Kazunobu Miura

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.

Transformation of NIH3T3 cells with synthetic c-Ha-ras genes

Synthetic human c‐Ha‐ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3′‐end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c‐Ha‐ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. colt. This expression vector system should he useful for studies on the structure‐function relationships of c‐Ha‐ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies.

Chemical modification of guanine residues of mouse 5 S ribosomal RNA with kethoxal. Nucleosides and nucleotides 46

Chemical modification of mouse 5 S rRNA with kethoxal was carried out to examine the secondary structure. The guanine residues located at positions 37, 41, 56, 66, 75 and 89 were modified. The relative rates of reaction are in the order G37, G56, G89, G66, G41, G75 at 28 degrees C and G37, G41, G56, G89, G75, G66 at 35 degrees C. These results support a secondary structure model containing 5 helices and 5 loops and indicate that the region around position 37 is the most exposed in higher-order structure.

Identification of specific amino acid residues of adenovirus 12 E1A involved in transformation and p300 binding.

The early region 1A (E1A) gene of highly oncogenic adenovirus type 12 (Ad12) was analyzed for transforming activity and protein binding using specific mutations. The Ad12 E1A proteins were found to bind p300 protein mainly within the CR1 region, although mutations that affect both p300 binding and transformation were identified in both the CR1 and the N-terminal region. The most critical mutation dlf89 located in the CR1 region was further dissected by point mutations and the results identified 68S as the most critical for transformation and 67E as the most critical for p300 binding. Specific mutations that retain p300 binding but impair transcriptional repression of a viral enhancer were also identified in both the N-terminal and CR1 regions.

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.

Monoclonal Antibodies with Affinity to Self-Complementary Left-Handed DNA Containing Cyclonucleosides with High Anti Conformation

Abstract Monoclonal antibodies specific for a self-complementary hexanucleotide with a high-anti conformation were found to bind another hexanucleotide with a high-anti conformation, but with a different sequence. A higher order structure seemed to be recognized in the reaction.

Chemical modification of cytosine residues of mouse 5 S ribosomal RNA with hydrogen sulfide (Nucleosides and nucleotides 43)

Cytosine residues of nucleic acids were converted to 4-thiouracil residues with hydrogen sulfide in pyridine and water to examine the secondary and tertiary structures of mouse 5 S rRNA. The cytosine residues at positions 10, 24, 34 (or 36), 39, 44 (or 46) and 63 were converted preferentially when the treatment was carried out at 28 degrees C. This result supports the model of the secondary structure of 5 S rRNA of Nishikawa, K. and Takemura, S. ((1974) FEBS Lett. 40, 106-109) consisting of five helices and five loops. As the temperature was increased to 35 degrees C, additional cytosine residues in positions 26, 52 and 78 were modified to moderate extents.

Crystallization and preliminary crystallographic data of a truncated derivative from the human c-Ha-ras protein

A truncated derivative of the human c-Ha-ras protein has been crystallized from polyethylene glycol 6000 solution by a vapour diffusion technique. The rectangular prism diffracts X-rays to at least 2.5 A resolution (1 A = 0.1 nm). The unit cell is monoclinic, space group P21, with unit cell parameters of a = 50.2 A, b = 110.9 A, c = 36.4 A, beta = 97.2 degrees. The unit cell contains four molecules.

Preparation and characterization of monoclonal antibodies specific for GoCodGdCGoCo duplex with high-anti conformation.

Monoclonal antibodies were prepared using a self-complementary oligonucleotide duplex containing cyclonucleosides with the high-anti conformation as an antigen. A competition ELISA assay showed that the monoclonal antibodies specifically recognized high-anti left-handed oligonucleotides but not oligonucleotides with B- or Z-conformation.