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PubMed1980

A prepriming DNA replication enzyme of Escherichia coli. I. Purification of protein n': a sequence-specific, DNA-dependent ATPase.

Protein n', an enzyme essential for in vitro conversion of single-stranded phiX174 DNA to the duplex replicative form, has been purified about 16,000-fold from Escherichia coli. The enzyme is a single polypeptide chain with a native molecular weight of 76,000; about 70 enzyme molecules are present in an E. coli cell. Nearly homogeneous preparations display an ATPase (dATPase) activity which depends on a unique sequence in the phiX174 DNA. Replicative activity of n' protein and its phiX174 DNA-dependent ATPase activity were present in a constant ratio during the latter stages of purification, upon sedimentation in a glycerol gradient, and during heat inactivation. Further studies of the properties of protein n' are presented in a succeeding paper.

Adenosine Triphosphatases↗

PubMed1984

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions.

The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis.

Bacteriophage phi X 174↗

PubMed1990

Simultaneous identification of viral proteins and nucleic acids in cells infected with Aleutian mink disease parvovirus.

A method combining in situ hybridization and immunohistochemistry was used to characterize cells infected with Aleutian mink disease parvovirus (ADV). Single-stranded RNA hybridization probes specific for obligate replicative intermediates and antisera specific for virion or non-structural proteins were employed. Crandell feline kidney cells in which the ADV-G strain of ADV was permissively replicating contained virion and non-structural proteins, large amounts of single stranded virion DNA, duplex replicative form (RF) DNA, and mRNA. Late in the infectious cycle, however, cells containing non-structural proteins but little nucleic acid were observed, probably representing cells in the end stage of viral cytopathology. Sections of lung prepared from mink kits infected with the ADV-Utah 1 strain were then examined. Alveolar type II cells permissively replicating ADV contained viral nucleic acids and proteins in patterns nearly identical to CRFK cells, suggesting that permissive ADV replication was similar in vitro and in vivo. Another population of ADV containing cells that had cytoplasmic virion antigen, but undetectable levels of non-structural protein was found in vivo. Furthermore, although virion DNA was present in the cytoplasm of these cells, RF DNA or mRNA could not be detected. These cells may have been alveolar macrophages sequestering viral particles.

Aleutian Mink Disease Virus↗

PubMed1977

RNA synthesized in calicivirus-infected cells is atypical of picornaviruses.

RNA labeled with [3H]uridine from Vero cells infected with San Miguel sea lion virus in the presence of actinomycin D was analyzed by glycerol density gradient sedimentation and polyacrylamide gel electrophoresis. The predominant single-stranded RNA (36S, 2.6 x 10(6) molecular weight) was genome size. There was also a prominent 22S, 1.1 x 10(6)-molecular weight, single-stranded component and one or more double-stranded or partially double-stranded classes. Replicative forms, sedimenting at 18S, contained single-stranded RNA corresponding to the larger-molecular-weight class. All classes of intracellular RNA and virion RNA were polyadenylated. These findings and results with pig kidney cells infected with vesicular exanthema of swine virus and feline cells infected with feline calicivirus indicate that caliciviruses exhibit a strategy of replication different from typical picornaviruses and supports removal of the caliciviruses from the family Picornaviridae.

Animals↗

PubMed1975

R17 RNA replicase. V. Rifamycin sensitivity of R17 RNA synthesis in vivo.

The effect of rifamycin on R14 phage growth in vivo was examined using rifamycin sensitive and resistant strains of E. coli as host cells. The following conclusions were obtained: (1) There is a time lag between the addition of rifamycin to the culture and the initiation of inhibitory action by the drug. At 25 mug rifamycin per ml, this time lag is approximately 30 min. At 50 mug per ml, it takes 10 min to exert 90% inhibition of RNA synthesis in both infected and non-infected cultures. (2) The rifamycin sensitive stage for R17 growth is the first 20 min of its infectious cycle during which time the synthesis of phage components, but not the assembly, takes place. (3) Of the phage component synthesis, RNA synthesis is definitely sensitive to rifamycin in contrast to the other RNA phage systems. Of the four phage specific RNA's, progeny plus strand synthesis is trongly inhibited. The synthesis of replicating forms is also sensitive to rifamycin, but their suppression appears to be incomplete.

Centrifugation, Density Gradient↗

PubMed2000

CTXphi contains a hybrid genome derived from tandemly integrated elements.

CTXphi is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5' prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3' element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXphi, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXphi DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXphi-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXphi does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXphi to new hosts.

Bacteriophages↗

PubMed1984

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

Acridines↗

PubMed1968

Protein synthesis directed by an arbovirus.

In contrast to chick embryo fibroblast protein synthesis, the bulk of the protein synthesis directed by Semliki Forest virus is carried out on membranes. Under conditions where more than 95% of cell protein synthesis was inhibited, viral polysomes could be demonstrated. Viral protein appeared to be produced on polysomes associated with nascent ribonucleic acid strands still attached to the base-paired, double-stranded replicative form of the virus. Very rapid incorporation of virus protein into 140S virus core particles was also demonstrated.

