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At least 685 records · Page 38Linked to original sources
PubMed2002

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system.

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.

Animals↗
PubMed2006

Probing structural effects on replication efficiency through comparative analyses of families of potential self-replicators.

A formidable synthetic apparatus for the creation of nanoscale molecular structures and supramolecular assemblies through molecular structures can potentially be created from systems that are capable of parallel automultiplication (self-replication). In order to achieve this goal, a detailed understanding of the relationship between molecular structure and replication efficiency is necessary. Diastereoisomeric templates that are capable of specific and simultaneous autocatalysis have been synthesised. A systematic experimental and theoretical evaluation of their behaviour and that of structurally-related systems reveals the key determinants that dictate the emergence of self-replicative function and defines the structural space within which this behaviour is observed.

Catalysis↗
PubMed1986

In the murine syngeneic mixed lymphocyte reaction, one T cell subset replicates in the presence of B cells or macrophages and replication is inhibited by simultaneous presence of both stimulator cells.

The murine T-non-T cell syngeneic mixed lymphocyte reaction has been examined to determine whether B cells and macrophages stimulate the same or different subpopulations of T cells. By using experiments in which replicating T cells were suicided, we found that the two different stimulators caused replication of what appears to be the same subset(s) of T cells. Since B cells and macrophages carry the same stimulating antigens (class II plus mls or others), one would expect them to stimulate the same T cell subpopulations were it not that they have been reported to stimulate two different subpopulations in humans. When B cells and macrophages were simultaneously used as stimulators, diminished T cell replication occurred. We have found the reduced response is not attributable to exhaustion of culture nutrients or to displacement of the response peak. Other possibilities to account for this marked reduction have been discussed from the viewpoint of suppression emanating from macrophages and/or T cells.

Animals↗
PubMed1985

In situ replication techniques: II. Quantitative methodologies for replicate materials.

Because replicate materials have requirements different from those of recording or impression materials, quantitative methodologies were sought using commercial impression materials. Two satisfactory objective techniques resulted, a laser-scattering and a capillary flow test. Using high-resolution gratings to stimulate tooth detail (less than 1 micron), the reproduction quality of 36 two-stage replicas was determined in diffraction, reflection, and in an unblazed state. Using precision bore glass tubes (0.25, 0.5, 1, and 2 mm diameters) to simulate the high-energy surface of enamel, the flow characteristics of nine elastomers (the first stage replicates) and four epoxies (the second stage replicates) were determined at isobaric conditions. Because the laser spot size was relatively large (0.6 mm) and the pressure differential was small (25 mm Hg), both the global resolution and the low shear rate characteristics could be measured. Of the commercial materials tested, Reprosil Light had the best combination of fluidity and resolution, regardless of which positive material was used. Although Permagum Low, Silene Wash, and Xantopren Blue scored high in one of the two tests, none of these materials could compare to Reprosil Light within the context described herein.

Capillary Action↗
PubMed1999

Replication pattern in cancer: asynchronous replication in multiple myeloma and in monoclonal gammopathy.

In this study we evaluated the replication pattern and cell-cycle dynamics of cells from patients considered to have a premalignant condition (monoclonal gammopathy, or MGUS) and patients with multiple myeloma (MM), as well as healthy controls. We applied the fluorescence in situ hybridization (FISH) technique with the TP53, RB-1 and 21q22 loci on the patient's cells. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronic replication was significantly higher in the cells from MM patients, with intermediate value in the cells from MGUS, while the lowest rate was in cells from controls. We suggest that these results may reflect the changes in gene replication and cell-cycle progression that occur in premalignant and malignant cells.

Aged↗
PubMed1969

Studies on chloroplast development and replication in Euglena. I. Vitamin B12 and chloroplast replication.

When Euglena gracilis is grown under vitamin B(12) deficiency conditions, the amount of protein and of chlorophyll per cell increase with decrease of B(12) in the medium and consequently in the cell. The increase in cell protein is proportional to and precedes an increase in the number of chloroplasts per cell. This replication of the chloroplasts under deficiency conditions is not accompanied by nuclear or cell division. It is concluded that chloroplast replication in Euglena gracilis is independent of nuclear and cellular replication, at least under B(12) deficiency conditions. We established a graph of the growth of Euglena under different concentrations of vitamin B(12) added to the growth medium, which permitted us to calculate that at least 22,000 molecules of vitamin B(12) per cell are required to give normal growth.

Cell Division↗
PubMed2000

Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosis.

Animals↗
PubMed2000

Potent inhibition of human immunodeficiency virus type 1 replication by conditionally replicating human immunodeficiency virus-based lentiviral vectors expressing envelope antisense mRNA.

