Search PubMed⌕ Search

Biomedical subjects

A Nakata

Publications and source records attributed to A Nakata.

At least 91 records · Page 5Linked to original sources
PubMed1992

A unique monoclonal antibody that recognizes mature p17 of HIV-1 but not its precursor.

The entire and partial gag regions of human immunodeficiency virus type 1 (HIV-1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17. We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety. Further analysis revealed that V17 recognizes the C-terminal 12-amino-acid region of p17 having free C-terminus. This monoclonal antibody may be useful for monitoring the maturation of virus particles.

Antibodies, Monoclonal↗
PubMed1992

A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1.

A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.

Chromobox Protein Homolog 5↗
PubMed1992

Molecular analysis of the Escherichia coli phoP-phoQ operon.

The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.

Amino Acid Sequence↗
PubMed1992

Suppressive effect of polyoxometalates on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and their inhibitory activity against HIV-1 reverse transcriptase.

One isopolyoxometalate and 42 heteropolyoxometalates consisting of 3 compounds with the trivacant Keggin structure, 2 with the lacunary Keggin structure, 30 with the Keggin structure, one with the Wells-Dawson structure and 6 with miscellaneous structures were tested for their suppressive effect on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and inhibitory activity against HIV-1 reverse transcriptase. In contrast to the leading interpretations which attribute the suppressive effect of polyoxometalates on the cytopathogenicity of HIV-1 to the inhibition of HIV-1 reverse transcriptase by these compounds, there was no distinct correlation observed between these two functions of polyoxometalates.

Antimony↗
PubMed1991

Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated alpha subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the alpha subunit, and the other not requiring it.

Bacterial Outer Membrane Proteins↗
PubMed1991

SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA.

The ruv operon is induced by treatments that damage DNA and is regulated by the LexA repressor. It encodes two proteins, RuvA and RuvB, that are involved in DNA repair, recombination in RecE and RecF pathways, and mutagenesis. RuvB protein was previously purified and has ATP-binding activity and weak ATPase activity. To study the biochemical properties of RuvA and its interaction with RuvB, we purified RuvA protein to near homogeneity from an over-producing strain. RuvA bound more efficiently to single-stranded DNA than to double-stranded DNA. RuvA bound to DNA greatly enhanced the ATPase activity of RuvB; the enhancing effect of various forms of DNA was in the order of supercoiled DNA greater than single-stranded DNA greater than linear double-stranded DNA. UV irradiation further enhanced the ATPase stimulatory effect of supercoiled DNA dose dependently. The RuvA-RuvB complex has an activity that renatures the cruciform structure in supercoiled DNA. From these experiments and previous work, we infer that the RuvA-RuvB complex may promote branch migration in recombination and may correct irregular structures in DNA, such as cruciforms and hairpins, to facilitate DNA repair using ATP as the energy source.

Adenosine Triphosphatases↗
PubMed1991

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.

Amino Acid Sequence↗
PubMed1991

Escherichia coli DNA polymerase II is homologous to alpha-like DNA polymerases.

The Escherichia coli polB gene encodes DNA polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89,917, and is structurally homologous to alpha-like DNA polymerases, which include eukaryotic replicative DNA polymerases. Comparison of the sequences of the alpha-like DNA polymerases including E. coli DNA polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA polymerases based on the differences in these conserved regions. The DNA polymerases of herpes groups viruses and the DNA polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA polymerases, including E. coli DNA polymerase II, human DNA polymerase alpha, and yeast DNA polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the -35 sequence of the polB promoter.

Amino Acid Sequence↗
PubMed1991

SOS-inducible DNA polymerase II of E coli is homologous to replicative DNA polymerase of eukaryotes.

The polB gene of Escherichia coli encodes DNA polymerase II whose role in vivo is not defined. The polB gene has been cloned and shown to be identical to a DNA damage-inducible gene dinA which is regulated by the LexA repressor. Nucleotide sequencing of polB reveals that E coli DNA polymerase II is highly homologous to replicative DNA polymerases of eukaryotes which include human DNA polymerase alpha and Saccharomyces cerevisiae DNA polymerases I, II and III. The polB gene is not required for growth, UV-repair and UV-mutagenesis.

Base Sequence↗
PubMed1991

Properties of the Escherichia coli RuvA and RuvB proteins involved in DNA repair, recombination and mutagenesis.

