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PubMed1991

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus.

A polymerase chain reaction (PCR) amplification method was developed and evaluated to detect porcine parvovirus (PPV). A pair of 20-base primers and an oligonucleotide probe were derived from the DNA sequences common to two isolates of PPV, NADL-8 and NADL-2. The primers flanked 118-bp nucleotides within the region coding for the major structural protein VP2. After DNA amplification of PPV replicative form (RF), a 158-bp fragment was detected in agarose gels. This amplified fragment was shown to be specific for PPV DNA after Southern transfer and hybridization to a 20-base internal probe. The amplified fragment also contained a single EcoRI cleavage site. Various conditions, such as number of cycles and annealing temperature, were examined to optimize the conditions for detecting viral DNAs from infected cell cultures and swine fetal tissues. Four different isolates of PPV, NADL-8, NADL-2, KBSH and Kresse, and two other viruses, canine parvovirus (CPV) and pseudorabies virus (PRV), were included to determine specificity of amplification. Slot blot hybridization with a radiolabeled probe was used to evaluate the sensitivity of PCR amplification. The optimized protocol was specific for PPV detecting equally all four strains of PPV, but failing to amplify CPV or PRV sequences. The PCR method could detect at least 100 fg of viral replicative form (RF) DNA or the equivalent of 1 PFU of infectious virus. The applications of this method include routine detection of PPV in clinical samples and as a contaminant in mammalian cell lines.

Animals↗
PubMed2004

Can modulation of viral fitness represent a target for anti-HIV-1 strategies?

Prolonged use of anti-retroviral compounds in human immunodeficiency virus type 1 (HIV-1) infection selects for drug-resistant and often mutidrug-resistant viral variants. Drug-resistance mutations may also affect viral fitness. Interestingly, recent research has indicated that some of the unfit drug-resistant variants may be less pathogenic, suggesting that decreased viral fitness is beneficial for the host and may be driven by specific treatments during anti-HIV-1 infection. A second potential antiviral strategy starting with profound inteference with viral fitness aims at forcing viruses towards lethal mutagenesis (the so-called "error catastrophe"). This review summarizes the methods for addressing HIV-1 fitness in vitro and ex vivo, the current understanding of clinical implications of reduced HIV-1 fitness, and the potential use of anti-HIV-1 strategies aiming at modulating viral fitness. Finally, it is emphasized how the peculiar features of HIV-1 quasispecies (displaying two different forms of memory, a replicative and a non-replicative form) may sharply influence the design of future diagnostic methodologies for fitness analysis.

Drug Resistance, Viral↗
PubMed2005

Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments.

Picornavirus infection of cells generally results in the production of membranous vesicles containing the viral proteins necessary for viral RNA synthesis. To determine whether foot-and-mouth disease virus (FMDV) infection induced similar structures, and which cellular components were involved, the subcellular distribution of FMDV proteins was compared with protein markers of cellular membrane compartments. Using immunofluorescence analysis and digital deconvolution, it was shown that FMDV structural and non-structural proteins co-localize to punctate structures in juxtanuclear virus assembly sites close to the Golgi complex. Significantly, viral protein 2C did not co-localize with marker proteins of the cis- or medial-Golgi compartments or trans-Golgi network. Furthermore, incubation of infected cells with brefeldin A caused a redistribution of Golgi proteins to the endoplasmic reticulum, but did not affect the distribution of 2C and, by inference, the integrity of the virus assembly site. Taken with the observation that 2C was membrane-associated, but failed to fractionate with Golgi markers on density gradients, it was possible to conclude that Golgi membranes were not a source of structures containing 2C. Further immunofluorescence analysis showed that 2C was also separate from marker proteins of the endoplasmic reticulum, endoplasmic reticulum intermediate compartment, endosomes and lysosomes. The results suggest that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication.

Animals↗
PubMed1987

Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus.

