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 883 records · Page 49Linked to original sources

PubMed2000

TT virus mRNAs detected in the bone marrow cells from an infected individual.

Although replicative forms of TT virus (TTV) DNA have been found in the liver and bone marrow cells, mRNAs of TTV have not yet been detected in these tissues. The entire nucleotide sequence of a TTV clone [TYM9 (3759 bases)] isolated from a patient with high TTV viremia (10(6) copies/ml) was determined, and the poly(A)(+) RNAs from bone marrow cells were subjected to reverse transcription-polymerase chain reaction with primers specific for the TYM9 sequence. Sequence analysis of the amplified products revealed the presence of three distinct species of spliced TTV mRNAs [2.9, 1.2, and 1.0 kilobases (kb)] with common 5' and 3' termini as well as splicing to bind nucleotide (nt) 181 to nt 283. The shorter mRNAs of 1.2 kb and 1.0 kb possessed another splicing to join nt 681 with nt 2341 or nt 2579. The transcription profile of TTV found in an infected human corroborates that observed in vitro.

3' Untranslated Regions↗

PubMed2000

Expression of the insect parvovirus GmDNV in vivo: the structural and nonstructural proteins are encoded by opposite DNA strands.

The nucleotide sequence of GmDNV, an insect parvovirus, reveals large open reading frames (ORFs) on both strands of the viral replicative form DNA. Previously, we identified two viral transcripts within the polyadenylated RNA fraction of infected host larvae (Gross et al., 1990, J. Invertebr. Pathol. 56, 175-180). In this work we used hybridization of single-stranded, unidirectional probes to RNA blots to show that the two transcripts, synthesized in vivo in GmDNV-infected Galleria mellonella larvae, are of antiparallel orientation. To determine their coding specificities, polyadenylated RNAs were isolated from hybrids with DNA from the left and right halves of the viral genome and translated in a rabbit reticulocyte system. The "right," 2.4-kb hybrid-selected RNA was shown to direct the synthesis of four polypeptides that comigrated with the four viral capsid proteins and were immunoprecipitated with anti-GmDNV serum. Translation of the "left," 1.8-kb RNA yielded three polypeptides, none of which was detected among the viral capsid proteins. This type of expression strategy is unique among vertebrate and most invertebrate parvoviruses, which use only one DNA strand to encode all their proteins. On the other hand, the basic organization of parvoviruses, in which the regulatory and structural proteins are encoded, respectively, by two clusters of ORFs located at the left and right halves of the genome, is conserved.

Animals↗

PubMed1999

The primary immunity determinant in modulating the lysogenic immunity of the filamentous bacteriophage cf.

Bacteriophage cf is the first single-stranded DNA phage that has been shown to set up a stable lysogenic state with its genome integrated into the host chromosome. From the isolation and characterization of a virulent mutant, cf-tv2, we report the first investigation into the mechanisms of the immunity established by the filamentous bacteriophage. The mutation in cf-tv2 enables the phage to produce plaques on lawns of cf lysogenic cells. The mutation was defined as a 49-nucleotide deletion located in a 0.59 kb NcoI/KpnI fragment of cf replicative form DNA. Two messages, cM1 and cM2, transcribed from the immunity region of wild-type cf but in opposite directions, were detected. In cf-tv2, the 49-nucleotide deletion abolishes cM2 transcription. The primer extension assay suggests a possible RNA-RNA interaction directed by base-pairing of the cM1 and cM2 RNAs. A frameshift mutation of the open reading frame ORF 165, encoded by cM2, resulted in a 10(5) plating efficiency on the cf lysogen. These observations suggest that both RNA-RNA interaction and repressor protein inhibition are involved in the mechanism of cf immunity. A model is proposed for the regulation of cf immunity.

Amino Acid Sequence↗

PubMed1998

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma.

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.

Animals↗

PubMed1995

The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion.

