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Coupling the T7 A1 promoter to the runaway-replication vector as an efficient method for stringent control and high-level expression of lacZ.

An expression vector characterized by tight regulation and high expression of cloned genes appears to be indispensable for the engineering need. To achieve this goal, in association with lacI the T7 A1 promoter containing two synthetic lac operators was constructed into a runaway-replication vector. To further examine this vector system, lacZ was subcloned and placed under the control of the T7 A1 promoter on the plasmid. With the application of the thermal induction alone, the Escherichia coli strain harboring the recombinant plasmid was able to produce 15,000 Miller units of beta-galactosidase, while it yielded the recombinant protein with 45,000-50,000 Miller units upon both thermal and chemical induction. In sharp contrast, only 60-90 Miller units of beta-galactosidase was obtained for the cell at an uninduced state. As a result, the production yield of beta-galactosidase over the background level is amplified approximately 170-fold by thermal induction and 500-fold by thermal and chemical induction. To produce the recombinant protein on a large scale, an approach by connecting two fermenters in series was newly developed. By applying the three-stage temperature shift in this dual fermenter system, 55,000 Miller units of beta-galactosidase was obtained. Overall, it shows the potential use of the vector system developed here for its tight control and high production of recombinant proteins.

Bacteriophage T7↗
PubMed1994

The role of 3-hydroxyethyldeoxyuridine in mutagenesis by ethylene oxide.

Ethylene oxide, a direct-acting mutagen and carcinogen, produces 3-hydroxyethyldeoxyuridine (3-HE-dU) after initial alkylation at N3 of dC, followed by rapid hydrolytic deamination. The significance of formation of 3-HE-dU in DNA was investigated by in vitro DNA replication of 3-HE-dU. A 55-nucleotide DNA template, containing 3-HE-dU at a single site, was constructed. DNA products, synthesized on the site-modified template, were analyzed and mutagenic bypass at 3-HE-dU estimated. The 3-HE-dU lesion blocked DNA replication by the Klenow fragment of Escherichia coli polymerase I (Kf Pol I) and bacteriophage T7 polymerase (T7 Pol) 3' to 3-HE-dU and after incorporating a nucleotide opposite 3-HE-dU. DNA synthesis past 3-HE-dU was negligible (< 3%). Substitution of Kf Pol I (exo-) and T7 Pol (exo-), polymerases lacking 3'-->5' exonuclease proofreading activity, for Kf Pol I and T7 Pol, respectively, facilitated DNA synthesis past 3-HE-dU. The bypass synthesis by Kf Pol I (exo-) was 60% and 90% by T7 Pol (exo-). These results suggest that the 3-HE-dU lesion could be bypassed, but that the extension at 3-HE-dU is rate-limiting. In the absence of proofreading, the nucleotide incorporated opposite 3-HE-dU is not excised and remains in position long enough for extension to occur. During post-lesion synthesis, both dA and dT were incorporated opposite 3-HE-dU. Since 3-HE-dU is derived from dC alkylation by ethylene oxide, incorporation of dA and dT opposite 3-HE-dU implicates this lesion in G.C-->A.T and G.C-->T.A mutagenesis.

Base Sequence↗
PubMed1986

Promoter and nonspecific DNA binding by the T7 RNA polymerase.

T7 RNA polymerase plays an important role in both the transcription and replication of bacteriophage T7. In this study we have used a nitrocellulose filter binding assay to examine the binding properties of the T7 RNA polymerase with T7 promoters cloned into plasmid DNAs. Promoter-specific binding was shown to be relatively insensitive to variations in the ionic strength of the incubation solution but dependent on the helical structure of the DNA. On the other hand, nonpromoter interior-site binding was independent of the superhelicity of the DNA but extremely sensitive to changes in the ionic strength. These results suggest that nonspecific binding results from ionic interactions between positively charged residues of the polymerase and the polyanionic backbone of the DNA, whereas promoter-specific binding is dependent upon base-specific contacts within the promoter sequence. A comparison between the transcriptional activity and binding strengths of the RNA polymerase to specific promoters indicates little correlation between these two properties. This suggests that differential promoter binding does not represent a major mechanism for regulating transcription in bacteriophage T7. Instead, factors which influence the efficiency or rate of formation of the polymerase-promoter open complex are found to have the major role in determining transcriptional levels in this system.

DNA, Viral↗
PubMed2007

Compensatory evolution in response to a novel RNA polymerase: orthologous replacement of a central network gene.

