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Biomedical subjects

A Nakata

Publications and source records attributed to A Nakata.

At least 109 records · Page 6Linked to original sources
PubMed1990

Regulation of the phosphate regulon of Escherichia coli: properties of phoR deletion mutants and subcellular localization of PhoR protein.

The phoR gene is a bifunctional regulatory gene for the phosphate regulon of Escherichia coli. It acts as a negative regulator in the presence of excess phosphate and as a positive regulator with limited phosphate, through modification of PhoB protein. We constructed several phoR genes, with various deletions in the 5' regions, which were regulated by the trp-lac hybrid promoter. The PhoR1084 and PhoR1159 proteins that lack the 83 and 158 N-terminal amino acids, respectively, retained the positive function for the expression of phoA that codes for alkaline phosphatase, but lacked the negative function. The PhoR1263 protein that lacks the 262 N-terminal amino acids was deficient in both functions. An antiserum against PhoR1084 protein was prepared. Western blot analysis of the subcellular fractions obtained by differential centrifugation indicated that the intact PhoR and PhoR1084 proteins are located in the inner membrane and cytoplasmic fractions, respectively. The results suggest that PhoR protein is anchored to the cytoplasmic membrane by the amino-terminal region.

Alkaline Phosphatase↗
PubMed1990

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli.

Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4-10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec+ host. Some deletions occurred at the same sites either in a recA host or a rec+ host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.

Base Sequence↗
PubMed1990

Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme.

Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.

Cloning, Molecular↗
PubMed1990

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Amino Acid Sequence↗
PubMed1990

The Escherichia coli polB gene, which encodes DNA polymerase II, is regulated by the SOS system.

The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the polB gene encoding DNA polymerase II was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dinA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method.

DNA Polymerase II↗
PubMed1990

Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2.

Transcription of the genes in the phosphate regulon in Escherichia coli is activated by PhoB protein, which is phosphorylated by PhoR protein under phosphate-limiting conditions. In the absence of the phoR function, the genes in the phosphate regulon are expressed constitutively and the expression is dependent on the function of phoM and phoB. We constructed a plasmid with a lacZ'-'phoM fusion gene, which encoded a hybrid protein (PhoM1206) in which the hydrophobic amino-terminal half of the native PhoM was replaced by beta-galactosidase. The phoM1206 gene could complement the phoM mutation in vivo. We purified PhoM1206 from the overproducing strain carrying the plasmid; it was autophosphorylated at a histidine residue in the presence of ATP, and the phospho-PhoM1206 phosphorylated PhoB. PhoM1206 could also transphosphorylate the product of phoM-orf2, which is structurally homologous to phoB and located immediately upstream of phoM. Although PhoR1084 that lacked the hydrophobic amino-terminal region of the native PhoR protein transphosphorylated PhoB, it could not phosphorylate PhoM-open reading frame 2. Therefore, cross talk by protein phosphorylation appears to occur from PhoM to PhoB but not from PhoR to PhoM-open reading frame 2.

Adenosine Triphosphate↗
PubMed1990

Overproduction of acetate kinase activates the phosphate regulon in the absence of the phoR and phoM functions in Escherichia coli.

A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain.

Acetate Kinase↗
PubMed1989

Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins.

PhoB protein is the transcriptional activator for genes in the phosphate regulon of Escherichia coli, such as phoA and pstS, that are induced by phosphate deprivation. PhoR protein activates PhoB when phosphate is limiting and inactivates it when phosphate is in excess. We constructed a plasmid with a mutant phoR gene (phoR1084), which encoded a PhoR protein (PhoR1084) lacking the amino-terminal hydrophobic region of the intact protein. The cells carrying the plasmid overproduced PhoR1084, which was recovered in the soluble fraction of the cell lysate. We purified the Phor1084 protein and showed that it was autophosphorylated in the presence of ATP, and the phosphate group on the protein was rapidly transferred to PhoB. The phosphorylation of PhoB protein occurred concurrently with the acquisition of the ability to activate transcription from the pstS promoter. On the basis of these findings, we propose that phosphorylated PhoB protein activates transcription from the promoters of the phosphate regulon, and that the role of PhoR is to catalyze the formation and breakdown of phosphorylated PhoB in response to phosphate concentrations in the medium.

Adenosine Triphosphate↗
PubMed1989

Cloning, nucleotide sequence, and expression of Achromobacter protease I gene.

Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini.

Alcaligenes↗
PubMed1989

DNA bending and binding factors of the human beta-actin promoter.

Transcription of the beta-actin gene is rapidly inducible in response to serum stimulation. To determine the regions responsible for serum inducible and basal level expression, the human beta-actin promoter was subjected to mutational analysis. Two distinct elements, the CCAAT homology and the beta-actin specific conserved sequences, were found by a chloramphenicol acetyltransferase expression assay and sequence comparisons, and then analyzed for possible functions. Using a DNA bend assay, it was shown that the conserved sequences included the core of a sequence-directed bend of DNA. Gel mobility shift and DNase I protection assays revealed that the conserved sequences and the CCAAT homology were recognized by binding factors in HeLa cell extracts.

Actins↗
PubMed1989

Involvement in DNA repair of the ruvA gene of Escherichia coli.

The ruv operon of Escherichia coli consists of two genes, orf1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orf1 and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.

Blotting, Southern↗
PubMed1989

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins.

Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, CysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.

Amino Acid Sequence↗
PubMed1989

Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene.

The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the -10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the -10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gel-mobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.

Base Sequence↗
PubMed1989

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.

Adenosine Diphosphate↗
PubMed1989

Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes.

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.

Amino Acid Sequence↗
PubMed1989

Phosphate regulon in members of the family Enterobacteriaceae: comparison of the phoB-phoR operons of Escherichia coli, Shigella dysenteriae, and Klebsiella pneumoniae.

The structure and function of the phoB and phoR genes of Shigella dysenteriae strains and Klebsiella pneumoniae, which are involved in regulation of the phosphate regulon, were analyzed. Complementation tests among the genes of Escherichia coli, S. dysenteriae strains, and K. pneumoniae for production of alkaline phosphatase indicate that S. dysenteriae serotype 2 and serotype 3 strains and K. pneumoniae are phoA+ phoB+ phoR+ but S. dysenteriae Sh and serotype 1 strains are phoA phoB+ phoR. Nucleotide sequences of phoB and phoR of S. dysenteriae Sh and K. pneumoniae are highly homologous to those of E. coli, except for a single base insertion found in phoR of S. dysenteriae Sh.

Alkaline Phosphatase↗
PubMed1989

Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.

Base Sequence↗