SEARCH · PubMed Health

Results for “replicative form”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources

PubMed2005

Rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor.

The events linking rhinovirus (RV) infection to airway symptoms are poorly understood. The virus initially infects airway epithelium followed by a vigorous inflammatory response that may entail spread of RV from epithelium to other cells in the airway wall. However, RV has fastidious growth characteristics and to date reproductive infection of primary cells other than human airway epithelium has not been confirmed. Airway fibroblasts are adjacent to and in contact with epithelial cells, play a key role in innate immune responses, and may participate in the evolution of inflammation. To investigate fibroblast actions, we first determined whether RV could infect and replicate in primary culture human lung fibroblasts. RV serotype 16 (RV16) was used to infect fibroblasts grown from lung tissue, and virus infection with replication was demonstrated by a combination of techniques. RT-PCR was used to show an increase in RV transcription; confocal microscopy demonstrated colocalization of the replicative form of RV genome (double-stranded RNA) and RV16 proteins; infectious virus was also recovered from the culture supernatant of infected fibroblasts. Functional consequences of RV infection were next examined. RV infection of fibroblasts was followed by an increase in epithelial neutrophil-activating peptide-78 (ENA-78) mRNA and protein. The permeability factor vascular endothelial growth factor (VEGF) was also induced over a similar time course. These data suggest that interactions between RV and human fibroblasts are feasible, may coordinate neutrophil chemoattraction with enhanced vascular permeability and that fibroblasts may contribute to inflammatory responses following RV infections.

Cells, Cultured↗

PubMed1976

Restriction-enzyme-cleavage maps of bacteriophage M13. Existence of an intergenic region on the M13 genome.

Replicative form DNA of bacteriophage M13 was cleaved into specific fragments by an endonuclease isolated from Hemophilus aegyptius (endoR.HaeII) and an endonuclease from Arthrobacter luteus (endoR.AluI). The fragments were ordered as to construct a circular map of the phage M13 genome by: (a) using each fragment as a primer for the synthesis in vitro of its respective neighbour and (b) digesting the isolated fragments with the Hemophilus aegyptius enzyme endoR.HaeII or the Hemophilus aphirophilus enzyme endoR.HapII and subsequent analysis of the overlapping sets of fragments. The resulting physical map was correlated with the M13 genetic map by marker rescue experiments with amber mutant phage DNAs and purified wild-type fragments. From the results of these analyses it has been concluded that gene II and gene V are contiguous on the genetic map. Evidence is provided that there is an internal start of RNA synthesis within the C-terminal region of gene II which then ultimately leads to the synthesis of X protein. Furthermore, we conclude that there is an intergenic space of considerable length (450-500 base pairs) which is located between gene II and gene IV on the M13 genome. The function of this intergenic region as the origin site for phage DNA replication is discussed.

Arthrobacter↗

PubMed1991

Effects of temperature on excluded volume-promoted cyclization and concatemerization of cohesive-ended DNA longer than 0.04 Mb.

The 0.048502 megabase (Mb), primarily double-stranded DNA of bacteriophage lambda has single-stranded, complementary termini (cohesive ends) that undergo either spontaneous intramolecular joining to form open circular DNA or spontaneous intermolecular joining to form linear, end-to-end oligomeric DNAs (concatemers); concatemers also cyclize. In the present study, the effects of polyethylene glycol (PEG) on the cyclization and concatemerization of lambda DNA are determined at temperatures that, in the absence of PEG, favor dissociation of cohesive ends. Circular and linear lambda DNA, monomeric and concatemeric, are observed by use of pulsed field agarose gel (PFG) electrophoresis. During preparation of lambda DNA for these studies, hydrodynamic shear-induced, partial dissociation of joined cohesive ends is fortuitously observed. Although joined lambda cohesive ends progressively dissociate as their temperature is raised in the buffer used here (0.1 M NaCl, 0.01 M sodium phosphate, pH 7.4, 0.001 M EDTA), when PEG is added to this buffer, raising the temperature sometimes promotes joining of cohesive ends. Conditions for promotion of primarily either cyclization or concatemerization are described. Open circular DNAs as long as a 7-mer are produced and resolved. The concentration of PEG required to promote joining of cohesive ends decreases as the molecular weight of the PEG increases. The rate of cyclization is brought, the first time, to values that are high enough to be comparable to the rate observed in vivo. For double-stranded DNA bacteriophages that have a linear replicative form of DNA (bacteriophage T7, for example), a suppression, sometimes observed here, of cyclization mimics a suppression of cyclization previously observed in vivo. The PEG, temperature effects on DNA joining are explained by both the excluded volume of PEG random coils and an increase in this excluded volume that occurs when temperature increases.

