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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 505 records · Page 28Linked to original sources
PubMed1983

Operational variables in high-performance gel filtration of DNA fragments and RNAs.

Double-stranded DNA fragments and ribosomal and transfer RNAs were measured by high-performance gel filtration on TSK-GEL G2000SW, G3000SW, G4000SW and G5000PW columns to investigate the separation range and resolution of these columns and the effects of eluent ionic strength and flow-rate on retention and resolution. These columns could separate double-stranded DNA fragments up to ca 1 X 10(6) and rRNAs up to ca 5 X 10(6) daltons in molecular weight. However, it was found that the selection of the column is very important to achieve optimum separation, depending on the molecular weight of the sample. Elution is delayed as the eluent ionic strength is increased. An eluent ionic strength of 0.3-0.5 seemed appropriate in most cases. Resolution is greatly increased as the flow-rate is decreased.

Chromatography, Gel↗
PubMed1983

Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.

Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of human gastrin precursor cDNA.

We cloned cDNA of gastrin mRNA from human gastric antrum. First we obtained a porcine gastrin precursor cDNA clone using a synthetic oligodeoxyribonucleotide, d(A-A-A-G-T-C-C-A-T-C-C-A-T-C-C-A-T) as a hybridization probe. Then, using this porcine clone as a hybridization probe, human gastrin precursor cDNA clones were obtained. Sequence analysis revealed 4, 303, and 98 nucleotides, respectively, in the 5' untranslated region, in the amino acid coding region, and in the 3' untranslated region. The deduced precursor molecule codes for big and small gastrin, surrounded by pairs of basic amino acids. When the sequences of porcine and human gastrin precursor are compared, a high degree of homology in the active peptide region and lower homology in other regions are observed.

Amino Acid Sequence↗
PubMed1983

Lambda phage DNA sequences affecting the packaging process.

Our previous work identified a minimal region of bacteriophage lambda DNA that is necessary for packaging into phage particles. It consists of 40 bp of the right arm and 45 bp of the left arm [Miwa and Matsubara, Gene 20 (1982)267-279]. A part of this region, 22 bp of the right arm and 38 bp of the left arm, is sufficient for cutting at cos lambda (the minimal sequence for cos lambda cutting). An 84-bp region to its right contains a binding site for lambda terminase, a complex of Nu1 and A gene products. This second region, which we called the enhancing region for packaging, stimulates cutting at cos lambda as well as packaging. This region is not active if it is physically separated from the minimal packaging region. The enhancing region has 15-bp inverted repeats. These sequences are conserved in the corresponding region of bacteriophage phi 80, which has the same packaging specificity as lambda.

Bacteriophage lambda↗
PubMed1983

Expression of hepatitis B surface antigen gene in yeast.

The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of autonomous replication in both yeast and Escherichia coli. Yeast transformed by this plasmid synthesized up to 5 X 10(5) molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg polypeptides produced in the yeast cells were assembled into 20- to 22-nm spherical or oval particles and were immunogenic.

Acid Phosphatase↗
PubMed1983

Instability of bacteriophage lambda initiator O and P proteins in DNA replication.

Lambda dv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant lambda phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressor-plus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulse-chase experiments demonstrated that O protein decays with a half-life of 8 min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.

Bacteriophage lambda↗
PubMed1983

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.

Bacteriophage lambda↗
PubMed1983

Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control.

