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At least 19 recordsLinked to original sources
PubMed1990

In vivo analysis of the initiation of bacteriophage T7 DNA replication.

We have examined the initiation of bacteriophage T7 DNA replication in vivo using a pulse-labeling technique. The pulse-labeling technique permits the rapid identification of initiation sites on the T7 chromosome and a determination of the rate of movement of the replication fork. This technique has been used to analyze a number of phage mutants having alterations in the nucleotide sequence of the primary origin. The experiments confirm the results obtained by electron microscope analysis on the mapping of the primary origin region and demonstrate the requirement for a T7 promoter in the primary origin. The secondary origins were found to be located near the center and at the right end of the genome. Analysis of T7 phage harboring mutations in the essential replication genes of T7 shows that they fell into three classes. The first, including those mutated in genes 4 and 5, do not initiate DNA synthesis. The second, in genes 3, 6, and 1.2, initiate and elongate as wild-type phage, albeit some with lower rates of synthesis, during the first round of replication and then cease DNA synthesis. Mutations in gene 2 have no apparent effect on initiation or elongation.

DNA Replication↗
PubMed1978

The structure and maturation of intermediates in bacteriophage T7 DNA replication.

Concatemeric DNA from T7-infected cells consists of phage genomes in a linear head-to-tail arrangement. Adjacent genomes within a concatemer overlap for the length of the terminal repetition. Fast-sedimenting T7 DNA contains single-stranded regions at roughly unit-lentth intervals but these interruptions are heterogeneously distributed and do not occur at the genetic termini. Mutations in either bacteriophage genes 9, 18, or 19 (required for DNA maturation and packaging) lead to the synthesis and persistence of DNA with fewer interruptions than normal.

Base Sequence↗
PubMed1979

Electron microscopic analysis of partially replicated bacteriophage T7 DNA.

Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144--6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460--476, 1961; Doermann et al., J. Cell. comp. Physiol. 45[Suppl.]:51--74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.

DNA Replication↗
PubMed1977

Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA.

A complex form of bacteriophage T7 DNA, containing up to several hundred phage equivalents of DNA, arises during replication of T7. The complex was stable to treatment with ionic detergent, Pronase, and phenol. The complex form normally exists for only a short time, corresponding to the phase of rapid T7 DNA synthesis. It is then converted to shorter molecules, both concatemers and unit-size DNA. The complex was stable up to the temperature of denaturation of the bihelix. It consisted of a series of loops amanating from a dense central core, as shownby electron microscopy. The complex form is similar to the relaxed Escherichia coli folded chromosome ('nucleoid'). The loops contained an average of 0.7 to 0.8 phage equivalent of DNA. During infection by phage with an amber mutation in gene 3 (endonuclease), formation of the complex occurred normally, but its maturation to unit-size DNA blocked. Before treatment with phenol, the complex contained short fragments of newly replicated DNA. These were released as single-stranded pieces during phenol treatment. A pathway for T7 DNA replication is indicated in which the flow of material is from unit-size DNA to linear concatemers to the complex form, and then back to unit-size DNA by way of linear concatemers.

Coliphages↗
PubMed1988

Initiation of DNA replication at cloned origins of bacteriophage T7.

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Base Sequence↗
PubMed1987

Relative roles of T7 RNA polymerase and gene 4 primase for the initiation of T7 phage DNA replication in vivo.

Initiation sites of T7 phage DNA replication in the presence and absence of T7 phage gene 4 primase have been analyzed by using Escherichia coli cells infected with T7 phage amber mutants, T73,6 and T73,4,6, respectively. Restriction analysis of the [3H]thymidine-labeled DNA, synthesized by the T73,4,6 phage-infected cells in the presence of 2',3'-dideoxy-3'-azidothymidine, has shown that only the light (L) strand of T7 DNA has been synthesized from the primary origin area to the right. Transition sites from RNA to DNA have been located precisely in the primary origin region of the T7 phage genome. In the gene 4- condition, greater than 20 transition sites have been detected only in the L strand. They scattered widely downstream from the phi 1.1 promoters and mostly downstream from the phi 1.3 promoter. The same transition sites have been detected in the gene 4+ condition, suggesting that the transcripts started from these promoters are used as primers of the rightward L-strand DNA synthesis in the gene 4+ condition. In addition, many heavy (H)- and L-strand transition sites have been detected at gene 4 primase sites in the gene 4+ condition. The relative roles of T7 phage RNA polymerase and primase at the primary origin have been discussed.

Base Sequence↗
PubMed1979

Gene 2 protein of bacteriophage T7: purification and requirement for packaging of T7 DNA in vitro.

The gene 2 protein of bacteriophage T7 is required for a late stage of T7 DNA replication because T7 gene 2 mutants fail to form normal concatemeric structures during the processing of newly synthesized T7 DNA. Extracts of gene 2 mutant phage-infected cells are unable to package T7 DNA into phage heads to form viable phage, as determined by an in vitro packaging assay for T7 DNA. Packaging activity can be stimulated greater than 100-fold in mutant extracts by the addition of extract prepared from cells infected with phage carrying a wild-type T7 gene 2, thus providing a complementation assay for the gene 2 protein. With this assay, the gene 2 protein has been purified to approximately 50% homogeneity. Purified preparations of the protein inhibit the activity of Escherichia coli RNA polymerase but have little effect on the activity of T7 RNA polymerase but have little effect on the activity of T7 RNA polymerase. The requirement for the gene 2 protein during T7 DNA replication may involve inactivation of E. coli RNA polymerase because the antibiotic rifampicin, a specific inhibitor of E. coli RNA polymerase, can substitute for the gene 2 protein in the in vitro packaging assay.

DNA, Viral↗
PubMed1978

Purification and structures of recombining and replicating bacteriophage T7 DNA.

During the infection of Escherichia coli by bacteriophage T7, there is a gradual conversion of host DNA to T7 DNA. Recombination and replication occur during this time. We have devised a new way of examining the physical structures of the intermediates of these processes. It is based on the observation that there are no sites in T7 DNA susceptible to cleavage by the restriction endonuclease EcoRI. E. coli DNA, on the other hand, is susceptible to degradation by EcoRI. Thus, phage and host DNA can be separated by sucrose gradient centrifugation after treatment with EcoRI. Concatemeric T7 DNA contains a high proportion of branched, gapped, and whiskered structures. These appear to be intermediates of replication and recombination. This approach also monitors the conversion process from host to T7 DNA.

Coliphages↗
PubMed1973

Association of replicating bacteriophage T7 DNA with bacterial membranes.

Infection of Escherichia coli with bacteriophage T7 leads to the formation of an association between host membranes and newly synthesized T7 DNA. Evidence for this conclusion is suggested by the findings that replicating T7 DNA cosediments with host membranes in sucrose and cesium chloride density gradients. Furthermore, the sedimentation rate of T7 DNA is dependent on the integrity of membrane structure. Finally, an association between DNA and membranes can be demonstrated by electron microscope studies.

Carbon Radioisotopes↗
PubMed1985

Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins. Site and direction of initial DNA synthesis.

In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.

Base Sequence↗