Bacteriophage lambda↗

PubMed1976

Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA.

Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.

Bacterial Proteins↗

PubMed1967

Replication of bacteriophage ribonucleic acid: some physical properties of single-stranded, double-stranded, and branched viral ribonucleic acid.

Replicative intermediate (RI) is considered to be the double-stranded ribo-nucleic acid (RNA) template for synthesis of viral RNA, with bound nascent single-stranded viral RNA. A theoretical description of RI is based on the analysis of a steady state of biopolymerization on a template which determines not only nucleotide sequence, but also chain length. The hydrodynamic properties of RI isolated from Escherichia coli infected with bacteriophage R17 are compared with those of RNA isolated from R17 (single-stranded RNA) and of replicative form (RF) isolated from E. coli infected with R17. RF is double-stranded RNA template without any single-stranded component. Whereas S for R17 RNA is a function of the ionic strength (Gamma/2) of the solvent, S is almost invariant with Gamma/2 for RF. By contrast S for RI lies between the sedimentation constants for R17 RNA and RF and S varies with Gamma/2 as does R17 RNA. The weight distribution of S for RI demonstrates the heterogeneity of this material, and the variation in the weight distribution with ionic strength demonstrates the duality of structure in RI. Using S and [eta], the Mw for RI is estimated to be 2.6 x 10(6) daltons, as compared with the theoretical value of 2.9 x 10(6) daltons.

Coliphages↗

PubMed1987

Comparison of porcine parvovirus to other parvoviruses by restriction site mapping and hybridization analysis of Southern Blots.

The genomic relationship between porcine parvovirus (PPV) and several other autonomous parvoviruses was examined by restriction site and hybridization analysis. Restriction site maps of the PPV genome were prepared by digesting the double-stranded replicative form of the viral DNA with each of eight restriction enzymes. Subsequent comparison of such maps with those previously reported for PPV, canine parvovirus (CPV), feline panleukopenia virus (FPV), minute virus of mice (MVM), H-1 virus (H-1) and bovine parvovirus (BPV) revealed that while the maps of CPV, FPV, MVM and H-1 had a number of features in common, those of PPV and BPV were substantially different. For hybridization analysis radioactive probes prepared by nick translation of PPV, CPV and BPV genomes were tested under conditions of both low and high stringency for homologous hybridization and for heterologous hybridization with each of the other two viruses and with FPV. The results of these tests indicated homology among the genomes of PPV, CPV and FPV, but little or no homology between the genome of BPV and those of any of the other viruses tested. Additional tests with restriction fragments of PPV and a CPV probe indicated that heterologous hybridization was confined primarily to a segment of the genome between 1.85 and 2.7 kb from the 3' end. Based on transcriptional maps previously determined for several of the rodent parvoviruses, this interval is likely to include part of the coding sequences for both non-structural and structural proteins and may be the genetic basis for the replicative as well as the antigenic similarities between PPV and both CPV and FPV.

Animals↗

PubMed2005

Establishment of effective methods for transducing genes into iris pigment epithelial cells by using adeno-associated virus type 2.