We describe an HIV-based lentiviral vector that expresses a 1-kb antisense mRNA directed against the HIV-1 mRNAs containing env sequences. The expression of antisense env mRNAs (envAS) does not inhibit the synthesis of p24 expressed from the HIV-1 helper plasmid used to package the vector, as this helper has a deletion in the env gene. This allows the production of high-titer VSV-G pseudotyped lentiviral particles. In challenge experiments using unselected populations of SupT1 cells transduced with this vector, a complete inhibition of HIV-1 replication was observed for long periods of in vitro culture, even at high HIV-1 infectious doses. The potent inhibition of HIV-1 replication by this vector correlated with a low occurrence of mobilization of the vector to previously untransduced cells. The infectivity of the wild-type HIV-1 that escapes inhibition was highly inhibited, suggesting that the vector is providing HIV-1 inhibition of replication not only due to its antisense effect but also by competing for encapsidation and mobilization to noninfected cells.

Blotting, Northern↗
PubMed1981

In vitro replication of bacteriophage GA RNA. Involvement of host factor(s) in GA RNA replication.

A certain factor(s) derived from Escherichia coli was found to extensively stimulate RNA synthesis by the RNA replicase of phage GA. This factor(s), named GA-HF (host factor(s) for GA RNA replication), was partially purified from an uninfected cell extract and characterized. In the presence of GA-HF, GA replicase synthesized 50-100 times more RNA than was synthesized in its absence, and was capable of synthesizing both the viral strand as well as its complementary strand. This factor(s) could not be replaced by HFI, which is necessary for the replication of Q beta RNA by Q beta replicase. In the presence of GA-HF, the GA RNA replication system has a characteristic template specificity. Group I and II phage RNAs, but none of the Group III and IV phage RNAs, showed template activity.

Carrier Proteins↗
PubMed1981

Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: comparison of amastigote replication in adherent and nonadherent macrophages.

Intracellular replication of Leishmania tropica was assessed in mouse peritoneal macrophage cultures. L. tropica replicated poorly in macrophage monolayers: both the percentage of cells infected and the number of intracellular amastigotes decreased with time in culture. In contrast, nonadherent macrophages supported continuous replication of the parasite, and intracellular amastigotes increased more than 10-fold in these cultures over 8 days.

Animals↗
PubMed2003

Intracellular replication of Mycobacterium marinum within Dictyostelium discoideum: efficient replication in the absence of host coronin.

Mycobacterium marinum causes tuberculosis-like disease in fish and amphibians and has been used as a model mycobacterial species because of its rapid growth and less stringent containment requirements relative to other mycobacterial species. We demonstrate here that M. marinum grows within Dictyostelium discoideum cells, allowing the genetic analysis of host factors that may modulate the replication of mycobacterial species. Intracellular growth of M. marinum was shown to mimic the properties previously observed for growth within cultured phagocytes. A defined bacterial mutant defective for growth within phagocytic cells was shown to be similarly defective for growth within D. discoideum. To test the role of host coronin, which was previously hypothesized to positively modulate mycobacterial growth within mouse macrophages, a defined D. discoideum coronin mutant was analyzed. Surprisingly, the absence of coronin resulted in enhanced intracellular replication of M. marinum relative to the control wild-type strain. Consistent with previous observations, some phagosomes showed persistence of coronin about the surface of the compartment, but colocalization of the protein was far from uniform. We conclude that in D. discoideum factors other than coronin support intracellular replication of M. marinum.

4-Butyrolactone↗
PubMed1970

Vegetative Replication and Transfer Replication of Deoxyribonucleic Acid in Temperature-Sensitive Mutants of Escherichia coli K-12.

Crosses were carried out at 34 C and 42 C between eight pairs of isogenic strains of Escherichia coli K-12. The donor and recipient of each pair carried the same mutation for temperature-sensitive deoxyribonucleic acid (DNA) synthesis; they differed only in the presence of F-lac in the donor and a spectinomycin-resistance marker in the recipient. A different temperature-sensitive mutation was present in each of the eight pairs, the eight temperature-sensitive mutations being located in at least two different genes. In all eight pairs, the transfer of F-lac occurred at high and equal rates at 34 C and 42 C, although vegetative DNA replication at 42 C was approximately 10(-4) of that at 34 C. The transfer of F-lac at 42 C was accompanied in seven of the eight crosses by an equivalent amount of DNA synthesis in excess of that observed in the unmated controls. The DNA synthesized during transfer at 42 C was characterized by equilibrium centrifugation in cesium chloride and by its sedimentation velocity in sucrose gradients. It was found to have a density and a molecular weight characteristic of F-lac DNA. A small proportion of the material labeled during transfer was recovered in the form of covalently closed DNA. It is concluded that vegetative replication of the chromosome and transfer replication of F are separate processes, the former requiring at least two gene products which are nonessential for the latter.