A complex "replication competent" holoenzyme form of DNA polymerase alpha (RC-alpha) was purified 10,000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-alpha form could partially replicate a double-stranded oligo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-alpha was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-alpha required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-alpha with the neutralizing anti-DNA polymerase alpha monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386-8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-alpha preparation following removal of specific polypeptides strongly suggested that the capacity of RC-alpha to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.

Adenosine Triphosphate↗
PubMed2003

Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms.

Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes. Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase. Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression. Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks. Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures. We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.

Cell Cycle Proteins↗
PubMed1972

Replication of mitochondrial DNA in mouse L cells and their thymidine kinase - derivatives: displacement replication on a covalently-closed circular template.

Thymidine kinase(-) lines of mouse L cells incorporate thymidine exclusively into mitochondrial DNA. This fact permits assessment of labeled mitochondrial DNA components in ethidium bromide-cesium chloride gradients. Contaminating nuclear DNA is unlabeled and need not be removed. Elimination of the DNase treatment of purified mitochondria reveals that the replicative forms that exhibit displacement replication up to full genome size are covalently-closed circular molecules. Denaturation followed by brief renaturation of these larger replicating molecules produces closed-circular DNA with a deficiency of Watson-Crick turns, appearing as single-strand loops. This result suggests that displacement replication proceeds with nicking and rapid closure of the covalently-closed circular template.

Animals↗
PubMed1973

Replication of bacteriophage phiX-174 in a mutant of Escherichia coli defective in the dnaE gene.

Strains of E. coli bearing a temperature-sensitive mutation at the dnaE locus were unable to support growth of bacteriophage varphiX-174 at 43 degrees while permitting normal growth at 30 degrees . The dnaE gene product was implicated in all stages of varphiX-174 DNA replication. Differences were observed between a double mutant (pol A(-), dnaE(ts)) and a single mutant (pol A(+), dnaE(ts)) in the ability to synthesize parental replicating form and its subsequent replication. The implications of these observations are discussed in terms of the relationship between DNA polymerases-I and -III.

Carbon Isotopes↗
PubMed1976

Characterization of an Acholeplasma laidlawii variant with a REP- phenotype.

An Acholeplasma laidlawii variant has been isolated that has a REP- phenotype. The properties of this variant, relative to parental cells, are: (i) it exhibits no change in cell growth kinetics; (ii) it does not propagate single-stranded deoxyribonucleic acid (DNA) mycoplasmaviruses but does propagate double-stranded DNA mycoplasmaviruses; (iii) it converts parental circular single-stranded mycoplasmavirus DNA to double-stranded replicative forms that are not replicated further; (iv) it exhibits no change in host modification and restriction; and (v) it has an increased ultraviolet light sensitivity. The REP- isolate is the first stable mycoplasma variant to which a physiological defect has been attributed.

Acholeplasma laidlawii↗
PubMed1974

Mechanism of replication of phi x174 single-stranded DNA. IX. Requirement for the Escherichia coli dnaG protein.

Escherichia coli NY73, possessing a temperature-sensitive mutation in the dnaG locus, was rendered sensitive to bacteriophage phiX174 by P1 transduction. phiX174 reproduces in this strain at 30 C but not at 40 C. All three stages of phiX174 replication, parental replicative form (RF) synthesis, RF replication, and progeny single-stranded DNA synthesis, are thermolabile in this mutant. Competition-annealing data show that both plus- and minus-strand synthesis are equally inhibited after shift up to 40 C during RF replication. We conclude that the dnaG gene product is required for the synthesis of both strands of phiX RF during RF replication and of the complementary strand and viral progeny strands during stages I and III, respectively.

Bacterial Proteins↗
PubMed1986

Denaturation of covalently closed circular DNA. Kinetics, comparison of several DNAs, mechanism and ionic effects.

