Genetic Analysis of Cytochrome b5 from Arachidonic Acid-Producing Fungus, Mortierella alpina 1S-4: Cloning, RNA Editing and Expression of the Gene in Escherichia coli, and Purification and Characterization ofthe Gene Product.

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Author(s)

    • Kobayashi Michihiko
    • Division of Applied Life Sciences. Graduate School of Agriculture, Kyoto University
    • Sakuradani Eiji
    • Division of Applied Life Sciences. Graduate School of Agriculture, Kyoto University
    • Shimizu Sakayu
    • Division of Applied Life Sciences. Graduate School of Agriculture, Kyoto University

Abstract

Information on the amino acid sequences of the internal peptide fragments of cytochrome <i>b</i><sub>5</sub> from <i>Mortierella hygrophila</i> was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from <i>Mortierella alpina</i> 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase <i>Sal</i>1-NruI genomic fragment coding for the whole <i>M. alpina</i> 1S-4 cytochrome <i>b</i><sub>5</sub>. On the basis of nucleotide sequences of both cytochrome <i>b</i><sub>5</sub> genomic gene and cDNA, the genomic cytochrome <i>b</i><sub>5</sub> gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine→guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of <i>M. alpina</i> 1S-4 cytochrome <i>b</i><sub>5</sub> showed significant similarities with those of cytochrome <i>b</i><sub>5</sub>s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome <i>b</i><sub>5</sub> gene was expressed to 16% of the total soluble protein in <i>Eseherichia coli.</i> The holo-cytochrome <i>b</i><sub>5</sub> accounted for 8% of the total cytochrome <i>b</i><sub>5</sub> in the transformants. The purified cytochrome <i>b</i><sub>5</sub> showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome <i>b</i><sub>5</sub>.

Journal

  • J Biochem (Tokyo)

    J Biochem (Tokyo) 125(6), 1094-1103, 1999

    The Japanese Biochemical Society

Codes

  • NII Article ID (NAID)
    130003417922
  • Text Lang
    EN
  • ISSN
    0021-924X
  • Data Source
    J-STAGE 
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