Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter

The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer. T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.     

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis.

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.     

Transfer of IncP Plasmids to Extremely Acidophilic Thiobacillus thiooxidans

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10^-5 to 10^-7 per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 × 10^-3 per recipient.     

The C-terminal residues of bacteriophage T7 gene 4 helicase-primase coordinate helicase and DNA polymerase activities

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.     

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.     

T7启动子-11碱基突变对转录影响的研究

T7噬菌体启动子能被T7RNA聚合酶和真核生物RNA聚合酶Ⅱ系统启动转录 ,为研究两个系统转录的关键碱基 ,将合成的T7噬菌体启动子 1 1变异体与报道基因CAT基因连在一起。体内CAT和体外狭缝RNA杂交实验显示 : 1 1碱基是T7RNA聚合酶和真核生物RNA聚合酶Ⅱ系统启动T7启动子的关键碱基之一。     

RNA polymerase binding sites on the broad host range plasmid RP4

Binding sites of Escherichia coli RNA polymerase on RP4 plasmid DNA were determined electron microscopically. Comparison of the RNA polymerase binding map and the genetic map of RP4 revealed several strong binding sites outside the well-known RP4 genes. RNA polymerase binding sites for the three antibiotic resistance genes were also detected. Two binding sites were observed for the tra-1 region, whereas the tra-2 and tra-3 regions showed no prominent affinity for RNA polymerase. The genomic regions for the replication origins, oriV (for vegetative replication) and oriT (for transfer replication, equivalent to rlx), both exhibited strong binding to RNA polymerase, as did genomic regions which code for trans-acting replication functions (trfA and trfB).     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system

Bacteriophage T7 lysozyme, a natural inhibitor of T7 RNA polymerase, can reduce basal activity from an inducible gene for T7 RNA polymerase and allow relatively toxic genes to be established in the same cell under control of a T7 promoter. Low levels of T7 lysozyme supplied by plasmids pLysS or pLysL, which are compatible with the pET vectors for expressing genes from a T7 promoter, are sufficient to stabilize many target plasmids and yet allow high levels of target protein to be produced upon induction of T7 RNA polymerase. Higher levels of lysozyme supplied by plasmids pLysE or pLysH reduce the fully induced activity of T7 RNA polymerase such that induced cells can continue to grow and produce innocuous target proteins indefinitely. Different configurations of the expression system can maintain several different steady-state levels of target gene expression. The presence of T7 lysozyme has the further advantage of facilitating the lysis of cells in preparing extracts for purification of target gene products.