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K Oeda

Publications and source records attributed to K Oeda.

27 records · Page 2Linked to original sources
PubMed1985

Expression of rat liver cytochrome P-450MC cDNA in Saccharomyces cerevisiae.

Rat liver cytochrome P-450MC cDNA was inserted between the ADH1 promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pAMC1, Saccharomyces cerevisiae cells synthesized up to 8 X 10(5) molecules per cell of the cytochrome P-450MC protein, most of which was localized in yeast microsomes. Approximately half of the synthesized cytochrome contained heme in the enzyme molecule. These formed a functional electron-transport chain in the microsomes which exhibited aryl hydrocarbon hydroxylase activity toward benzo[a]pyrene.

Alcohol Dehydrogenase↗
PubMed1985

Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae.

Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC.

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.

Amino Acid Sequence↗
PubMed1984

Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450.

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.

Amino Acid Sequence↗
PubMed1982

Cloning of mutator genes and identification of their products.

To elucidate molecular mechanisms leading to the increment of mutation frequency by mutator genes, we have cloned the dnaQ and uvrD genes of Escherichia coli and identified their gene products. By in vitro recombination hybrid plasmids capable of complementing a dnaQ mutation were constructed. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one with a molecular weight of 25,000 and the other with a weight of 21,000. Because insertion of the gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25,000-dalton protein, it was concluded that that protein is the dnaQ gene product. The 21,000-dalton protein was identified as RNase H. The uvrD (uvrE, recL, mutU, and pdeB) gene has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light, high mutability and conditional lethality of uvrD- strains and their derivatives were all suppressed by lysogenization of the mutant cells with lambda uvrD+. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg2+, Mn2+, and Co2+ through the cell membrane. By analyzing proteins produced by the transducing phages, the uvrD and corA gene products were identified as a 75,000-dalton protein and a 37,000-dalton protein, respectively.

Bacterial Proteins↗
PubMed1981

Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency.

The uvrD gene of Escherichia coli that controls UV sensitivity and spontaneous mutation frequency has been cloned with phage lambda as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and supl26 mutations were all suppressed by lysogenization of the mutant cells with lambda uvrD+. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvrD gene, lambda uvrD+ carried the corA gene that controls transport of Mg++, Mn++, and Co++ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD- as well as CorA- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the gamma delta sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.

Bacteriophage lambda↗
PubMed1978

An enzyme activity specific for nitrous acid-treated DNA in Escherichia coli.

An enzyme activity specifically active on nitrous acid-treated DNA was found in an extract of Escherichia coli. The enzyme acts on both double- and single-stranded DNAs, treated with nitrous acid, in the presence of EDTA, although the former DNA is a better substrate. Evidence is presented that nitrous acid- and bisulfite-induced types of damage in DNA are recognized by different enzymes: (1) Uracil-DNA glycosylase, purified 250-fold from E. coli 1100, attacks bisulfite-treated DNA but not nitrous acid-treated DNA. (2) Almost equal levels of activity toward nitrous acid-treated DNA were found in wild-type and uracil-DNA glycosylase-deficient strains of E. coli.

Coliphages↗