Journal Article↗
PubMed1978

Replication of bacteriophage M13. XIV. Differential inhibition of the replication of M13 and M13 miniphage in a mutant of Escherichia coli defective in the 5' leads to 3' exonuclease associated with DNA polymerase I.

Previous studies have shown that M13 single-strand synthesis is inhibited at nonpermissive temperature in Escherichia coli polAexl, a temperature-sensitive mutant defective in the 5' leads to 3' exonuclease activity of polymerase I (T.-C. Chen and D. S. Ray, J. Mol. Biol. 106:589-604, 1976). Under these conditions the formation of covalently closed replicative form (RF) molecules is greatly reduced, and miniature forms of RF accumulate. We show here that the accumulation of mini-RFs is the consequence of a differential inhibition of the replication of unit-length phage and preexisting miniphage rather than a de novo production of miniphage. Mini-RFs do not accumulate even after as many as nine cycles of growth in the mutant host infected only with unit-length phage. Mixed infections of the mutant host with plaque-purified unit-length phage and a single cloned miniphage show that discontinuities in the mini-RFs are joined with higher efficiency than are those contained in unit-length RFs. After a shift to nonpermissive temperature during single-strand synthesis in cells infected with plaque-purified phage alone, M13 RFs are found largely as RFII molecules (RF form having one or more single-strand discontinuities) containing only a single discontinuity in the viral strand. The inability of the accumulated unit-length RFII molecules to actively replicate may reflect the presence of either a bound protein or RNA primer on the 5' terminus of the viral strand and provides further support for the existence of distinct initiation and termination events in the synthesis of the viral strand.

Coliphages↗
PubMed1998

Graded Autocatalysis Replication Domain (GARD): kinetic analysis of self-replication in mutually catalytic sets.

A Graded Autocatalysis Replication Domain (GARD) model is proposed, which provides a rigorous kinetic analysis of simple chemical sets that manifest mutual catalysis. It is shown that catalytic closure can sustain self replication up to a critical dilution rate, lambda c, related to the graded extent of mutual catalysis. We explore the behavior of vesicles containing GARD species whose mutual catalysis is governed by a previously published statistical distribution. In the population thus generated, some GARD vesicles display a significantly higher replication efficiency than most others. GARD thus represents a simple model for primordial chemical selection of mutually catalytic sets.

Catalysis↗
PubMed1996

Conservation of the genetic switch between replication and transfer genes of IncP plasmids but divergence of the replication functions which are major host-range determinants.

The trfA operon of broad-host-range IncP plasmids is essential to activate the origin of vegetative replication in diverse species. The trb operon encodes most of the apparatus for mating pair formation, the first step in conjugative transfer. Comparison of the nucleotide sequence of the IncP beta plasmid R751 presented here with the equivalent IncP alpha sequence identifies conserved features of the organization and regulation of the trfA operon and the region controlling expression of the trb operon. As in IncP alpha plasmids, these operons are transcribed from a bidirectional promoter region consisting of trfAp for the trfA operon and trbAp and trbBp for the trb operon. The KorA-dependent switch between the trfA and trbA promoters is conserved as is the trbA gene encoding the third IncP global regulator. The intergenic region between trbA and trbB shows very little sequence identity between the two plasmids but the spacing, the KorB operator, the trbB promoter, and the existence of a hairpin loop (albeit of different actual sequence) which sequesters the trbB ribosome binding site are all conserved. The trfA operon encodes two ORFs. The first ORF is highly conserved and encodes a putative single-stranded DNA binding protein (Ssb). The second, trfA, contains two translational starts as in the IncP alpha plasmids, generating related polypeptides of 406 (TrfA1) and 282 (TrfA2) amino acids. TrfA2 is very similar to the IncP alpha product, whereas the N-terminal region of TrfA1 shows very little similarity to the equivalent region of IncP alpha TrfA1. This region has been implicated in the ability of IncP alpha plasmids to replicate efficiently in Pseudomonas aeruginosa. A TcR derivative of R751 was constructed and shown not to establish itself efficiently in P. aeruginosa at 37 degrees C, although it did establish itself inefficiently at lower temperatures, underlining the importance of this region in the adaptation of the plasmid to the host.

Amino Acid Sequence↗
PubMed2004

Identification and autonomous replication capability of a chromosomal replication origin from the archaeon Sulfolobus solfataricus.

Here, we describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). By means of a cumulative GC-skew analysis of the Sulfolobus genome sequence, a candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.

Archaea↗
PubMed1981

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA.

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.

Bacteriophage phi X 174↗
PubMed1984

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine.

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Animals↗