The ruvA and ruvB genes constitute an operon, which is regulated by the SOS system and involved in DNA repair, recombination and mutagenesis. RuvA protein binds to both single-stranded and double-stranded DNA. RuvB protein has weak ATPase activity. RuvA bound to DNA greatly enhances ATPase activity of RuvB. UV-irradiation to supercoiled DNA further enhances the stimulatory effect of RuvA on the RuvB ATPase activity. In the presence of ATP the RuvA-RuvB complex has an activity that renatures cruciform structures formed by heating and gradually cooling supercoiled DNA with an inverted repeat. These findings suggest that the RuvA-RuvB complex interacts with an irregular conformation in damaged DNA and induces conformational changes in DNA using energy provided by ATP hydrolysis, so that it facilitates DNA repair, recombination and error prone replication.

Bacterial Proteins↗
PubMed1991

Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease.

The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orf17-orf26-ruvC-orf23 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orf17, orf26, and ruvC (orf19) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.

Amino Acid Sequence↗
PubMed1991

Dual regulation of the ugp operon by phosphate and carbon starvation at two interspaced promoters.

The ugp operon of Escherichia coli includes genes involved in the uptake of sn-glycerol-3-phosphate and glycerophosphoryl diesters and belongs to the pho regulon which is induced by phosphate limitation. This operon has two transcriptional initiation sites, as determined by S1 nuclease mapping of the in vivo transcripts. The downstream promoter has multiple copies of the pho box, the consensus sequence shared by the pho promoters; the upstream promoter has a consensus sequence for the promoters regulated by cyclic AMP and its receptor protein, CRP. PhoB protein, which is the transcriptional activator for the pho regulon, protected the regulatory region with the pho boxes in DNase I footprinting experiments and activated transcription from the downstream promoter in vitro. Studies with transcriptional fusions between ugp and a promoterless gene for chloramphenicol acetyltransferase show that the upstream promoter is induced by carbon starvation in a manner that required the cya and crp genes. PhoB protein may act as a repressor for this upstream promoter, which also overlaps the upstream third pho box. The downstream promoter was induced by phosphate starvation and requires the PhoB protein for its activation as do the other pho regulon promoters. These results suggest that the two promoters function alternately in responding to phosphate or carbon starvation, thus providing the cell with a means to adapt to these physiological stresses.

Base Sequence↗
PubMed1991

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.

Acid Phosphatase↗
PubMed1991

Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12.

We cloned the cryptic phn operon of a K-12 strain, phn(EcoK), and analyzed the nucleotide sequence of the phn region (11,672 bp). An mRNA start site upstream of the phnC gene was identified by S1 nuclease mapping. The pho regulon activator PhoB protects a pho box region near the mRNA start in DNase I footprinting and methylation protection experiments. The sequence of the cryptic phn(EcoK) operon was very similar to that of the functional phn operon of an Escherichia coli B strain, phn(EcoB) (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990). The phnE(EcoK) gene has an 8-bp insertion, absent from the phnE(EcoB) gene, which causes a frameshift mutation. The spontaneous activation of the cryptic phn(EcoK) operon is accompanied by loss of this additional 8-bp insertion. Studies of the structure, regulation, and function of the phn region suggest that the phosphate starvation-inducible phn operon consists of 14 cistrons from phnC to phnP.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of the Escherichia coli OxyR protein, the positive regulator for a hydrogen peroxide-inducible regulon.

The Escherichia coli oxyR gene is required for the induction of a regulon that is inducible by hydrogen peroxide and confers resistance to oxidative stresses. We constructed a plasmid system that greatly overproduced OxyR protein and purified the protein. OxyR protein specifically bound to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the gel-retardation assay and the DNase I footprinting experiment, and activated the transcription initiation of the katG gene in vitro. Using a plasmid carrying an oxyR'-'lacZ fusion gene, we studied the regulation of oxyR expression in vivo. The expression of oxyR was not induced by the treatment with a low concentration of hydrogen peroxide which induces the genes in the oxyR regulon. The expression of the oxyR'-'lacZ gene was higher in an oxyR-deletion strain than in the oxyR+ strain, and was repressed by overexpressing the OxyR protein. These results suggest that OxyR protein functions as a repressor for oxyR, in addition to its known function as a transcriptional activator for the genes in the oxyR regulon.

Bacterial Proteins↗
PubMed1990

Proteolytic activation of UmuD and MucA proteins for SOS mutagenesis.

SOS mutagenesis in Escherichia coli requires the functions of the umuD, C genes, or their functional analogues mucA, B derived from a plasmid pKM101, and the recA gene. However, mere derepression of these SOS genes does not increase the ability of the cell to perform mutagenesis. Activation of RecA protein to a form (RecA*) that mediates cleavage of the LexA repressor is required for mutagenesis. We present evidence that UmuD and MucA are proteolytically processed by RecA* and that the processed products are the active forms involved in mutagenesis.

Bacterial Proteins↗
PubMed1990

Proteolytic processing of MucA protein in SOS mutagenesis: both processed and unprocessed MucA may be active in the mutagenesis.

The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ'-'MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA+ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis.

Bacterial Proteins↗