the SV40 origin of replication was reacted with N-methyl-N-nitrosourea and used as a substrate for SV40 T antigen dependent replication by HeLa cell extracts. The plasmid was methylated with a range of N-methyl-N-nitrosourea concentrations that introduced an average of 0.3-2.5 O6-methylguanine and equal amounts of 3-methyladenine lesions per DNA molecule. When methylated plasmid was incubated with extract of Mex-HeLaMR cells under conditions favoring DNA replication, an impairment of replication was observed as the accumulation of incompletely replicated form II plasmid molecules. These extracts simultaneously performed a T antigen independent, DpnI-sensitive DNA repair synthesis that increased with increasing DNA damage. Subtraction of this repair DNA synthesis revealed that methylation inhibited overall replication. At low levels of methylation (< or = 1 O6-methylguanine and < or = 1 3-methyladenine lesion per plasmid), inhibition was transient, while more extensive damage resulted in apparently irreversible inhibition of replication. Removal of O6-methylguanine by pretreatment of the methylated plasmid with purified human O6-methylguanine-DNA methyltransferase restored replication to almost normal levels. When the methylated plasmid was replicated by extracts of Mex+ HeLaS3 cells proficient in the repair of O6-methylguanine, a lower level of inhibition and less repair DNA synthesis was observed. The inhibition of DNA synthesis and the stimulation of repair DNA synthesis are thus both largely due to the presence of O6-methylguanine in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Polyomavirus Transforming↗

PubMed1980

Indiscriminate recombination in simian virus 40-infected monkey cells.

DNA transfection of African green monkey BSC-1 cells with simian virus 40 (SV40) DNA and bacterial virus phi X174 replicative form DNA ("cotransfection") yielded stocks containing SV40/phi X174 recombinant virus, which was detected by an infectious-center in situ plaque hybridization procedure and which was sensitive to anti-SV40 antiserum. The recombinant virus replicated during serial passage. Restriction endonuclease cleavage of the SV40/phi X174 DNA indicated that several different types of recombinant DNA structures had arisen. Similar SV40 DNA cotransfection experiments with polyoma virus DNA, bacterial plasmid (pBR322) DNA, and a plasmid-cloned segment of the mouse genome (coding for intracisternal type A particles) yielded stocks that generated recombinant plaques as judged by in situ plaque hybridization with the appropriate labeled probe. It appears, therefore, that an active indiscriminate recombination process, incapable of distinguishing between diverse DNAs of prokaryotic and eukaryotic origin, occurs in SV40-infected monkey cells.

Animals↗

PubMed1991

[Cellular immunity in viral hepatitis B and its transition into chronic forms].

The authors studied population and subpopulation composition of peripheral blood lymphocytes and HBV circulation markers in 126 patients in the acute period of the disease and prophylactic screening within 6 months. It was established that in case of complete recovery the changes of immunocompetent cells had a transitory character. Replication forms of chronic hepatitis the immune status showed marked T-lymphopenia with prevailing reduction of T-suppressors. The role of cellular immunity in the formation of chronic liver disease is discussed.

Adolescent↗

PubMed1980

Bacteriophage T7 DNA replication in vitro. Electron micrographic analysis of T7 DNA synthesized with purified proteins.

Extensive replication of duplex T7 DNA is catalyzed in reactions contining T7 DNA polymerase, T7 gene 4 protein, and T7 RNA polymerase. When the product of this reaction is analyzed in the electron microscope, many eye form and Y form replication intermediates are observed. Replication in vitro is not initiated at a single region of the T7 genome. However, we tentatively conclude that initiation does occur preferentially at a few specific sites along the DNA, and that these sites may be near promoters at which the T7 RNA polymerase initiates transcription.

DNA Replication↗

PubMed2004

Presence of TT virus DNA in bone marrow cells from hematologic patients.

Recent observations suggest that TT virus (TTV), in addition to liver, may also infect bone marrow. In this study, bone marrow samples and sera from 33 patients with haematological disorders and sera from 16 healthy controls were investigated for TTV DNA presence. Altogether TTV DNA sequences were demonstrated in bone marrow cells of 84.84% of patients. Moreover TTV DNA was detected in sera from 72.72% of patients and from 93.75% of controls. N22 sequences amplified from bone marrow cells and serum of 3 patients were analysed, after cloning: all these isolates were of type 2c and 2 or 3 variants were present in each isolate. After single strand DNA degradation, replicative forms were detectable in BM cells. This finding, in addition to the detection of variants similar in the BM and in the serum of the same patient could suggest that BM is a site of TTV replication (or one of the sites) from which the virus is spread in blood.

Antibodies, Viral↗

PubMed1977

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.

Coliphages↗

PubMed1987

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1.

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.

Animals↗

PubMed1982

Selective cloning of Co1E1 DNA initiation sequences using the cloning vector M13 delta E101.

DNA sequences required to direct single-strand to double-strand conversion by a phi X174-type mechanism have been detected in Co1E1 plasmid DNA. The sites responsible for this DNA strand initiation, named rriA and rriB, have been localized to the L strand of the HaeII-E fragment and to the H strand of the HaeII-C fragment of Co1E1. To define rriA and rriB more precisely, random fragments of Co1E1 DNA have been cloned into the single-stranded phage cloning vector M13 delta E101. This phage contains a viable deletion of the M13 complementary strand origin and makes small turbid plaques. Cloning of DNA initiation determinants into the unique EcoRI site of M13 delta E101 rescues the mutant phage and restores the ability to form clear plaques. DNA sequence analysis of the random, 100-400 bp inserts in clear plaque isolates has localized the rriA and rriB determinants within DNA fragments of about 100 bp. These initiation determinants promote assembly of a functional, multiprotein priming complex, "primosome", identical to that utilized in the in vitro conversion of phi X174 single-stranded DNA to the duplex replicative form.

