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Biomedical subjects

A Ishihama

Publications and source records attributed to A Ishihama.

At least 19 recordsLinked to original sources
PubMed1992

Stimulation of the phage lambda pL promoter by integration host factor requires the carboxy terminus of the alpha-subunit of RNA polymerase.

Escherichia coli integration host factor (IHF) binds with high affinity to two tandem IHF consensus sequences located upstream from the pL promoter of bacteriophage lambda. IHF was shown to stimulate transcription initiation from the pL promoter by increasing close complex formation (KB). We show here, by the use of reconstituted mutant RNA polymerases, that the C-terminal portion of the alpha subunit of RNA polymerase plays an essential role in the stimulation of transcription by IHF. Our results are in agreement with the hypothesis that IHF, like the cAMP-CRP activator, increases the affinity of RNA polymerase to the promoter by protein-protein interaction.

Bacterial Proteins↗
PubMed1992

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus.

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.

Animals↗
PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗
PubMed1992

Identification of Escherichia coli proteins cross-reacting with antibodies against region 2.2 peptide of RNA polymerase sigma subunit.

Antisera against a synthetic tetradecameric peptide with the sequence DLIQEGNIGLMKAV, which is present in region 2.2 of both sigma 70 and sigma 32 subunits of Escherichia coli RNA polymerase, cross-reacted with more than 10 E. coli proteins including these two sigma subunits. Four major species of these cross-reacting proteins (SCRPs) were purified. N-Terminal amino acid sequence analysis revealed that one of them (SCRP-27A) was an as yet unidentified protein while the other three (SCRP-34, SCRP-27B and SCRP-23) were thioredoxin reductase, ribosomal protein S2, and alkyl hydroperoxide reductase, respectively. Immunological competition experiments with various fragments of this sigma region 2.2 peptide indicated that the anti-sigma peptide serum contained at least three different species of antibodies. All the four SCRPs analyzed here reacted with an antibody against a C-terminus-proximal epitope.

Amino Acid Sequence↗
PubMed1992

Promoter selectivity of the stationary-phase forms of Escherichia coli RNA polymerase and conversion in vitro of the S1 form enzyme into a log-phase enzyme-like form.

Upon growth transition of Escherichia coli cells from exponential to stationary phase, RNA polymerase is converted into at least three different forms (S1, S2 and S3), which could be separately isolated by phosphocellulose column chromatography (Ozaki et al., 1991 (2)). Here, the promoter selectivity of these three stationary-phase enzymes was examined using an in vitro mixed transcription system and an E. coli promoter collection. These altered forms of RNA polymerase showed different recognition properties of promoters from that by the log-phase holoenzyme (L1). One of the stationary-phase RNA polymerases, S1, was found to be converted in vitro into an enzyme like the log-phase form following incubation with nucleotides or pyrophosphate. The conversion was indicated by not only the shift of elution position from a phosphocellulose column but also the change in the promoter selectivity. These results may suggest that RNA polymerase is interconvertible between different forms with different promoter selectivity by interaction with a phosphorylated compound(s).

Adenosine Triphosphate↗
PubMed1992

Sequence determination of cDNA clones of transcripts from the tumor-associated region of the Marek's disease virus genome.

The number of 132-bp tandem direct repeats within the long inverted repeat region of the Marek's disease virus type 1 (MDV1) genome increases concomitantly with the loss of oncogenicity during serial passages in cultured cells. Twelve clones carrying the 132-bp sequence were isolated from a cDNA library constructed from chicken embryo fibroblasts infected with the MDV1 Md5 strain. Through sequence analysis of a cDNA clone and primer extension analysis, the corresponding mRNA was found to be a linear transcript which included the two 132-bp tandem direct repeats. Two open reading frames were found in this transcript. One had a week homology with v-fms. The other should increase its size concomitantly with expansion of the 132-bp tandem direct repeat. PCR analysis of both cDNA clones and RNA gave amplified products which were as large as that produced from the genomic clone, indicating that a majority of mRNA from this region is composed of unspliced transcripts.

Amino Acid Sequence↗
PubMed1992

Enzymatic properties of the mouse Mx1 protein-associated GTPase.

Murine Mx1 protein, an interferon-inducible nuclear protein present in inbred mouse Mx+ strains, confers resistance to influenza virus infection. The purified Mx1 protein was found to carry the activities of both GTPase and GTP-binding. Enzymatic properties of the Mx1-associated GTPase were examined using the Mx1 protein purified from Escherichia coli expressing Mx1 cDNA. The Mx1 protein exhibited a substrate preference for GTP. The Vmax of ATP hydrolysis was about 7.6% the rate of GTP hydrolysis. The hydrolysis of CTP and UTP was virtually negligible. The Km for GTP hydrolysis was 667 microM and the rate was 13.8 mol GTP hydrolysis per min per mol Mx1 protein. The enzymatic properties of Mx1 protein-associated GTPase were compared with those of the GTPase super-gene family and the Mx-related family.

Adenosine Triphosphate↗
PubMed1992

Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system.

Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.

Animals↗
PubMed1992

Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.

The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.

Base Sequence↗
PubMed1992

Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes.

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.

Animals↗
PubMed1992

Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase.

The C-terminal region (amino acid residues 236-329) of the Escherichia coli RNA polymerase alpha subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C-terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant alpha subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP-CRP. DNA sequence determination of several 'Lac-' mutant rpoA genes revealed that all had mutations clustered within a short segment near the C-terminus of alpha, between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).

Bacterial Proteins↗
PubMed1992

Role of the RNA polymerase alpha subunit in transcription activation.

The N-terminal two-thirds of the alpha subunit of Escherichia coli RNA polymerase plays an essential role in the initiation of subunit assembly, by gathering two large subunits, beta and beta', together into a core-enzyme complex. One group of RNA polymerase mutants deficient in response to transcription activation carries mutations in the C-terminal region of the alpha subunit, indicating that the C-terminal region of the alpha subunit is involved in protein-protein contact in positive control of transcription. A set of activators (class I transcription factors) which make contact with this contact site I region on RNA polymerase alpha subunit bind in most cases to DNA upstream of the promoter -35 signal. Genetic fine mapping indicates that a cluster of subsites exists in the contact site I region, each interacting with a set of the class I factors and each consisting of a structure formed by only 5-10 amino acid residues.

Bacterial Proteins↗
PubMed1992

Activation defects caused by mutations in Escherichia coli rpoA are promoter specific.

Escherichia coli RNA polymerases containing mutated alpha subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two alpha (rpoA) mutants, alpha-256 and alpha-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the lambda pRM promoter by cI protein (repressor) was nearly normal, activation of the lambda pRE promoter by cII protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.

Binding Sites↗
PubMed1992

Solubilization and promoter analysis of RNA polymerase from rice stripe virus.

The RNA-dependent RNA polymerase associated with rice stripe virus was dissociated from viral RNA (vRNA) by CsCl centrifugation. The solubilized RNA-free RNA polymerase transcribed a model RNA template 50 nucleotides in length carrying the 5'- and 3'-terminal conserved sequences of all four genome RNA segments. A 3'-terminal half molecule of the model template was also active as a template. Hence, we propose that the 3'-terminal conserved sequence serves as a promoter for the rice stripe virus-associated RNA polymerase. The solubilized enzyme, however, was unable to transcribe vRNA. The failure of the solubilized enzyme to transcribe vRNA is discussed in relation to the apparent loss of RNA polymerase activity after treatment of virions with high concentrations of salt.

Base Sequence↗