Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication.

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.     

Stimulation of T7 DNA polymerase by a new phage-coded protein

Summary A bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton unwinding protein of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.     

Host-dependent modification of bacteriophage T7 and SAMase-negative T3 derivatives affecting their adsorption ability

Summary When passaging phage T7 and SAMase-negative T3 mutants betweenE. coli strains with identical (EcoB) or without (EcoO) DNA host specificity, phenotypically a host-controlled modification and restriction is observed. This phenomenon is not due to classical modification and restriction of the bacteriophage DNA but depends on the reversibly altered adsorption capacity of the phages on the different host strains.     

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

Summary 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 subunit of RNA polymerase. When a single copy of the wildtype rpoC allele is introduced into the mutant using the transducing phage drif ^d18, the mutant allele is dominant over wildtype. The drif ^d18 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.     

Active protection by bacteriophages T3 and T7 againstE. coli B-and K-specific restriction of their DNA

Summary The bacteriophages T3 and T7 are not modified and restricted byE. coli strains with different host specificity (E. coli B, K, O) in vivo. The phages code for a gene product with the ability toovercomeclassicalrestriction (ocr):ocr ^– mutants are subject to modification and restriction via DNA methylation vs cleavage. The T3 genome possesses recognition sites for the restriction endonuclease R.EcoB which, unless the DNA is B-specifically modified, trigger 5–7 DNA cleavages. Theocr gene function of T3 and T7 is located within the gene 0.3 region of these phages and is not identical with thesam (SAMase) function of T3. The mechanisms ofocr protection remains unclear, while it is certain that this protection by the gene 0.3 protein is exerted in the infected cell and not through over-all modification in the preceding growth cycle of the phage.     

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced β-myosin heavy chain gene expression and cardiomyocyte hypertrophy

Endothelin-1 (ET-1) has been found to increase cardiac -myosin heavy chain (-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced -MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced -MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for ³H-leucine incorporation and -MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and -MyHC gene expression. ET-1 increased ³H-leucine incorporation and -MyHC promoter activities, which were blocked by the specific ET_A receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced ³H-leucine incorporation, -MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH₂ -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased ³H-leucine incorporation and -MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced -MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the -MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced -MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and -MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and -MyHC expression. Tzu-Hurng Cheng, Neng-Lang Shih: These authors have equally contributed to this work     

Elevated temperature and chemical modification selectively abolishes levan forming activity of levansucrase of Zymomonas mobilis

A levansucrase (SacB) of Zymomonas mobilis was purified to electrophoretic homogeneity from a recombinant Escherichia coli. The 55 kDa enzyme hydrolysed -fructosides but not -glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity (30°C ) was lower than that for hydrolase activity (50°C ). In contrast to other levansucrases, polymerase activity of levansucrase was inhibited by para- chloromercuribenzoate (1 mM) but with little or no effect on hydrolase activity. Selective modulation of polymerase activity by this inhibitor will be useful in revealing the mechanism of levansucrase catalysis.     

In vitro studies on the role of phage T7 gene 4 product in DNA replication

Summary A soluble enzyme fraction prepared from T7-infected E. coli is able to initiate DNA synthesis on circular single-stranded phage DNA. The product synthesized in vitro is a full-length linear complementary strand as judged by alkaline sucrose gradient analysis. DNA synthesis requires the products of the phage genes 4 and 5, Mg⁺⁺, dNTPs and rNTPs; however, ATP by itself can almost completely satisfy the rNTP requirement. The gene 4 product is essential for DNA chain initiation on unprimed single-stranded DNA, but is dispensable for the replication of a X174 DNA-RNA hybrid. The enzyme system from T7-infected cells does not discriminate between the DNA templates from phages X174, M13 or fd and is also capable of replicating native T7 DNA. However, a striking difference with regard to the template DNA is revealed by complementation analysis. Extracts of T7 mutant-infected cells complement each other only with T7 DNA but not with X174 DNA as template.Abbreviations rNTP ribonucleoside triphosphate - dNTP deoxyribonucleoside triphosphate - BSA bovine serum albumin     

In vivo and in vitro "phenotypic mixing" with amber mutants of phages T3 and T7

Summary Gene 17 of phage T7, and a homologous gene of T3, were shown to code for the serum blocking power protein (SBP). Noninfective T7 particles lacking gene 17 product (SBP-less particles) could be rendered infective by incubation with extracts of nonpermissive host bacteria infected with amber mutants of T7, as well as of T3, defective in other genes. SBP-less T7 particles activated by extracts of T3-infected cells were characterized as coat chimeras by their specificity towards anti-T3 and anti-T7 sera.     