A bacteriophage genome was forced to evolve a new system of regulation by replacing its RNA polymerase (RNAP) gene, a central component of the phage developmental pathway, with that of a relative. The experiment used the obligate lytic phage T7 and the RNAP gene of phage T3. T7 RNAP uses 17 phage promoters, which are responsible for all middle and late gene expression, DNA replication, and progeny maturation, but the enzyme has known physical contacts with only 2 other phage proteins. T3 RNAP was supplied in trans by the bacterial host to a T7 genome lacking its own RNAP gene and the phage population was continually propagated on naive bacteria throughout the adaptation. Evolution of the T3 RNAP gene was thereby prevented, and selection was for the evolution of regulatory signals throughout the phage genome. T3 RNAP transcribes from T7 promoters only at low levels, but a single mutation in the promoter confers high expression, providing a ready mechanism for reevolution of gene expression in this system. When selected for rapid growth, fitness of the engineered phage evolved from a low of 5 doublings/h to 33 doublings/h, close to the expected maximum of 37 doublings/h. However, the experiment was terminated before it could be determined accurately that fitness had reached an obvious plateau, and it is not known whether further adaptation could have resulted in complete recovery of fitness. More than 30 mutations were observed in the evolved genome, but changes were found in only 9 of the 16 promoters, and several coding changes occurred in genes with no known contacts with the RNAP. Surprisingly, the T7 genome adapted to T3 RNAP also maintained high fitness when using T7 RNAP, suggesting that the extreme incompatibility of T7 elements with T3 RNAP is not an invariant property of divergence in these expression systems.

Bacteriophage T3↗
PubMed2005

A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.

A set of ligation-independent expression vectors system has been developed for co-expression of proteins in Escherichia coli. These vectors contain a strong T7 promoter, different drug resistant genes, and an origin of DNA replication from a different incompatibility group, allowing combinations of these plasmids to be stably maintained together. In addition, these plasmids also contain the lacI gene, a transcriptional terminator, and a 3' polyhistidine (6x His) affinity tag (H6) for easy purification of target proteins. All of these vectors contain an identical transportable cassette flanked by suitable restriction enzyme cleavage sites for easy cloning and shuttling among different vectors. This cassette incorporates a ligation-independent cloning (LIC) site for LIC manipulations, an optimal ribosome binding site for efficient protein translation, and a 6x His affinity tag for protein purification Therefore, any E. coli expression vector of choice can be easily converted to LIC type expression vectors by shuttling the cassette using the restriction enzyme cleavage sites at the ends. We have demonstrated the expression capabilities of these vectors by co-expressing three bacterial (dsbA, dsbG, and Trx) and also two other mammalian proteins (KChIP1 and Kv4.3). We further show that co-expressed KChIP1/Kv4.3 forms soluble protein complexes that can be purified for further studies.

Base Sequence↗
PubMed1994

Use of single-turnover kinetics to study bulky adduct bypass by T7 DNA polymerase.

The mechanism by which T7 DNA polymerase (exo-) bypasses N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) adducts was studied by single-turnover kinetics. These adducts are known to be mutagenic in several cell types, and their bypass was studied in the framework of understanding how they promote mutations. Synthetic primer/templates were made from a template sequence containing a single guanine, to which the adducts were covalently attached, and one of three primers whose 3' ends were various distances from the adduct in the annealed substrates. Upon approaching the site of either adduct, the polymerase was found to add nucleotides as rapidly as to unmodified primer/templates, until just opposite the lesion. The incorporation rate of dCTP (at 100 microM) opposite AF-dG or AAF-dG was approximately 5 x 10(4)- and 4 x 10(6)-fold slower, respectively, than incorporation at the same position into an unmodified primer/template. The polymerase dissociated from the sites of the adducts at approximately the same rate that it dissociated from unmodified DNA. Correct nucleotide incorporation was favored both opposite and immediately after AF-dG. However, at both positions, dATP was the most rapidly misincorporated nucleotide. Misincorporation of dATP was more rapid than correct nucleotide incorporation both opposite and immediately after AAF-dG. These results are discussed in terms of the effects of AF and AAF adducts in vivo.

2-Acetylaminofluorene↗
PubMed1992

Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.

The helicase and primase activities of bacteriophage T7 are distributed between the 56- and 63-kDa gene 4 proteins. The 56-kDa gene 4 protein lacks 63 amino acids found at the N terminus of the colinear 63-kDa protein and catalyzes helicase activity. The 63-kDa gene 4 protein catalyzes both primase and helicase activities. A bacteriophage deleted for gene 4, T7 delta 4-1, has been tested for growth by complementation on Escherichia coli strains that contain plasmids expressing either one or both of the gene 4 proteins. T7 delta 4-1 cannot grow (efficiency of plating, 10(-7)) on E. coli cells that express only 56-kDa gene 4 protein. In contrast, T7 delta 4-1 has an efficiency of plating of 0.1 on an E. coli strain that expresses only 63-kDa gene 4 protein in which glycine is substituted for methionine at position 64. A bacteriophage, T7 4B-, in which methionine at residue 64 is replaced by glycine, expresses only 63-kDa gene 4 protein. The burst sizes, latency periods, and Okazaki fragment sizes of T7 4B- are similar in the presence and absence of the 56-kDa gene 4 protein; however, T7 4B- has a reduced rate of DNA synthesis when compared with a phage that synthesizes both gene 4 proteins.

Amino Acid Sequence↗
PubMed1994

Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase.

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.

Base Sequence↗
PubMed1989

Mitochondrial RNA polymerase: dual role in transcription and replication.