The rates of the alkaline denaturation of the covalently closed, circular DNAs (form I) of the replicative forms (RF) of phages G4, phi X174, and fd, and of plasmid pBR322 and phage PM2 have been measured at 0 degrees C and some at higher temperatures. These rates are orders of magnitude slower than the denaturation of linear DNA because of the increased stability of the helix to deprotonation that results from the accumulating positive superhelicity during denaturation. Denaturation reactions were initiated by rapid, infrasonic mixing (Camien, M.N., and Warner, R.C. (1984) Anal. Biochem. 138, 329-334), and their progress was measured by analytical ultracentrifugal analysis for the amounts of form I and denatured (Id) DNA after neutralization of the alkaline reaction. The comparative rates of the five DNAs varied over a wide range; the fastest, G4-RF, denatured at 500-fold the rate of the slowest, fd-RF. The differences are accounted for by the interaction of positive superhelicity with the sequence-dependent regions of relative helix stability in the various DNAs. Renaturation rates of Id DNAs varied similarly for Ids prepared at 0 degrees C, but only a few-fold for Ids prepared at 50 degrees C. The rate of denaturation of G4-RF was determined over a wide range of NaOH and NaCl concentrations at 0 degrees C, and the pHm was determined as a function of ionic strength and temperature. The effects of ionic strength have been analyzed in an application of the Manning ion condensation-screening theory (Manning, G.S. (1978) Q. Rev. Biophys. 11, 179-246) which is shown to account for the large destablizing effect of salts on the helix. The pH region of transition at 50 degrees C from renaturation to denaturation was examined, and it was found that the maximum rate of renaturation occurred at a pH about 0.05 units below the pHm.

Coliphages↗
PubMed1995

ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome.

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

Cell Cycle Proteins↗
PubMed1975

Fate of polyoma form IDNA during replication.

The fate of polyoma form IDNA generated during replication was investigated in resting BALB-3T3 cells. The experiments showed that there was extensive re-entry of such molecules into replication. This process took place over a period of several hours and appeared to be random. Progeny form I molecules must, therefore, spend some time in a nonreplicating pool before reinitiating replication. We propose that two factors affect the fate of progeny form IDNA. (i) The rate of reinitiation of progeny molecules is determined by the capacity of the initiation machinery. (ii) The extent of re-entry is determined by the availability of maturation proteins which divert form I from replication.

Animals↗
PubMed1995

Serodiagnosis of leishmaniasis.

Leishmaniasis is a spectrum of diseases ranging in severity from cutaneous (CL), post-kala-azar dermal (PKDL), and diffuse cutaneous (DCL) to mucocutaneous (MCL) and visceral (VL) infections that are endemic in 86 tropical and subtropical countries around the world, accounting for 75,000 deaths per year. Different forms of leishmaniases are generally caused by different distinct species of Leishmania having a digenetic life cycle alternating between an aflagellated amastigote form replicative within the macrophages of the host and a flagellated promastigote form that multiplies within the gut of the sandfly. VL, MCL, PKDL, DCL, and CL forms of the disease can be arranged on a priority basis in accordance with the humoral immune responses of host. Generally, the cell-mediated immunity, particularly the delayed-type hypersensitivity to leishmanial antigens, is associated with CL, MCL, PKDL, and cured VL cases. The serodiagnosis of leishmaniasis appears to be an alternative to parasite detection in biopsy samples either by the staining of amastigotes or by culturing the amastigotes, which transform to a promastigote form and replicate. A battery of immunological procedures have been developed or adapted to demonstrate either humoral or cell-mediated immune responses against Leishmania for diagnosis and epidemiological survey. The sensitivity and specificity of such diagnostic methods depend on the type, source, and purity of antigen employed, as some of the leishmanial antigens have common cross-reactive epitopes shared with other microorganisms, particularly Trypanosoma, Mycobacteria, Plasmodia, and Schistosoma. Serodiagnostic techniques for the detection of antileishmanial antibodies have been employed with about 72 to 100, 23 to 90, 83, and 33 to 100% success in VL, CL, MCL, and PKDL patients, respectively. The Leishmanin skin test (LST) is useful to detect MCL and CL, with about 100 and 84% success, respectively. In PKDL, the gradual fall of antileishmanial antibody titer to some extent and the rise of delayed hypersensitivity to the parasite antigen are the characteristic features associated with the chronicity of the disease. The use of whole promastigote as the source of antigens in the direct agglutination test (DAT) and immunofluorescent test (IFAT) gave cross-reactions with the sera of leprosy, tuberculosis, and African trypanosomiasis patients. Again, the use of cell-free extracts of promastigotes generally gave false positive results with the sera of normal human and Chagas' disease, leprosy, tuberculosis, and malaria patients in enzyme-linked immunosorbent assay (ELISA), dot ELISA, immunodiffusion, immunoelectrophoresis, and counter-current immunoelectrophoresis tests.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

