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PubMed2002

Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases.

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

Antineoplastic Agents↗
PubMed1984

Plasmid-phage recombination in T7 infected Escherichia coli.

Recombination between genetically marked T7 bacteriophage and plasmids containing inserts of T7 DNA has been studied in order to gain some insight into the phage recombination process. The results suggest that plasmid-phage recombination requires the products of T7 genes 3 (endonuclease), 4 (DNA primase), 5 (DNA polymerase), and 6 (exonuclease), as has been demonstrated previously for phage-phage recombination. Plasmid replication does not compensate for a complete block in phage polymerase synthesis, suggesting a direct role for this enzyme in recombination, rather than an indirect role, by means of producing replicative structures that are recombinogenic. In most respects, plasmid-phage recombination appears to be similar to phage-phage recombination. The participation of two autonomous, structurally dissimilar, homologues, however, might render certain aspects of the recombination process more amenable to analysis. As examples, the characterization of an apparent marker effect and the demonstration of genetic heterozygotes among the products of plasmid-phage recombination are presented.

DNA Primase↗
PubMed1991

A matched set of cat vectors for rapid mutational analysis of eukaryotic promoters and enhancers.

The eukaryotic cat expression vectors, pBRAMScat1 and pBRAMScat2, were constructed to simplify the analysis of genomic fragments containing putative transcriptional regulatory elements. These vectors contain the f1 filamentous phage origin of replication for single-stranded DNA rescue, and permit site-directed mutagenesis, and dideoxy sequencing of nested deletion mutants using commercial T3, T7 and M13 universal forward/reverse primers. The above features eliminate the need to shuttle back and forth between a conventional cloning vector and the cat expression vector during the analysis of putative eukaryotic gene regulatory elements. Plasmid pBRAMScat1 contains the bacterial chloramphenicol acetyltransferase-encoding gene (cat) and no eukaryotic promoter and was designed for the analysis of eukaryotic promoters. Plasmid pBRAMScat2 contains the cat gene under the control of the Herpes simplex virus thymidine kinase promoter and was designed for the analysis of eukaryotic enhancers.

Animals↗
PubMed1987

Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.

Cloning, Molecular↗
PubMed1988

Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC.

Transcription by RNA polymerase preceding the initiation of replication from the E. coli chromosomal origin (oriC) in vitro enables dnaA protein to open the DNA duplex under conditions when its action alone is insufficient. The RNA polymerases of phages T7 and T3 are as effective as the E. coli enzyme in activating initiation. The persistent RNA transcript hybridized to the template creates an R-loop that is responsible for activation. The activating RNA need not cross oriC, but must be less then 500 bp away. Transcripts lacking a 3' OH group are effective, proving that priming of DNA synthesis is not involved in the activation. Thus, transcription activates the origin of an otherwise inert plasmid by altering the local DNA structure, facilitating its opening by dnaA protein during the assembly of replication forks.

Bacterial Proteins↗
PubMed1998

A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.

A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

Blotting, Northern↗
PubMed1991

Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein.

Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.

Amino Acid Sequence↗
PubMed2002

Cloning and characterization of replication protein A p32 complementary DNA in zebrafish (Danio rerio).

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (70, 32, and 14 kDa) that is an essential component of the DNA replication fork. A complementary DNA encoding zebrafish RPA 32-kDa subunit was isolated by screening a zebrafish embryo lambda APII cDNA library with a human RPA p32 cDNA probe. The zebrafish RPA p32 cDNA consisted of 1097 bp encoding 272 amino acid residues. The deduced amino acid sequence shows high similarity to mouse and human RPA p32. In vitro phosphorylation of zebrafish RPA protein by Cdc2 kinase was shown. A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography. In this article, cloning of the zebrafish RPA p32 cDNA is reported in relation to the study of DNA replication in fish.

Journal Article↗
PubMed1996

A replication-deficient adenovirus enhances liposome-mediated nucleic acid transfer into a stable cell line expressing T7 RNA polymerase.

Liposome-mediated transfer of nucleic acids into a cell line expressing bacteriophage T7 RNA polymerase was enhanced by addition of a replication-deficient adenovirus (Ad5-259A) to transfection mixtures. Increasing quantities of Ad5-259A resulted in a dose-related (up to 30-fold) enhancement of reporter gene activity expressed in BT7-H cells transfected with plasmid DNA containing the reporter sequence fused to the internal ribosome entry site of encephalomyocarditis virus. Similarly, Ad5-259A enhanced reporter gene expression 7-fold following transfection of DNA containing the reporter sequence under transcriptional control of the Rous sarcoma virus long terminal repeat. Addition of Ad5-295A to transfection mixtures increased the proportion of cells staining positively for reporter gene activity, from 2 to 25% when the reporter was expressed via the T7 polymerase and from 20 to 50% when the reporter was under the control of a eucaryotic promoter. Thus, Ad5-259A enhanced reporter protein activities expressed by cytoplasmic T7-directed transcription and cap-independent initiation of translation, or nuclear transcription and cap-dependent translation. Transfection enhancement was blocked by neutralizing antibody to Ad5, and is most likely related to the endosome-disrupting activities of the virus. Adenovirus enhancement of liposome-mediated transfection provides a useful method for efficient nucleic acid transfer into eucaryotic cells.

Adenoviruses, Human↗
PubMed1991

pEXPRESS: a family of expression vectors containing a single transcription unit active in prokaryotes, eukaryotes and in vitro.