Optimizing recombinant protein expression in the T7 system under the control of the proUp promoter

-Galactosidase and streptokinase expression was tested under the control of the T7 promoter in batch and fed-batch cultures. An Escherichia coli host GJ1158, which contained the T7 RNA polymerase gene under the osmo-responsive proUp promoter, was used for expression studies. -Galactosidase expression was enhanced from 26 mg l^–1 to 127 mg l^–1 in batch culture when a combination of sucrose and sorbitol was used instead of salt as an inducer. Similarly in fed-batch cultures 140 mg streptokinase l^–1 was formed with sucrose and sorbitol induction which was higher than that achieved with IPTG induced cultures.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Effect of busulphan treatment and elevated temperature on the expression of the β-pol gene in rat testis

Changes in the expression pattern of the DNA polymerase gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of -pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of -pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptochidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of -pol mRNA. Involvement of DNA polymerase in meiotic recombination is discussed.     

Transfection of epithelioma papulosum cyprini (EPC) carp cells

The variables involved in the transfection of epithelioma papulosum cyprini (EPC) cells (a representative carp fish cell line) with the genes for -galactosidase from E. coli, for luciferases from firefly or renilla, for the G protein of viral haemorrhagic septicemia virus or for green fluorescent protein under the cytomegalovirus, the SV40 or the T7 polymerase promoters have been studied. Fugene was selected among 10 transfection different reagents because it is simpler to use and it induced maximum efficiences of transfection of 37% equivalent to 10–15 ng -galactosidase per 500000 EPC cells.     

Structural and Functional Rearrangements of Mesophyll as a Probable Basis for the Cytokinin-Dependent Assimilate Translocation in Detached Leaves

The effects of benzyladenine (BA) on the mesophyll functioning, such as osmotic potential (), the effect of the inhibitors of ⁺-ATPase on the influx of ¹⁴C-sucrose, the direction of carbon metabolism, and the rate of dark respiration, were followed in the detached leaves of pumpkin (Cucurbita pepo L.) and broad beans (Vicia faba L.). BA elevated and established a gradient of (_p) between the treated and untreated leaf regions. The inhibitors of H⁺-ATPase did not affect the BA-induced influx of ¹⁴C-sucrose. The changes were accompanied with the elevated synthesis of starch and other polymeric compounds and the diminished synthesis of the substances of relatively low molecular weight. The stimulation of dark respiration was short and inconsiderable. The author concludes that the BA-induced transport was a passive process related to a increase. Leaf expansion accompanied by the synthesis of high-molecular-weight substances essential for cell growth and by starch synthesis apparently increased the sink capacity of the BA-treated detached leaves. The diminished efflux from the leaf blade was probably related to a lowered level of the transportable carbon compounds restricting their entry into the phloem. The influx induction could result from the activation of growth and metabolic processes, the decline in the number of organic molecules per cell volume unit, and the development of _p between the source and sink leaf regions.     

Neisseria gonorrhoeae IgA protease. Secretion and implications for pathogenesis

A cloned 5 bk DNA fragment from Neisseria gonorrhoeae strain MS11 promotes expression and excretion of IgA protease in E. coli and other Gram-negative hosts. DNA sequencing reveals a large open reading frame coding for a prcursor molecule of 169 kd. The 106 kd mature IgA protease is released from the bacteria in conjunction with a 15 kd soluble precursor segment, the -protein. In contrast, the carboxy terminal portion of the precursor, the -protein (45 kd), remains associated with the outer bacterial membrane. The three proteins result form autoproteolytic cleavage at sites in the precursor which are similar to the target site in IgA1. Consensus sequences of the specific cleavage sites are found in a number of relevant human proteins. IgA protease may therefore have other natural substrates besides IgA1. The soluble -protein as well as the membrane bound -protein, both associated with IgA protease, may confer additional virulence functions to the gonococcus.     

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila

Summary We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the -amanitinresistant allele, RpII215 ^C4 , includes all sequences required to produce amanitin-resistant transformants.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

Gene 4 protein of bacteriophage T7. Purification physical properties, and stimulation of T7 DNA polymerase during the elongation of polynucleotide chains.

With the use of an in vitro complementation assay to measure activity, the gene 4 protein of bacteriophage T7 has been purified 1000-fold to yield a nearly homogeneous protein. The purified gene 4 protein is a single polypeptide having a molecular weight of 58,000. In addition to being essential for T7 DNA replication in vivo and in vitro, the gene 4 protein is required for DNA synthesis by the purified T7 DNA polymerase on duplex T7 DNA templates. In the absence of ribonucleoside 5'-triphosphates, DNA synthesis by the gene 4 protein and the T7 DNA polymerase is dependent on phosphodiester bond interruptions containing 3'-hydroxyl groups (nicks) in the duplex DNA. The reaction is specific for the T7 DNA polymerase, but any duplex DNA containing nicks can serve as template. The Km for nicks in the reaction is 3 x 10(-10) M.