Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L

Kotaro Kirimura, Masashi Yoda, Shoji Usami

    研究成果: Article

    45 引用 (Scopus)

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    A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.

    元の言語English
    ページ(範囲)472-477
    ページ数6
    ジャーナルCurrent Genetics
    34
    発行部数6
    DOI
    出版物ステータスPublished - 1999

    Fingerprint

    Aspergillus niger
    Open Reading Frames
    Organism Cloning
    Complementary DNA
    Genes
    Cyanides
    Respiration
    Clone Cells
    Escherichia coli
    Isopropyl Thiogalactoside
    Plant Genes
    Neurospora crassa
    Conserved Sequence
    DNA
    Gene Library
    Citric Acid
    alternative oxidase
    Amino Acid Sequence
    Plasmids
    Fungi

    ASJC Scopus subject areas

    • Genetics

    これを引用

    Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L. / Kirimura, Kotaro; Yoda, Masashi; Usami, Shoji.

    :: Current Genetics, 巻 34, 番号 6, 1999, p. 472-477.

    研究成果: Article

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    abstract = "A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50{\%}, 55{\%}, and 52{\%} homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.",
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