[Pathogenesis and virulence determinants of the family Legionellaceae].

Legionellae are Gram-negative obligate intracellular parasites of unicellular animal organisms, some of which are sometimes able to cause an acute and sever pneumonia in humans. Legionellae, are capable of surviving for long periods in water when between hosts. Their adaptation to these quite diverse environments seems to be accomplished by morphological and biochemical pathway changes. It has been well established that humans coexisted with these bacteria for a long period of time, and it is only recently that our industrial technology provided these organisms with the means of causing infection. This review describes the invasion process and the life cycle of Legionellae in both protozoan and mammalian cells. Our understanding of the infection cycle of Legionellae is primarily based on observations by transmission and scanning EM and by contrast phase and fluorescence microscopy. The identification of virulence determinants by molecular techniques is also discussed. The bacteria enter the host cell mainly by coiling phagocytosis and reside within unique phagosomes which, during the first hours of infection, are isolated from the endosomal pathway. Within the protected vacuole the mature infectious forms of Legionellae convert to replicative forms that no longer express virulence traits. Evidence is provided on how the biogenesis of the replication niche is determined. The virulence factors that arrest phagosome maturation during intracellular replication are also described. The status of our current knowledge on the means by which Legionellae successfully infect the host cells to cause disease is discussed.

Animals↗
PubMed1978

The relationship of SV40 replicating chromosomes to two forms of the non-replicating SV40.

SV40 replicating chromosomes were extracted from infected cells using a detergent free extraction method. This procedure also extracts 2 forms of the non-replicating chromosome, one of which corresponds to the well characterized 50-55S SV40 minichromosome. The other is a more compact structure which has a sedimentation coefficient of 80-85S. The replicating chromosomes sediment between the 2 conformations of the mature chromosome. Electron microscopy of the replicating chromosomes suggests an overall conformation that resembles the 50-55S form of the mature chromosome rather than that of the 80-85S structure. Nucleosomes are present on both sides of the replication forks. When the replicating chromosomes were incubated in an in vitro DNA synthesis assay all regions of the SV40 genome were synthesized and a significant fraction of the replicating chromosomes completed replication. The progeny chromosomes co-sedimented with the 50-55S chromosomes which were present prior to the incubation. The sedimentation coefficients and relative amounts of the two forms of the mature chromosome were unaffected by the incubation.

Centrifugation, Density Gradient↗
PubMed1997

pH-independent uptake of hepatitis B virus in primary human hepatocytes.

The replication cycle of the hepatitis B virus (HBV) is still incompletely understood. In particular, the early steps of the viral life cycle, such as absorption, penetration, uncoating, and nuclear translocation require further clarification. In this study we performed infection experiments with HBV in primary human hepatocyte cultures. To further elucidate the possible mechanism of virus uptake, infection experiments were performed at different pH levels, after pretreatment of viral particles with acidic buffers and in the presence of lysosomotropic agents (chloroquine and ammonium chloride, respectively). Using a selective PCR technique which discriminates between input virus DNA and the earliest replicative form, we could demonstrate viral replication 36 hr after inoculation. HBV was taken up most efficiently at a pH of 7.4. Infection was still successful after pretreatment of viral particles at low pH and was unaffected by the presence of lysosomotropic agents. In conclusion, this suggests HBV to be a pH-independent virus.

Cells, Cultured↗