PURPOSE: To establish an efficient method of transferring the human brain-derived neurotrophic-factor (hBDNF) gene into human iris pigment epithelial (hIPE) cells by using recombinant adeno-associated virus type 2 (rAAV2). METHODS: Cultured hIPE cells were treated with either hydroxyurea-sodium butyrate (HUSB; DNA synthesis inhibitor), or tyrphostin-1 (Tyr; epidermal growth factor receptor [EGFR] tyrosine kinase inhibitor), or a combination of HUSB and Tyr (HUSB-Tyr). After each treatment, cells were exposed to rAAV2 (rAAV-LacZ or rAAV-hBDNF). The levels of BDNF were measured by ELISA and also determined by Western blot analysis. Southern blot analysis was performed on each type of treated cell. The neuroprotective effect of BDNF on the retinal ganglion cells (RGCs) was quantitatively assessed by culturing rAAV-hBDNF-hIPE with RGCs. RESULTS: The infection of hIPE cells was significantly lower than ARPE and HT1080 cells, which are highly permissive cells for rAAV2. The treatment of HUSB-Tyr enhanced the transgene expression more than that after treatment with one of these agents in rAAV-hIPE cells. Southern hybridization revealed that the amount of replicative form monomer (RFm) was less in Tyr than in HUSB or HUSB-Tyr treatment and there was no difference in conversion of virus genome to double stranded form after HUSB and HUSB-Tyr treatment. However, adding Tyr treatment stimulated the JNK1/2 and p38 pathways and modified the target transgene expression. BDNF had a significantly greater rescue effect of RGCs with the HUSB-Tyr-treated rAAV-hBDNF-hIPE cells (P < 0.01) than that with the HUSB-treated rAAV-hBDNF-hIPE cells (P > 0.05) compared with noninfected hIPE cells. CONCLUSIONS: The combined treatment of HUSB-Try is an effective method of increasing transgene expression with the AAV-mediated gene transfer. The role of HUSB and Tyr in the increase of gene expression may be different and related to the conversion of virus into the host genome and the enhancement of the transcription, respectively.

Blotting, Southern↗

PubMed1976

Replicating DNA of herpes simplex virus type 1.

Newly synthesized herpes simplex virus type 1 DNA yielded a heterogeneous sedimentation profile in neutral sucrose gradients, with the main peak occurring at approximately 40S. Components sedimenting slower than virion DNA and a rapidly sedimenting intracellular HSV DNA were also observed. Both the low-molecular weight and the rapidly sedimenting components seemed to be precursors of virion DNA: they almost completely disappeared after a 60-min chase of a 3-min pulse of 3H-thymidine, and were converted into DNA which cosedimented with virion 32P-labeled DNA. However, sedimentation analysis in alkaline sucrose gradients showed that a 60-min period was insufficient for completing the maturation of HSV DNA. Cleavage of parental DNA molecules was observed in neutral sucrose gradients after infection with 3H-thymidine-labeled virions. No evidence for the formation of covalently closed circles during the replication process was obtained. The presence of single-stranded regions in the replicative form of HSV DNA was revealed. Some of the short-pulse (30 sec) labeled HSV DNA (26.1%) was eluted from hydroxylapatite columns with the properties of single-stranded DNA, and 22% of its trichloroacetic acid precipitability was susceptible to single-strand specific S1 nuclease treatment. Pulse-chase experiments indicated that the life-time of this single-stranded component in nascent DNA was probably not longer than 3 min. A small proportion of single-stranded regions, however, survived for longer periods. Almost all of the newly synthesized short-pulse-labeled HSV DNA exhibited an affinity for nitrocellulose filters. This affinity, which was S1 nuclease-sensitive, gradually decreased with prolongation of the time of the chase. After chasing the pulse for 1 h, the attachment of newly synthesized DNA was comparable with virion DNA.

Cell Line↗

PubMed1985

By-pass of the major aminofluorene-DNA adduct during in vivo replication of single- and double-stranded phi X174 DNA treated with N-hydroxy-2-aminofluorene.