Base Sequence↗

PubMed1988

Detection of Aleutian disease virus DNA in tissues of naturally infected mink.

Organs of naturally infected mink were examined for the presence of Aleutian disease virus (ADV) DNA by in situ hybridization. Spleen, lymph nodes, thymus, bone marrow, kidney, liver, lung and small intestine were found to be positive for ADV to differing extents. Infected lymphoid organs showed a focal distribution of positive cells. Southern blot analysis of DNA extracted from infected organs revealed replicative forms of viral DNA in spleen and bone marrow samples only. These findings are consistent with a lymphotropism of ADV in vivo. Compared to the situation after experimental infection of mink these results indicate additional sites of virus replication and/or persistence of the naturally occurring disease.

Aleutian Mink Disease↗

PubMed1991

DNA replication of wheat dwarf virus, a geminivirus, requires two cis-acting signals.

Replication of the single-stranded DNA genome of wheat dwarf virus (WDV) leads to the accumulation of covalently closed double-stranded DNA of genome length in infected cells. By studying the replication properties of a naturally occurring deletion mutant of WDV isolated from infected plants and of deletion mutants constructed in vitro, we have defined cis-acting regions required for viral DNA replication. The results show that two distinct regions are required in cis to yield the normal replicative forms of WDV-DNA.

Blotting, Southern↗

PubMed2005

Genome packaging sense is controlled by the efficiency of the nick site in the right-end replication origin of parvoviruses minute virus of mice and LuIII.

The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5'-CTAT(black triangle down)TCA-3', while in LuIII a two-base insertion (underlined) changes it to 5'-CTATAT(black triangle down)TCA-3'. Matched LuIII genomes differing only at this position (designated LuIII and LuDelta2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for LuDelta2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical "kinetic hairpin transfer" model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and LuDelta2 infections, we extend this model, showing that positive-sense strands do accumulate in LuDelta2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.

Animals↗

PubMed1986

[Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli].

The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E. coli spheroplasts. With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved. The majority of DNA macromolecules were released in the form of single and connected rosettes. Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm). The mean length of the rosette loops was 1.06 +/- 0.4 micron. Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation. Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops. Linear, cyclic and various replicative forms of DNA have also been observed. It is assumed that rosettes of the extrachromosomal elements of E. coli reflect one of the levels of organization of prokaryotic genetic material.

DNA, Bacterial↗

PubMed1990

The priA gene encoding the primosomal replicative n' protein of Escherichia coli.

The Escherichia coli gene encoding protein n' has been isolated and named priA for primosomal protein A. Protein n' is absolutely required for the conversion of single-stranded phi X174 DNA to the duplex replicative form in an in vitro-reconstituted system. The gene maps to 88.7 minutes on the chromosome adjacent to the cytR locus. Soluble protein extracts from cells harboring the priA gene on a multicopy plasmid contained 45-fold more n' replication activity than wild-type extracts. Enhanced overproduction of greater than 1000-fold was achieved by replacing the natural Shine-Dalgarno sequence with that of the phage T7 phi 10 gene and placing this priA under the control of the T7 phage promoter and RNA polymerase. The priA sequence reveals a 732-amino acid open reading frame and a nucleotide-binding consensus site consistent with the size and ATPase activity of the purified protein. The gene for protein n has been named priB and the putative gene for protein n", priC.

Amino Acid Sequence↗

PubMed1993

Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells.

Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or beta-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (-) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.

Blotting, Southern↗

PubMed1996

Cytokine inhibition of the hepatitis B virus core promoter.

Hepatitis B virus (HBV) DNA contains consensus elements for transactivating proteins whose binding activity in other systems is regulated by inflammatory cytokines. Because HBV replicates within an environment of provoked inflammation, we speculated that the HBV core/pregenomic promoter may be regulated by cytokines produced in response to infection. To evaluate this hypothesis, the HBV core/pregenomic (C/P) promoter and associated cis-acting elements were placed upstream of a luciferase-encoding plasmid. This reporter construct was transfected into cytokine-sensitive hepatoma cells permissive for HBV replication, which were exposed to stimulated mononuclear cell-conditioned medium or human recombinant cytokines. Conditioned medium reduced luciferase expression by 80%. Tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and interferon alfa (IFN-alpha) each reduced luciferase activity by 40%. Combinations of TNF-alpha and interferons mimicked the extent of conditioned medium inhibition. Non-specific effects from diminished cellular viability or growth were not responsible for decreased luciferase activity. Retention of HBV DNA 330 basepairs upstream of the C/P transcription start site was required to maintain the TNF-alpha effect. A 60% reduction in HBV replicative forms within intracellular core particles was demonstrated with TNF-alpha treatment of Hep G2 cells stably transfected with HBV DNA. The inhibitory action of these cytokines implicates a noncytolytic mechanism by which antigen-nonspecific immune responses in part regulate HBV replication in infected hepatocytes. This function may be beneficial in accelerating viral clearance, but in alternative circumstances could contribute to viral persistence by attenuating immunogen recognition.

Carcinoma, Hepatocellular↗