Yabusaki Y, Murakami H, Nakamura K, Nomura N, Shimizu M, Oeda K, Ohkawa H
J Biochem. 1984 Sep;96(3):793-804. doi: 10.1093/oxfordjournals.jbchem.a134897.
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.
构建了与从经3 - 甲基胆蒽（MC）处理的大鼠肝脏中制备的部分纯化mRNA互补的双链DNA，并将其克隆到大肠杆菌中。通过阳性杂交翻译分析和用抗细胞色素P - 450抗体的免疫化学分析，验证了20个克隆携带编码MC诱导型细胞色素P - 450的互补DNA（cDNA）插入片段。根据cDNA插入片段的限制性图谱比较，将鉴定出的cDNA克隆至少分为两组。一个cDNA插入片段长度约为2.7 kb的克隆pAU157包含细胞色素P - 450MC或P - 450c几乎全长的mRNA信息，细胞色素P - 450MC或P - 450c是MC诱导型细胞色素P - 450的主要形式。其他cDNA克隆pTZ286 - pTZ330包含与细胞色素P - 450d mRNA互补的1.2 kb序列。RNA印迹分析表明，pAU157和pTZ286 - pTZ330 cDNA克隆分别来源于22S和18S mRNA，这两种mRNA在MC处理的大鼠肝脏中均被诱导。序列分析表明，pAU157和pTZ286 - pTZ330 cDNA插入片段中存在密切同源的序列区域，并且大多数同源序列位于两种细胞色素P - 450物种的两个有限编码区域内。pAU157编码细胞色素P - 450MC或P - 450c的总氨基酸序列，pTZ286 - pTZ330编码细胞色素P - 450d的C末端368个氨基酸残基。在这些细胞色素P - 450物种的氨基酸序列中发现了两个高度同源的区域。