To examine the effects of aminofluorene-DNA adduct formation on the biological activity of DNA, single-stranded (ss) phi X174 DNA and phi X174 replicative form (RF) DNA were modified to different extents with 3H-labeled N-hydroxy-2-aminofluorene and subsequently transfected to Escherichia coli spheroplasts with different repair capabilities. When the fraction of active ss phi X174 DNA molecules was measured as a function of the mean number of adducts per molecule, exponential survival curves were obtained from which it could be deduced that in wild-type, uvrA- and recA- cells at least 86%, and in uvrC- cells at least 82% of the introduced adducts do not cause inactivation. In the case of RF DNA the survival curves are non-exponential, but they nevertheless show that an exceptionally high number of adducts per RF molecule must be introduced to destroy its biological activity. On average 52 adducts per RF molecule were needed to reduce the survival to 37%, irrespective of whether wild-type, uvrA- or recA- cells were used. On the other hand, the survival of the uvrC- cells was considerably lower, but even in these cells a majority of the adducts is not lethal. By h.p.l.c. analysis of the modified DNA after hydrolysis with trifluoroacetic acid, 81 and 84% of the adducts in ss- and RF DNA, respectively, could be identified as N-(guanin-8-yl)-2-aminofluorene. The results strongly indicate that this type of major modification product is very frequently by-passed during replication of both single- and double-stranded DNA. The results together with the data obtained by sucrose gradient analysis both before and after an alkali treatment and those obtained by h.p.l.c. analysis suggest that inactivation of ssDNA is mainly due to minor modifications such as secondary lesions consisting of chain breaks and alkali-labile sites together with unidentified interaction products.

Bacteriophage phi X 174↗

PubMed1972

Isolation of phi X174 specific messenger ribonucleic acids in vivo and identification of their 5' terminal nucleotides.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of PhiX174 replicative form DNA and in vivo PhiX174 specific messenger RNA (mRNA) with some modification. We found that PhiX174 mRNA up to molecular weight 1.2 x 10(6) could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5' terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo PhiX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5' terminal nucleotides and identified them as 2'- or 3'-monophosphate guanosine 5'-triphosphate (pppGp) and 2'- or 3'-monophosphate adenosine 5'-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized PhiX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.

Adenosine Monophosphate↗

PubMed1984

Analysis of the mechanism of ATP stimulation of calf thymus DNA alpha-polymerase.

Biochemical kinetic analyses of the ATP stimulation of the A2 form of calf DNA alpha-polymerase show that when DNA or primer termini are the variable substrates, maximum reaction velocity is independent of ATP concentration. When dNTP concentration is the variable substrate, the apparent Km is invariant with ATP. Such results indicate that the increase in the synthetic rate caused by ATP results from an improvement in synthesis initiation at primer termini. The effect of ATP on the DNA binding affinity of alpha-A2-polymerase was examined by using column chromatography. Passage of the polymerase through native DNA-cellulose at 70 mM ionic strength resulted in 40% binding of the enzyme. In the presence of 4 mM ATP, binding increased to 80%. In both cases, the bound polymerase could be eluted by a 370 mM ionic strength wash. An elution profile similar to that observed in the absence of ATP was obtained with 0.1 mM ATP, 4 mM GTP, or 4 mM each of the nonhydrolyzable ATP analogues adenyl-5'-yl imidodiphosphate or adenosine 5'-O-(3-thiotriphosphate). These results suggest that hydrolysis of the gamma-phosphate occurs at millimolar levels of ATP and leads to a higher affinity of polymerase for DNA. To distinguish the effects of ATP on RNA priming from those on DNA synthesis, products synthesized processively by alpha-A2-polymerase were sized by gel filtration. Results indicate that essentially all products made on a gapped fd replicative form template in the presence of four dNTPs and 4 mM ATP result from the extension of preexisting DNA primers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1988

Differential stability of filamentous phage genomes in Xanthomonas campestris pv citri.