Mitochondrial RNA polymerases from humans, Xenopus laevis and Saccharomyces cerevisiae are very similar in protein composition and function. They consist of a nonspecific core RNA polymerase and a protein factor that confers promoter selectivity on the core component, and they participate in transcription as well as in DNA replication. Amino acid sequence comparisons indicate that the yeast mitochondrial core component is related to bacteriophage T3 and T7 RNA polymerases; mitochondrial and phage polymerases may therefore belong to a family of related polymerases.

Animals↗
PubMed2004

Genome function--a virus-world view.

By studying viruses one may begin to understand how static genomes can define dynamic processes of development. This talk will describe some of the approaches we are taking, using computer simulations and laboratory experiments, to account for the many molecular-level processes and interactions that occur when a common bacterium, E. coli, is infected by one of its viruses, phage T7. We accounted for processes of phage genome entry, transcription, translation, and DNA replication, including protein-DNA and protein-protein regulatory interactions, and we predicted the dynamics of phage progeny formation. The simulations have enabled us to identify limiting host-cell resources in phage growth, discover novel anti-viral strategies, and suggest frameworks for mining data from global mRNA and protein studies.

Bacteriophage T7↗
PubMed1994

Continuous in vitro evolution of bacteriophage RNA polymerase promoters.

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Bacteriophage T3↗
PubMed1991

Overproduction of adenovirus DNA polymerase and preterminal protein in HeLa cells.

Adenovirus (Ad) DNA polymerase (AdPol) and the preterminal protein (pTP) form a complex that is involved in the in vitro initiation of Ad DNA replication. Recombinant vaccinia viruses (vv) were constructed in which the genes encoding AdPol and pTP were cloned into a vaccinia/T7 hybrid expression-based vector downstream from the T7 promoter (pT7)/encephalomyocarditis virus (EMCV) 5'-untranslated region (UTR). HeLa cells infected with the recombinant vv-AdPol or vv-pTP or a mixture of both, together with the vv expressing T7 RNA polymerase produced significant levels of pTP and AdPol which were biologically active in the in vitro initiation of Ad DNA replication. These amounts of pTP and AdPol were only about two-fold less than the levels produced in insect cells infected with the recombinant baculovirus constructs expressing AdPol and pTP.

Adenoviruses, Human↗
PubMed1998

Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydroguanine base pair mismatches by bacteriophage T7 DNA polymerase exo-.

8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases. The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair. Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation. Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation. Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively. Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue. Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair. Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.

Bacteriophage T7↗
PubMed1992

The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules.

A DNA copy of DI RNA of cymbidium ringspot tombusvirus was cloned downstream of a phage T7 promoter. In vitro-transcribed RNA replicated in Nicotiana clevelandii when co-inoculated with full-length viral genomic RNA transcripts and protected plants from apical necrosis. Artificial deletion mutants derived from the DI RNA clone showed that most of the central sequence block is necessary for replication. Hybrid DI RNA-satRNA clones were prepared and in vitro-synthesized RNA was inoculated to plants in the presence of helper viral RNA. There was replication only of in vitro transcripts derived from hybrid clones where satRNA sequences were inserted upstream or downstream from the central block, but not of those derived from clones where satRNA sequence replaced the central block. Progeny RNA of biologically active clones was either full-length or showed deletions depending on the insertion of satRNA sequences in DI RNA. DI RNA-satRNA constructs having part of the 5' region exchanged were not replicated.

Base Sequence↗
PubMed2000

Heteroduplex resolution using T7 endonuclease I in microbial community analyses.

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.

Artifacts↗
PubMed1992

N-acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded phi X174 in Escherichia coli.

Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP). The modified DNAs were used as templates for in vitro DNA synthesis. DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types of adducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase. In addition, all three types of adducts completely blocked replication of ss phi X174 in an E. coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage. Polyacrylamide gel electrophoresis of DNA fragments synthesized by E. coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template. However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-trifluoroacetyl-N-acetoxy esters) were frequently bypassed during replication of ss phi X174 in E. coli, though with different efficiencies: 1 out 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation. Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.

Aminobiphenyl Compounds↗
PubMed1982

Genetic analysis of two bacterial RNA polymerase mutants that inhibit the growth of bacteriophage T7.

The Escherichia coli mutants 7009 and BR3 are defective in the growth of bacteriophage T7. We have previously shown that both of these mutant hosts produce an altered RNA polymerase which is resistant to inhibition by the T7 gene 2 protein (De Wyngaert and Hinkle 1979). In both strains, the mutation which prevents T7 growth is closely linked to rifA (rpoB). Both mutants are complemented by transformation with a multicopy plasmid carrying rpoB and rpoC but not by a plasmid carrying only rpoB. This indicates that the mutations reside in rpoC, the structural gene for the beta' subunit of RNA polymerase. When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage lambda drifd18, the mutant allele is dominant over wildtype. The lambda drifd18 transductant also remains unable to support the growth of T7 in the presence of rifampin. This supports our conclusion that the mutation is in rpoC. We have measured the growth of T7 phage, the kinetics of phage DNA synthesis, and the structure of replicative DNA intermediates in several transductants, and compared these results with those obtained in the original mutant strains.

DNA↗