We have constructed a family of expression vectors containing a single transcription unit that is active in Escherichia coli, eukaryotic cells, and in coupled in vitro transcription-translation systems. These vectors use the Rous sarcoma virus-long terminal repeat (RSV-LTR) as the promoter/enhancer for eukaryotic cells. In vitro transcription is made possible by inclusion of a bacteriophage T7 promoter. This same promoter is actively transcribed in E. coli that produce T7 RNA polymerase. Other features of this transcription unit include a high-efficiency eukaryotic translation start codon, a phage f1 origin of DNA replication for site-directed mutagenesis and a three-frame stop codon that facilitates C-terminal deletion mutagenesis. We term this vector family, pEXPRESS.

Avian Sarcoma Viruses↗
PubMedDate not supplied

[Discontinuous transfer of phage T7 DNA molecules into Escherichia coli cells during infection].

HpaI restriction analysis of the part of T7 DNA molecule which comes off from E. coli after ultrasonic desorption of virion had been carried out. In such a way it was possible to follow the transfer of labelled T7 DNA into the host cell after the phage adsorption under different conditions. It was established that in the presence of chloramphenicol the left 60% of T7 chromosome is gradually (during 20 min) transferred into the cell and further transport is stopped. This suggests that some T7 gene(s) of I or (and) II class(es) is (are) necessary to transfer the last 40% of T7 DNA molecule containing the genes encoding capsid proteins. Also some new results are obtained which support thr idea about the tight coupling of the processes of T7 DNA transport and its transcription, and about the possibility for RNA polymerase to carry a mechanical function as well. All these results suggest a rather complicated mechanism of the process of T7 DNA transfer into the host cell consisting of at least three stages tightly connected with T7 gene expression temporal control. Some probable consequences of this model as well as its agreement with functional structure of T7 chromosome and with T7 development are discussed.

Chloramphenicol↗
PubMed1987

Computer simulation of T3/T7 phage infection using lag times.

A minimal mechanism is proposed which describes the transcriptional and translational processes for four phage proteins (RNA polymerase, DNase, primase and DNA polymerase) involved in T3/T7 DNA replication. Phage DNA replication is also included. It is shown how lag times may be incorporated into a kinetic mechanism. The distinct three-stage transport of phage DNA into the bacterial host (E. coli) is considered. DNA transport is assumed to be rate-determining for the transcription of class I and II proteins. Transcriptional and translational lag times have been calculated on the basis of available gene mapping of T7 phages. The kinetic behavior of T7 and T3 phage infection is practically identical. The hydrolysis of bacterial DNA by phage DNase (endonculease and exonuclease) as well as the subsequent phosphorylation to the deoxymononucleoside triphosphates are assumed to be rate-determining in phage DNA replication. Good agreement with experiment is obtained in our computer simulations.

Computer Simulation↗
PubMed1992

Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.

Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.

Avian Myeloblastosis Virus↗
PubMed2005

Dipeptide alcohol-based inhibitors of eukaryotic DNA polymerase alpha.

We reported previously that a novel dipeptide alcohol, l-homoserylaminoethanol (Hse-Gly-ol), is a selective inhibitor of eukaryotic DNA polymerase epsilon (pol epsilon) [Bioorg. Med. Chem.2004, 12, 957-962]. The discovery suggests that the dipeptide structure could be a chemical frame for a DNA polymerase inhibitor. Therefore, we chemically synthesized 27 different species of dipeptide alcohols, and tested this inhibitory capability. Compound 6 (l-aspartylaminoethanol, Asp-Gly-ol) was found to be the strongest pol alpha inhibitor. Compound 6 did not influence the activities of other replicative DNA polymerases such as delta and epsilon, and had no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. The inhibitory effect of compound 6 on pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 33.5 microM. Compound 6-induced inhibition of pol alpha activity was non-competitive with both the DNA template-primer and the dNTP substrate. This is the first report on a water-soluble pol alpha-specific inhibitor, sought for precise biochemical studies of pol alpha. The relationships between the structures of dipeptide alcohols and the inhibition of eukaryotic DNA polymerases are discussed.

Alcohols↗
PubMed2004

Protein interaction networks in bacteria.

The complement of expressed cellular proteins - the proteome - is organized into functional, structured networks of protein interactions that mediate assembly of molecular machines and dynamic cellular pathways. Recent studies reveal the biological roles of protein interactions in bacteriophage T7 and Helicobacter pylori, and new methods allow to compare and to predict interaction networks in other species. Smaller scale networks provide biological insights into DNA replication and chromosome dynamics in Bacillus subtilis and Archeoglobus fulgidus, and into the assembly of multiprotein complexes such as the type IV secretion system of Agrobacterium tumefaciens, and the cell division machinery of Escherichia coli. Genome-wide interaction networks in several species are needed to obtain a biologically meaningful view of the higher order organization of the proteome in bacteria.

Bacteria↗
PubMed2001

An error-prone T7 RNA polymerase mutant generated by directed evolution.

Viruses replicate their genomes at exceptionally high mutation rates. Their offspring evolve rapidly and therefore, are able to evade common immunological and chemical antiviral agents. In parallel, virus genomes cannot tolerate a further increase in mutation rate: Experimental evidence exists that even few additional mutations are sufficient for the extinction of a viral population. A future antiviral strategy might therefore aim at increasing the error-producing capacity of viral replication enzymes. We employed the principles of directed evolution and developed a scheme for the stringent positive selection of error-prone polymerase activity. A mutant T7 RNA polymerase with a nucleotide substitution error rate at least 20-fold greater than that of the wild-type was selected. This enzyme synthesized highly heterogeneous RNA products in vitro or in vivo and also decreased the replication efficiency of wild-type bacteriophage T7 during infection.

Bacteriophage T7↗