Resolution of the covalently closed terminus of adeno-associated Virus (AAV) DNA is mediated by viral replication protein Rep78 or Rep68. In vitro studies with purified Rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5' end of the viral genome. The in vivo existence and fate of the covalently associated Rep protein during the virus life cycle has not yet been elucidated. In this report, we use immunoprecipitation analyses to demonstrate that the Rep78 protein is covalently attached to viral DNA in a preformed virion. The attached Rep78 is susceptible to antibody binding and protease digestion, and the DNA linkage is susceptible to nuclease digestion, therefore Rep78 is probably located on the outside of the particle. Rep proteins are also attached to double-stranded replicative-form monomer (RFM) DNA in extracts from AAV and adenovirus coinfected cells. Rep protein attachment to RFM and encapsidated AAV DNA suggest that the covalent complex is an intermediate in virus assembly. These observations are similar to those noted by others for the autonomous parvoviruses and provide additional insights into parvovirus assembly.

Animals↗

PubMed1996

A murine host cell factor required for nicking of the dimer bridge of MVM recognizes two CG nucleotides displaced by 10 basepairs.

During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

Animals↗

PubMed1997

Regulation of a geminivirus coat protein promoter by AL2 protein (TrAP): evidence for activation and derepression mechanisms.

Tomato golden mosaic virus (TGMV) is a bipartite member of the subgroup III Geminiviridae. Like all geminiviruses, TGMV replicates in the nucleus of susceptible cells by rolling circle replication (RCR). Double-stranded replicative form DNA generated during RCR serves as template for the transcription of viral genes by RNA polymerase II and the associated cellular transcription machinery. Previous studies in tobacco protoplasts and Nicotiana benthamiana leaf discs have shown that the viral AL2 gene product transactivates expression of the coat protein (CP) and BR1 movement protein genes, and that activation occurs at the level of transcription. Because of its function and properties, we propose the name TrAP, transcriptional activator protein, for the AL2 gene product. Using transgenes consisting of complete and truncated versions of the CP promoter fused to the GUS reporter gene, we show in the studies presented here that TrAP is required for CP gene expression in both mesophyll and phloem tissues. Surprisingly, TrAP appears to induce CP expression by different mechanisms in different cell types: it may activate the CP promoter in mesophyll cells, and acts to derepress the promoter in phloem tissue. In addition, TrAP is clearly capable of inducing the expression of responsive chromosomal promoters and could, in principle, activate host genes. Distinct viral sequence elements mediate expression and derepression in phloem and activation in mesophyll, suggesting that TrAP interacts with different components of the cellular transcription machinery to accomplish CP gene expression in different cell types, and underscoring the intricacy and complexity of virus-host interactions.

Capsid↗

PubMed1998

Repetitive DNA insertion in a protein kinase ORF of a latent FSV (Feldmannia sp. virus) genome.

We describe the structure of a Feldmannia sp. virus (FsV) genome integrated in the brown alga, Feldmannia. This integrated FsV genome appears to be permanently inactivated and lost its ability to excise and replicate. Unlike the replicated form of FsV, this integrated FsV genome contains a large (>50 kb) repeat region inserted in a protein kinase open reading frame. While related to the 173-bp repeats previously characterized in the FsV genome (Lee et al., 1995), Southern blot analysis indicates that the repeats in the inactive, integrated FsV genome are distinct from those previously characterized. Fine structural analysis of the repeat-insertion sites in the protein kinase gene indicates that there are 8- and 10-bp palindromic sequences present in multiple locations located near the repeat-insertion site. The translated protein kinase contains all of the catalytic motifs conserved in most serine/threonine protein kinases and a potential autophosphorylation site. This protein kinase gene is expressed as RNA in sporophyte plants where virus production is active but not in gametophyte plants where the virus genome is latent. The structure of the integrated virus genome is discussed.

Amino Acid Sequence↗

PubMed1998

Psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus.

Cloning and sequencing of the circular, single-stranded DNA of one isolate of psittacine beak and feather disease virus (BFDV) demonstrate a genome composed of a circular molecule of 1993 nucleotide bases. An analysis of the assembled replicative form demonstrated seven open reading frames (ORFs) (three in the virion strand and four in the complementary strand), potentially encoding seven viral proteins of >8.7 kDa. High amino acid sequence similarity was demonstrated between a potential 33.3-kDa protein product of ORF1 of BFDV and the replicase-associated protein of porcine circovirus (PCV), subterranean clover stunt virus, and faba bean necrotic yellows virus. However, significant similarity in nucleotide or amino acid sequences was not present between BFDV and chicken anaemia virus. A potential stem-loop structure similar to that found in PCV and plant circoviruses was present in the putative encapsidated strand of the BFDV genome. At the top of this structure, a nonanucleotide motif (TAGTATTAC) similar to that of PCV, plant circoviruses, and geminiviruses also was recognised. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1% identity, and in both viruses, ORF2 is located on the complementary strand, beginning close to or within the hairpin stem. Our findings provide further evidence of a close relationship among BFDV, PCV, and plant circoviruses but not chicken anaemia virus.