Stability of carrier state in filamentous phage-infected Xanthomonas campestris pv citri varied drastically even for closely related phage types. The spontaneous curing frequency for cells infected with Cf16-12, Cf16, Cf16-v1 and Cf was 1, 5, 96 and 100%, respectively. The size of the phage replicative-form (RF) pool which built up rapidly at the onset of Cf16 infection was critical to the maintenance of the carrier state and the eventual integration of the prophage. A correlation in stability between phage carrier state in infected cells and lysogeny was found for these phages. The past history of a phage infection altered the response of the cured host to reinfection. In those cured cells which remained susceptible, the stability of the reinfecting phage genome was not altered appreciably, while a distinctive new plaque morphology appeared in greater than 50% of these cells. These alterations were not dependent on the prophage integration prior to curing, and no phage DNA was detected in cured cells by blot hybridisation. Notwithstanding these changes, the sites of phage integration in cured cells remained the same as the original infection on both the phage RF and the host chromosome.

Bacteriophages↗

PubMed1991

Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗

PubMed1979

Heteroduplexes of phiX174 and G4 DNAs: orientation to genetic map and comparison with predictions from nucleotide sequences.

Heteroduplexes between the viral DNA of phiX174 and DNA from the replicative form (RF) of phage G4 were examined by electron microscopy. The single Eco RI site of G4-RF was utilized as a physical marker by preparing the heteroduplexes from the denatured, linear DNA obtained by restricting G4-RF with Eco RI endonuclease. Restriction fragments of phiX were used in a separate series of heteroduplexes to align the heteroduplex map and the G4 Eco RI site with the similar genetic maps of the two phages. The positions of the branch migrating junctions of recombinant phiX-G4 figure-8s, previously located only with respect to the G4-Eco RI site, have now been located with high proability within the gene A region of the two genomes. The degree of mismatch between the known nucleotide sequences of phi X and G4 accounts for positions of all of the regions of single-strandedness in the observed heteroduplexes, but unexplained discrepancies were also found.

Base Sequence↗

PubMed1981

Bacteriophage fd gene-2 protein. Processing of phage fd viral strands replicated by phage T7 enzymes.

Bacteriophage T7 gene 4 protein and DNA polymerase of the phage were used to study the viral strand synthesis of bacteriophage fd in vitro. Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand. The cleaved double-stranded DNA was unwound by T7 gene 4 protein and T7 DNA polymerase and the 3' end of the nicked strand simultaneously extended according to the rolling circle mechanism. After a complete round of DNA synthesis fd gene 2 protein cleaved the viral strand presumably at the same site, where the endonuclease cuts fd RF I, and subsequently sealed the single-stranded linear DNA into a circle. The reaction products were analyzed by velocity sedimentation, gel electrophoresis and electron microscopy. Most of the single-stranded DNA synthesized was circular. No host proteins were required for the formation of the single-stranded circles. Strand switching of the T7 DNA polymerase indicated by double-stranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzyme system.

DNA Replication↗

PubMed1987

Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA.

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.

Cell Nucleus↗

PubMed1979

[Antiviral activity of interferon and its inducers in human lymphoblastoid and somatic cells].

The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells.

Adamantane↗

PubMed1975

Poliovirus-induced infectious double-stranded RNA: Effect of RNA-degrading enzymes.

The infectivity of replicative form RNA (RF-RNA) isolated from poliovirus-infected HeLa cells is completely resistant to the action of T-1 RNase but decreases after exposure to RNase A in the presence of 0.3 M NaCl. Under these conditions neither enzyme produces single-stranded nicks in RF-RNA. Three endonuclease-free exonuleases (RNase II, polynucleotide phosphorylase and spleen phosphodiesterase) rapidly destroy the infectivity of single-stranded RNA, but do not alter the infectivity of RF-RNA. It is concluded that RF-RNA does not contain single-stranded ends essential for infectivity. Indirect evidence suggests that all or most of the poly A region at the 3' end of the plus strand of infectious RF-RNA is base-paired to a poly U region at the 5 end of the minus strand.

Base Sequence↗