Animals↗

PubMed1971

Requirement of protein synthesis for bacteriophage phi X174 superinfection exclusion.

Within 5 to 10 min at 37 C, bacteria infected by bacteriophage phiX174 acquire the ability to exclude superinfecting phiX particles from reproducing themselves. The superinfecting phage are blocked at a stage prior to synthesis of the parental replicative form molecule; the superinfecting deoxyribonucleic acid remains as intact (infective) single strands. Establishment of superinfection exclusion and its maintenance require protein synthesis.

Bacteria↗

PubMed1990

Mechanisms of mutagenesis by the vinyl chloride metabolite chloroacetaldehyde. Effect of gene-targeted in vitro adduction of M13 DNA on DNA template activity in vivo and in vitro.

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogenic industrial chemical vinyl chloride, reacts with single-stranded DNA to form the cyclic etheno lesions predominantly at adenine and cytosine. In both ethenoadenine and ethenocytosine, normal Watson-Crick hydrogen-bonding atoms are compromised. We have recently shown that CAA adduction leads to efficient mutagenesis in Escherichia coli predominantly at cytosines, and less efficiently at adenines. About 80% of the mutations at cytosines were C-to-T transitions, and the remainder were C-to-A transversions, a result similar to that of many noninstructional DNA lesions opposite which adenine residues are preferentially incorporated. It is widely believed that noninstructional lesions stop replication and depend on SOS functions for efficient mutagenesis. We have examined the effects of in vitro CAA adduction of the lacZ alpha gene of phage M13AB28 on in vivo mutagenesis in SOS-(UV)-induced E. coli. CAA adduction was specifically directed to a part of the lacZ sequence within M13 replicative form DNA by a simple experimental strategy, and the DNA was transfected into appropriate unirradiated or UV-irradiated cells. Mutant progeny were defined by DNA sequencing. In parallel in vitro experiments, the effects of CAA adduction on DNA replication by E. coli DNA polymerase I large (Klenow) fragment were examined. Our data do not suggest a strong SOS dependence for mutagenesis at cytosine lesions. While adenine lesions remain much less mutagenic than cytosine lesions, mutation frequency at adenines is increased by SOS. SOS induction does not significantly alter the specificity of base changes at cytosines or adenines.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde↗

PubMed1997

Growth of the parvovirus minute virus of mice MVMp3 in EL4 lymphocytes is restricted after cell entry and before viral DNA amplification: cell-specific differences in virus uncoating in vitro.

Two murine parvoviruses with genomic sequences differing only in 33 nucleotides (8 amino acids) in the region coding for the capsid proteins show different host cell specificities: MVMi grows in EL4 T lymphocytes and MVMp3 grows in A9 fibroblasts. In this study we compared the courses of infections with these two viruses in EL4 cells in order to investigate at which step(s) the infection process of MVMp3 is interrupted. The two viruses bound equally well to EL4 cells, and similar amounts of MVMi and MVMp3 input virion DNA appeared in the nuclear fractions of EL4 cells 1 h after infection. However, double-stranded replicative-form (RF) DNA of the two viruses appeared at different times, at 10 h postinfection with MVMi and at 24 h postinfection with MVMp3. The amount of MVMp3 RF DNA detected at 24 h was very small because it was produced only in a tiny subset of the population of EL4 cells that proved to be permissive for MVMp3. Replication of double-stranded viral DNA in EL4 cells was measured after transfection of purified RF DNA, cloned viral DNA, and cloned viral DNA with a mutation preventing synthesis of the capsid proteins. In each of these cases, DNA replication was comparable for MVMi and MVMp3. Production of virus particles also appeared to be similar after transfection of the two types of RF DNA into EL4 cells. Conversion of incoming 32P-labeled single-stranded MVM DNA to 32P-labeled double-stranded RF DNA was detected only after RF DNA amplification, indicating that few molecules serve as templates for viral DNA amplification. We showed that extracts of EL4 cells contain a factor which can destabilize MVMi virions but not MVMp3 by testing the sensitivity of viral DNA to DNase and by CsCl gradient analyses of viral particles. We therefore conclude that the MVMp3 life cycle is arrested after the transport of virions to the nucleus and prior to the replication of RF DNA, most likely at the stage of viral decapsidation.

Animals↗

PubMed1996

Gene expression and active virus replication in the liver after injection of duck hepatitis B virus DNA into the peripheral vein of ducklings.

