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At least 127 records · Page 7Linked to original sources
PubMed1976

Site of cleavage of superhelical phiX174 replicative form DNA by the single strand-specific Neurospora crassa endonuclease.

Experiments with the Neurospora crassa single strand-specific endonuclease have provided evidence for the existence of regions of partially single-stranded character in covalently closed superhelical replicative form DNA of phiX174. The nuclease converts the superhelical molecules to either singly hit relaxed circular or doubly hit linear molecules. We show that the initial cleavage of phiX174 superhelical DNA is a "nick" bounded by a 5'-phosphate and a 3'-hydroxyl; no nucleotides are excised as evidenced by the ability of T4-polynucleotide ligase to reform the phosphodiester bond. The nick can be found in either strand of the double-stranded DNA and is either randomly distributed or at least can be found at any one of many possible locations in the genome. Thus, the regions in phiX174 superhelical molecules that are sensitive to the N. crassa nuclease do not occur at highly specific sites in the genome.

Amino Acids↗
PubMed1985

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome.

Animals↗
PubMed2001

Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTX(Phi) genome instead of CTX(Phi) lysogens in adult rabbits.

Toxigenic Vibrio cholerae strains are lysogens of CTX(Phi), a filamentous bacteriophage which encodes cholera toxin (CT). Following infection of recipient V. cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid. We infected naturally occurring attRS-negative nontoxigenic V. cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTX(Phi) genome using the adult rabbit diarrhea model. Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits. However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTX(Phi) genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTX(Phi) produced severe fatal diarrhea. Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative. These results suggested that strains carrying the RF of CTX(Phi) are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTX(Phi) genome. These results may have implications for the development of live V. cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi). These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V. cholerae strains.

Animals↗
PubMedDate not supplied

[Protection of segments of replicative form I of phiX174 phage DNA recognized by HindII, BspRI, and AluI restrictases by Escherichia coli RNA-polymerase].

The preincubation of a DNA with E. coli RNA polymerase provides its partial protection against the HindII, BspRI and AluI cleavage giving possibility to determine the location of RNA polymerase tight-binding sites. Using this approach about 16 RNA polymerase tight-binding sites were detected on replicative form of phiX174 phage DNA. The protection degree of each of these sites depended on the preincubation conditions. Some of the protected sites hit the known phiX174 promoters and rho-dependent terminators and the other were distributed along the whole phiX174 DNA molecule. Many of them could be considered as potential promoters because they contain all the necessary elements specifying the real promoter sequences. At least some of the intrinsic promoter elements could be observed next to the rest of protected sites. One of the protected sites (R6b/l) is located in phiX174 DNA region which is very similar to the cAMP-CRP-controlled promoter sequences. It was confirmed that phiX174 DNA has two B promoters positioned by Sanger on the phiX174 nucleotide map, according to our data obtained by RNA polymerase protection experiments along with RNA product analysis of the R8 DNA fragment transcription in vitro.

Bacteriophage phi X 174↗
PubMed1972

Characterization of bacteriophage S13 suN15 single-strand and replicative-form deoxyribonucleic acid.

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 x 10(6) daltons, an S degrees (20) of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 x 10(6) daltons, an S([ill]) of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than phiX174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.

Centrifugation, Density Gradient↗
PubMed1996

Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs.

In addition to the genomic RNA, a 3' coterminal nested set of six subgenomic mRNAs is produced in equine arteritis virus (EAV)-infected cells. The seven viral RNAs are also 5' coterminal, since they all contain a 206-nucleotide common leader sequence which is identical to the 5' end of the genome. A conserved penta-nucleotide sequence motif, 5' UCAAC 3', was shown to be present at the junctions between the leader and body sequences in each of the mRNAs. In addition, two alternative junction sites were detected for mRNA 3. Seven replicative-form (RF) RNAs (RFs I to VII), corresponding to the genomic RNA and each of the subgenomic EAV mRNAs, could be prepared from lysates of infected cells. The minus-strand RNA contents of these RF RNAs were analyzed by using an RNase protection assay with an RNA probe containing the mRNA 2 leader-body junction. It was established that RF II contained a negative-stranded copy of mRNA 2, including a complementary leader sequence. The presence of subgenomic minus-strand RNA in RFs is indicative of a function as a transcription template during the production of EAV subgenomic mRNAs.

Animals↗
PubMed1988

Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome.