Amino Acid Sequence↗

PubMed1999

Transcription of the genome of the filamentous bacteriophage cf from both plus and minus DNA strands.

The filamentous bacteriophage cf infects the bacterium Xanthomonas campestris pv. citri. Northern blot analysis with probes derived from various restriction fragments of cf replicative form (RF) DNA has revealed the presence of five major phage-specific transcripts in infected cells. Four of these transcripts were shown to be derived from the region of the cf genome extending from gene II to gene VIII and are consistent with the cascade model of transcription proposed for Ff coliphages. These transcripts overlap with each other and terminate upstream of an efficient Rho-independent transcription terminator. Unlike the well-characterized Ff phages, in which only the minus strand of viral DNA serves as a transcription template, both strands of the RF DNA of phage cf appeared to be transcribed. Thus one of the five major cf transcripts was shown to be derived from a region of the viral minus strand that contains an open reading frame encoding a putative polypeptide of 165 amino acids. Primer extension analysis mapped the transcriptional initiation site of this RNA to a cytosine residue at position 870. A partial transcription map of phage cf revealed two independent regions of transcriptional activity. The region with the highest activity coincides with that encoding the polypeptides required in the largest amounts during the cf infection cycle.

5' Untranslated Regions↗

PubMed2000

Nascent synthesis of leader sequence-containing subgenomic mRNAs in coronavirus genome-length replicative intermediate RNA.

Infection with coronavirus results in the accumulation of genomic-sized mRNA and six to eight subgenomic mRNAs that make up a 3' coterminal nested-set structure. Genome-length negative-strand RNA and subgenomic-length negative-strand RNAs, each of which corresponds to each of the subgenomic mRNAs, also accumulate in infected cells. The present study examined whether the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis. Genome-length replicative intermediate (RI) RNA was purified by two-dimensional gel electrophoresis of intracellular RNAs from cells infected with mouse hepatitis virus. RNase A treatment of the purified genome-length RI resulted in the production of the genome-length replicative form RNA, indicating that the genome-length RI included genome-length template RNA. RNase protection assays using the purified genome-length RI and two probes, which corresponded to the 5' 300-nt region of mRNA 6 and to the same region of mRNA 7, showed the presence of nascent leader sequence-containing subgenomic mRNAs in the genome-length RI. These data demonstrated that the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis.

Animals↗

PubMed1991

Escherichia coli DNA polymerase II is homologous to alpha-like DNA polymerases.

The Escherichia coli polB gene encodes DNA polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89,917, and is structurally homologous to alpha-like DNA polymerases, which include eukaryotic replicative DNA polymerases. Comparison of the sequences of the alpha-like DNA polymerases including E. coli DNA polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA polymerases based on the differences in these conserved regions. The DNA polymerases of herpes groups viruses and the DNA polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA polymerases, including E. coli DNA polymerase II, human DNA polymerase alpha, and yeast DNA polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the -35 sequence of the polB promoter.

Amino Acid Sequence↗

PubMed1989

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans.

Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408.1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.

Base Sequence↗

PubMed1976

Discrimination of competent Bacillus subtilis with respect to ribonucleic acids.

A study has been made of the affinity of double-stranded helical RNA for DNA receptors in competent Bacillus subtilis. In competition experiments, using homologous and heterologous DNA samples which had been sheared to molecular weights comparable to that of the RNA (about 2 x 10(6)), and which still exhibited appreciable competition in DNA uptake experiments, the replicative form of phage f2 RNA showed no evidence of affinity for receptor sites. A second double-stranded RNA preperation from a widely different source, a mycophage of Penicillium chrysogenum, behaved similarly to the f2 RNA. Transfer RNA and 23S ribosomal RNA, which reduce transformation frequencies in pneumococcus, did not compete for B. subtilis receptors. Lack of competition was not due to enzymatic degradation of the RNA, since the latter was recovered intact following exposure to competent cells. Under conditions where homologous native DNA undergoes normal uptake, there was virtually no uptake of native double-stranded RNA. The results are examined in the light of reports on transformation by RNA and DNA-RNA hybrids, and also in relation to the characterization of the specificity of cell-nucleic acid interactions.

