Nariko Shinriki

Effect of ozone on plasmid DNA of Escherichia coli in situ

Abstract The cell suspension of Escherichia coli HB101 harboring pBR322 plasmid DNA was exposed to ozone, and the damage of plasmid DNA in vivo and also cell survival were investigated. Closed circular plasmid DNA (ccDNA) converted to open circular DNA (ocDNA) by ozone treatment. The mechanism of the conversion seemed to be similar to that observed in vitro ozone treatment of plasmid DNA [Sawadaishi et al. (1985) Nucleic Acids Res. 13 , 7183; idem. (1986) ibid. 14 , 1159]. These findings indicate that ozone penetrates cell membrane and reacts with cytoplasmic substances. Therefore, chromosomal DNA may be one of targets of the ozone degradation, and its damage may be one of the factors responsible for the cell killing.

Mechanism of inactivation of tobacco mosaic virus with ozone

Abstract The inactivation mechanism of tobacco mosaic virus (TMV) in a phosphate buffer (pH 6.9) by ozone was studied. We previously reported that the damage of naked TMV-RNA occurred at the guanine moiety of RNA (Shinriki et al., Biochim. biophys. Acta655, 323, 1981). In this paper, we clarified the mode of the inactivation of TMV by using tritium-labeled TMV (TMV∗) prepared by the reconstitution of tritium-labeled TMV-RNA (TMV-RNA∗) and coat protein of TMV. It was found that the amount of extracted TMV-RNA∗ from ozone-treated TMV∗ decreased with the advance of ozonization, and that there was good correlation between the loss of infectivity and the decrease of recovery of TMV-RNA∗. When TMV lost its infectivity due to ozone, tryptophan and tyrosine of the coat protein were also degraded by ozone. Polyacrylamide gel electrophoretic analysis of the substance produced during ozonization showed that the coat protein subunits were aggregated with each other and cross-linked with TMV-RNA∗. From these results, it was concluded that the inability of uncoating is the major cause of the TMV inactivation by ozone.

Molecular cloning of guinea pig CYP1A1 : complete primary structure and fast mobility of expressed protein on electrophoresis

Guinea pig CYP1A1 cDNA was isolated from a liver cDNA library of guinea pig treated with 3-methylcholanthrene. The cDNA, named GPc1, was 2674 bp long and contained an entire coding region for 516 amino acids. The amino acid sequence of guinea pig CYP1A1 shared 74-78% identity with those of the other mammalian CYP1A1s. RNA blot and immunoblot analyses revealed that CYP1A1 was constitutively expressed and was induced by 3-methylcholanthrene in guinea pig liver. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, guinea pig CYP1A1 expressed in yeast had a significantly smaller apparent molecular mass than expressed mouse CYP1A1. An alignment of the amino acid sequences of mammalian CYP1A1s demonstrated that guinea pig CYP1A1 was several residues shorter than the counterparts in the N-terminal region. Thus, to clarify the contribution of the N-terminal sequence of guinea pig CYP1A1 to the fast mobility on the electrophoresis, mouse-guinea pig chimeric CYP1A1 was prepared through cDNA-directed expression in yeast. The chimeric CYP1A1 protein had an intermediate molecular mass between mouse and guinea pig CYP1A1s indicating that the anomalous mobility of guinea pig CYP1A1 is in part due to the shortened N-terminal amino acid sequence of the protein.

Molecular cloning and sequence analysis of mouse NADPH-cytochrome P-450 oxidoreductase

Abstract A cDNA clone coding for cytochrome P -450 oxidoreductase was isolated from a mouse liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the mouse cytochrome P -450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function.

Upgrading of coal-derived liquids: 3. Characterization of upgraded liquids by thin-layer chromatography combined with flame-ionization detection

Abstract Four components, namely, saturates, aromatics, resins and asphaltenes, in upgraded coal-derived liquids were determined by thin-layer chromatography combined with flame-ionization detection (t.l.c./FID method). The results agree with previous results using various analytical methods. The t.l.c./FID method was found to be a simple and effective method for screening of hydroconversion catalysts and also for a follow-up investigation of the chemical changes of coal-derived liquids during the hydroconversion process.

Thermal transition of plasmid pBR322 closed circular, open circular and linear DNAs

Abstract Thermal transitions of pBR322 closed circular (cc), open circular (oc) and linear (l) DNAs have been measured by calorimetry. Six or eight peaks were observed for l and oc DNAs in 0.1 × SSC and 1 × SSC buffer solutions in the temperature ranges 67–84°C and 82–97°C, respectively, while ccDNA showed one broad peak at 103°C. Thermal reversibility was excellent for 1DNA and good for ocDNA but the DSC curve of ccDNA at the consequent second heating was quite different from that of the first run. The morphological changes induced by heating cc and ocDNAs have been examined by electrophoresis of the preheated samples. ccDNA which was heated above 75°C was found to be converted partly to ocDNA, the amount of ocDNA increasing with increasing temperature. The DSC curve of lDNA in 0.1 × SSC has been deconvoluted to a 24-state transition, and thermodynamic functions for the multistep transitions have been estimated; the results are discussed in comparison with its theoretical melting curve and its genetic map.