The complete nucleotide sequence of virion DNA from two isolates of woodchuck hepatitis virus (WHV 7 and WHV 59) was determined along with the sequence of supercoiled DNA from one of those isolates (WHV 7). The sequences of the two WHV isolates were compared with the previously published sequences of two other isolates (WHV 1 and WHV 8). The range of nucleotide sequence variation of the four isolates (1 to 3.5%) was similar to those of HBVs of the same subtype (1.5 to 2%), but less than those of HBVs of different subtypes (8 to 10%). When amino acid sequences from the four WHV isolates were aligned, a consensus sequence could not be obtained at 13 sites. At 11 of 13 sites (7 in polymerase, 2 in presurface, 1 in surface, and 1 in the X region) the viruses could be grouped into pairs, so that WHV 1 and WHV 59 shared one amino acid and/or WHV 7 and WHV 8 shared a different amino acid. WHV 7 was passaged twice in woodchucks and supercoiled DNA was isolated. The nucleotide sequence of WHV 7 supercoiled DNA (derived from liver) was compared to that of WHV 7 virion DNA (derived from serum). Sequence comparison of virion and free supercoiled DNA from WHV 7 showed no nucleotide changes, except for a single nucleotide deletion thought to represent an error during molecular cloning or a defective viral genome. Thus, the nucleotide sequence of two different replicative forms of WHV DNA, separated by two in vivo passages, were virtually identical.

Amino Acid Sequence↗
PubMed1993

The major capsid protein VP2 of minute virus of mice (MVM) can form particles which bind to the 3'-terminal hairpin of MVM replicative-form DNA and package single-stranded viral progeny DNA.

The capsids of minute virus of mice (MVM) consist of two closely related proteins, VP1 and VP2. We inactivated the VP1 gene in an infectious clone of MVM DNA by frameshift mutation. After transfection of mutated DNA, capsids consisting of VP2 only were made. They can package negative-strand DNA, and they specifically bind MVM 3'-terminal hairpin DNA.

Animals↗
PubMed1978

[Interferonogenic and antiviral activity of the double-stranded replicative form of phage f2 RNA in tissue culture and on animals].

Biological activity of a new natural interferon inductor, the replicative RNA form of phage f2 (RFf2) was studied. A possibility of using RFf2 for production of highly active interferon under conditions of superinduction providing an increase in the interferon yield by to 256--512 times as compared to the control samples was shown. The protective interferonogenic and antiviral effect of RFf2 in mice infected with Semliki forest virus (SFV) and tickborne encephalitis virus (TBEV) was studied on administration of the inductor by various routes. It was found that intraperitoneal administration of RFf2 in a dose of 10 gamma per a mouse protected the infected animals from death. It was accompanied by production of up to 1280 units/ml of interferon in the blood serum of the animals. Maximum protection of the animals from death under conditions of the experiment (80 per cent on infection with SFV and 65 per cent on infection with TBEV) was observed when the preparation was administered twice: 4 hours after the infection. Combined use of RFf2 with chemotherapeutics (rimantadine) increased the protective effect both in the tissue culture and in vivo.

Adamantane↗
PubMed2000

Replicative forms of TT virus DNA in bone marrow cells.

TT virus (TTV) is a human virus consisting of a single-stranded, circular DNA genome of 3.8 kilobases (kb). To examine whether TTV replicates in peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs), DNA was extracted from the PBMCs and/or BMCs of six TTV-infected individuals and separated by agarose gel electrophoresis. The TTV DNAs from the PBMCs migrated to the 2.0- to 2.5-kb region. The TTV DNAs from the BMCs migrated to the 2.0- to 2. 5-kb and 3.3- to 6.1-kb regions. The faster-migrating TTV DNAs were sensitive to S1 nuclease, while the slower-migrating TTV DNAs were resistant and their position on the agarose gel shifted to the position of the full genomic size upon digestion with restriction enzyme PstI. Full-length inverted polymerase chain reaction on the slower-migrating, double-stranded TTV DNAs from the BMCs amplified a 3.8-kb product. Replicative intermediate forms of TTV DNA are present in BMCs but not in PBMCs.

Base Sequence↗
PubMed1978

Structural and replicative forms of mitochondrial DNA from human leukocytes in relation to age.

The structure and replication of human leukocyte mitochondrial DNA (mtDNA) was investigated in healthy young adult males (23--37 years old), middle-aged males (42--52 years old) with secondary polycythemia, and elderly males (80--89 years old) who exhibited different degrees of age-related disease syndromes. The distribution of the various cell types within the white cell population was within normal limits in all samples. Total mtDNA was isolated in ethidium bromide--CsCl gradients and examined by electron microscopy after spreading by the aqueous and formamide techniques. The individual frequencies of catenated forms ranged from 2 to 6% but showed relatively little change (declining slightly) with age. The individual frequencies of circular dimers varied from 0 to 0.1% in the young adult and polycythemic groups and in 10 out of 12 elderly individuals. One elderly individual had a circular dimer frequency of 0.3% (including a circular molecular of tetramer size) and another had 4.5%. This finding suggest that agerelated cellular pathology may exist in the blood-forming system in some cases. The mode of replication of leukocyte mtDNA agrees well with that described for mouse L cells. There was no evidence of aberrant mtDNA replication as a result of aging.

Adult↗
PubMed1980

The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA.

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.

Bacterial Proteins↗