Bacillus subtilis↗

PubMed1980

Folded chromosomes of Saccharomyces cerevisiae: comparison of response to sporulation medium, arrest at start, and G0 arrest.

Folded chromosome phenotypes have been examined and compared in four cell-division-cycle (cdc) mutants during transitions between cycling and non-cycling states. The two start mutants, cdc 28 and cdc 25, can undergo G0 arrest at the restrictive temperature. Arrest at start, defined by the cdc 28 and cdc 25 block points, is distinguishable from G0 arrest. Arrest at the cdc 28 and cdc 25 block points can also be distinguished from each other: folded chromosomes appear to be destabilized at the cdc 25 block, but are stable at the cdc 28 arrest point. On the other hand, folded chromosomes from cdc 28 in sporulation medium at the restrictive temperature appear unstable, while chromosomes from cdc 25 are stable. The G1 arrest mutants, cdc 4 and cdc 7, can undergo G0 arrest at the restrictive temperature. In sporulation medium no meiotic replication form is detected at the restrictive temperature, although incorporation of labeled precursors into nuclear DNA does take place. A schematic model incorporating these various findings is presented.

Chromosomes↗

PubMed1987

Size distributions of circular molecules in plant mitochondrial DNAs.

Some physicochemical properties of the mitochondrial DNAs (mtDNA) from plants of flax, broad bean and mung bean, and from tissue culture cells of jimson weed, soybean, petunia and tobacco were determined. Circular molecules were observed in electron microscope preparations of each mtDNA. In soybean, petunia, broad bean and mung bean mtDNAs, the circular molecules had a continuous distribution of lengths (ranges between 1 to 36 kb, and 1 to 126 kb), heavily skewed toward smaller molecules. Eighty-six percent of the flax circular molecules were from 27 to 54 kb in size, and 78% of the jimson weed circular molecules were from 4 to 15 kb. Replicative forms of 1.2-1.6 kb circular molecules were observed in electron microscope preparations of broad bean mtDNA.

DNA Restriction Enzymes↗

PubMed1992

Detection of chicken anaemia agent DNA sequences by the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of chicken anaemia agent (CAA) DNA. The assay used a single set of 20-base primers complementary to sequences located in the coding regions of the CAA replicative form (RF) DNA genome at positions 485 to 504 and 1048 to 1067. The observed amplification product had the expected size of 583 bp and was confirmed to derive from CAA RF DNA by a unique Hind III restriction enzyme cleavage pattern. The amplified fragment was shown to be specific for CAA RF DNA after chemiluminescence dot blot hybridisation with a digoxigenin-labelled 25-base internal probe. The optimised PCR assay was specific for CAA and highly sensitive, being able to detect a single CAA-infected MDCC-MSB1 cell and at least 100 fg of CAA RF DNA. Preliminary results also showed that the PCR assay can detect CAA DNA in clinical specimens from chicks experimentally infected with CAA.

Animals↗

PubMed1992

Partial DNA cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease.

The cloning and sequencing of an Eco RI-PstI fragment derived from the replicative form of a canine parvovirus (CPV) vaccine strain are reported. The variability of the 5' end of NS 1 protein gene in the genome is confirmed by comparison with previously determined DNA sequences. A 15 nucleotide deletion was also observed in this vaccine strain. In order to improve CPV diagnosis, radioactively labelled RNA or DNA and biotin labelled DNA obtained by random priming of the recombinant plasmid were used as probes mainly on gut or stool samples from naturally infected dogs. Results of filter hybridization correlated well with histopathological diagnosis of parvovirus infection and with hemagglutination tests performed on dog faeces. We propose that nucleic acid hybridization may be an alternative diagnostic method to ascertain the presence of CPV, especially in frozen samples.

Amino Acid Sequence↗