Kojima Y, Tsukuda Y, Kawai Y, Tsukamoto A, Sugiura J, Sakaino M, Kita Y
Central Research Laboratory, Oji Paper Co., Ltd., Tokyo, Japan.
J Biol Chem. 1990 Sep 5;265(25):15224-30.
Two cDNAs and two genomic DNAs coding for the allelic forms of the ligninolytic phenoloxidase were isolated from the white-rot fungus Coriolus hirsutus. The cloned genes were identified in genetic libraries by hybridization screening using four deoxyoligonucleotide probes which corresponded to the partial amino acid sequence of the purified enzyme. Each cDNA encoded the full-length of the phenoloxidase, a protein consisting of 499 amino acid residues, and its putative signal peptide of 21 amino acid residues. The nucleotide sequences of the two alleles differed by 18 single base changes within the open reading frames resulting in one amino acid substitution. Ten small introns interrupted both genomic DNAs as indicated by direct comparison with the corresponding cDNAs. Putative eukaryotic regulatory sequences, "CAAT" and "TATA," were observed in the 5'-flanking region of both genomic DNAs. Each of the phenoloxidase cDNAs was successfully expressed in an active form in Saccharomyces cerevisiae using the useful yeast expression vector YEp51.
从白腐真菌糙皮侧耳中分离出两个编码木质素分解酚氧化酶等位形式的cDNA和两个基因组DNA。使用四个与纯化酶的部分氨基酸序列相对应的脱氧寡核苷酸探针，通过杂交筛选在基因文库中鉴定出克隆的基因。每个cDNA编码酚氧化酶的全长，该蛋白由499个氨基酸残基组成，其推定的信号肽由21个氨基酸残基组成。两个等位基因的核苷酸序列在开放阅读框内有18个单碱基变化，导致一个氨基酸替换。通过与相应的cDNA直接比较表明，十个小内含子中断了两个基因组DNA。在两个基因组DNA的5'侧翼区域观察到推定的真核调控序列“CAAT”和“TATA”。使用有用的酵母表达载体YEp51，每个酚氧化酶cDNA在酿酒酵母中成功以活性形式表达。