BACKGROUND/AIMS: Duck hepatitis B virus is a member of the hepadnavirus family, which possesses strong hepatotropism. Duck hepatitis B virus DNA serves as a replicative template for producing biologically active virus particles after transfection into cell lines established from human hepatocellular carcinoma or into duck liver by direct injection of calcium phosphate-precipitated DNA. Our aim was to develop a new method of liver-specific gene expression after intravenous DNA delivery. METHODS/RESULTS: We inoculated duck hepatitis B virus DNA with and without cationic liposomes, Lipofectin or LipofectAMINE, as DNA carries. Two weeks after a single intravenous injection of 10 or 50 micrograms of plasmid DNA containing a head-to-tail dimer of duck hepatitis B virus DNA into 25 one-day old ducklings, duck hepatitis B virus RNA transcripts including the pregenome replicative intermediate were detected by Northern blot in the liver of eight ducks (100%) of the Lipofectin group, five ducks (63%) of the LipofectAMINE group, and three ducks (50%) of the group which received DNA without carrier. Duck hepatitis B virus RNA transcription was almost exclusively liver specific, even though the liposomes had no tissue specificity. Replicative forms of duck hepatitis B virus DNA were detected in the liver and DHBsAg was observed in the cytoplasm of the hepatocytes by immunostaining. The serum of transfected ducklings contained virus particles which were infectious in other ducklings. CONCLUSION: The efficient and liver-specific expression of inoculated DNA was due to the amplification of nucleic acids by active virus replication process under the control of hepatocyte specific regulation.

Animals↗

PubMedDate not supplied

Preferential transfection with M13mp2 RF DNA synthesized in vitro.

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS leads to DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and S1 endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.

Base Composition↗

PubMed1982

Enzymatic synthesis of bacteriophage fd viral DNA.

An enzyme system with requirements similar to those for replication of phage fd replicative form (RF) DNA in bacteriophage fd-infected cells has been reconstituted with purified fd gene 2 protein, and DNA polymerase III holoenzyme, DNA binding protein I and rep-protein (rep-helicase) of Escherichia coli. The system generates viral circular single strands, which are infective for E. coli spheroplasts. Parental and newly synthesized DNA are covalently connected in early stages of replication, as expected for DNA replication using the rolling circle mechanism. Single-stranded tails of the rolling circle intermediates are cleaved after a full round of replication by gene 2 protein and circularized by the same enzyme molecule.

Carrier Proteins↗

PubMed1996

Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5')-deoxyadenosine, in Escherichia coli under SOS conditions.

The biological activity of TA*, the major photoproduct of thymidylyl-(3',5')-deoxyadenosine, has remained speculative since it was identified a decade ago. To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil-containing (+)-strand of the phage. The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand. TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic. Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants. The most abundant mutation was a 3'A-->T substitution (31/46, 56%). The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*.

Bacteriophage M13↗

PubMed1993

Complete nucleotide sequence of sugarcane streak Monogeminivirus.

The complete nucleotide sequence of the genome of the Monogeminivirus sugarcane streak virus (SSV) was determined from cloned replicative form DNA. The genome is contained in one DNA circle of 2,758 nucleotides, and has four open reading frames with the potential to encode proteins of MW > 10 kDa: two in the viral (+) sense and two in the complementary (-) sense. Each open reading frame has a counterpart among the open reading frames reported for other Monogeminiviruses. A potential binding site for a DNA replication primer and potential transcriptional control sequences were identified on the (+) strand, and a possible intron on the (-) strand. Phylogenetic analysis of coat protein and replication-associated protein sequences of SSV and other grass-infecting geminiviruses indicate that SSV, although distinct from any other virus, is part of an "African streak virus subgroup" of Monogeminiviruses.

Base Sequence↗

PubMed1984

Examination of the mode of action of ribavirin against tobacco mosaic virus.

Ribavirin inhibited a specific step of tobacco mosaic virus (TMV) replication. Time-courses of sensitivity of TMV multiplication in asynchronous and synchronous infections demonstrated that ribavirin inhibited an early function that occurs prior to the period of viral RNA and protein synthesis. In the synchronous infection, ribavirin inhibited the synthesis of both single-stranded genomic RNA and double-stranded replicative form RNA when treatment began during the first 4 h, but there was no inhibition when treatment began at 12 h or later, even though most RNA synthesis occurred after 12 h. Viral protein synthesis also was inhibited when treatment began during the first 4 h and not when treatment began later. Unlike the mechanisms proposed against animal viruses, ribavirin appears to inhibit TMV replication by preventing an early function that is necessary to initiate viral RNA synthesis.

Kinetics↗

PubMed1976

Bacteriophage phiX174 single-stranded viral DNA synthesis in temperature-sensitive dnaB and dna C mutants of Escherichia coli.

We asked if phiX174 single-stranded DNA synthesis could reinitiate at the nonpermissive temperature in dnaB and dnaC temperature-sensitive host mutants. The rates of single-stranded DNA synthesis were measured after the removal of chlorampheicol that had been added at various times after infection to specifically stop this stage of phiX174 DNA synthesis. Reinitiation was not defective in either mutant host. Our data suggested that the reinitiation of the single-stranded stage of phiX174 DNA synthesis in these experiments was analogous to the normal initiation of this stage of phiX174 DNA synthesis in infections without chloramphenicol. Assuming this to be the case, we conclude that the host cell dnaB and dnaC proteins are not essential for the normal initiation of the single-stranded synthesis stage of phiX174 DNA synthesis. In related experiments we observed that in the dnaC mutant host at the permissive temperature, phiX174 replicative form DNA synthesis continued at its initial rate even during the single-stranded DNA synthesis stage. This indicates that these two stages of phiX174 DNA synthesis are not necessarily mutually exclusive.

Bacterial Proteins↗