An autonomously replicating 2,248-base-pair DNA segment of the mini-F plasmid carries nine 19-base-pair repeating sequences. Five of the repeats are arranged in one direction and form the right cluster, whereas the remaining four repeats are arranged in the opposite direction and form the left cluster (Murotsu et al., Gene 15:257-271, 1981). Each cluster, cloned separately into the multicopy plasmid vector pBR322, exhibited a strong F-specific incompatibility phenotype (FIP). These clusters were thought to be responsible for the expression of IncB and IncC phenotypes, causing a switchoff function on mini-F replication. Mini-F DNA fragments containing two, three, or more than four repeats were inserted into pBR322. Cells carrying these recombinant plasmids exhibited, respectively, no, intermediate, and strong FIP intensity. Cloning of five repeats into pSC101, whose copy number is about 6 in contrast to 20 for pBR322, resulted in an FIP of intermediate intensity. Thus, the intensity of FIP reflects the dosage of repeats in a cell. The five repeats in the right cluster were eliminated from the mini-F derivative without impairing its autonomous-replicating ability (Bergquist et al., J. Bacteriol. 147:888-889, 1981; Kline and Palchavdhuri, Plasmid 4:281-289). Such deletion, however, caused a sixfold elevation of the copy number. When the eliminated cluster of repeats was reinserted in the derivative, the copy number was lowered to the original value, viz., 1 to 2. The position and orientation of this insertion was not important in the copy number control. Thus, the repeats are also related to copy number control. A model to account for the role of the repeating sequences in the control of copy number and FIP is discussed.

Base Composition↗
PubMed1983

Chromophoric labeling of cannabinoids with 4-dimethylaminoazobenzene-4'-sulfonyl chloride.

A simple and sensitive assay for the cannabinoids is presented using a dabsylation procedure. Dabsyl derivatives of delta 9-tetrahydrocannabinol (delta 9-THC) and cannabinol (CBN) were prepared by reacting with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) in acetone in the presence of sodium carbonate-sodium bicarbonate buffer (pH 10). Crystalline dabsylcannabinoids gave intense absorption in the visible region. With these derivatives, analysis by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) were tested. These techniques gave good separation and nanogram detection of dabsyl-THC and -CBN by using n-hexane-ethyl acetate-diethylamine (20:5:1) for TLC and MeOH--H2O (95:5) at 450 nm for HPLC.

Cannabinoids↗
PubMed1982

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments.

Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or lambda dv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.

Bacterial Proteins↗
PubMed1982

Bacteriophage lambda initiators: preparation from a strain that overproduces the O and P proteins.

A recombinant plasmid was constructed which carries bacteriophage lambda initiator genes O and P under control of tandemly arranged PL and PR promoters. These promoters were repressed by a thermosensitive repressor, cI857, at low temperature, but became active when the culture was incubated at 42 degrees C. Upon elevation of the temperature, the O and P proteins were overproduced to the extent that they constituted several per cent of the total E. coli cellular proteins. Both the O and P proteins have been purified to apparent homogeneity, and were shown to consist of 298 and 233 amino acid residues, respectively. The amino acid composition and the terminal partial amino acid sequence of each protein were determined. Through these analyses, the locations of the O and P genes in the known lambda DNA sequence were determined. The termination codon for the O gene overlaps with the initiation codon for the P gene. The purified O protein binds specifically to the replication origin of lambda (lambda ori) in accordance with our previous observations. The purified P protein inhibits an ATPase activity of dnaB protein.

Adenosine Triphosphatases↗
PubMed1982

Changes in ambulatory activities and muscle relaxation in rats after repeated doses of diazepam.

Changes in rat ambulatory activity and muscle tone after diazepam treatments (2.5, 5, 10, and 20 mg/kg orally) were compared under single and repeated (for 2, 4, 7, and 14 days) administration schedules. Ambulatory activity in the open field test was enhanced by a single dose of 2.5 mg/kg and reduced by 20 mg/kg. However, after all repeated treatment schedules, diazepam resulted in dose-dependent elevations of activity. Even with a treatment period of only 2 days, 20 mg/kg diazepam produced a marked increase in ambulation, which became more conspicuous with increases in the treatment periods. Acutely administered diazepam produced a dose-dependent reduction in muscle tone, but tolerance was noted to this effect during repeated administrations. Thus, time- and dose-dependent increases in ambulation during repeated treatments with diazepam can be partially explained by time-dependent tolerance development to its muscle relaxant effects. The enhancement in ambulation after short-term repetitive dosings of benzodiazepines can be used as a simple indicator for detecting their disinhibitory potentials.

Animals↗
PubMed1982

Identification of sequences necessary for packaging DNA into lambda phage heads.

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.

Bacteriophage lambda↗