Atchison M, Adesnik M
J Biol Chem. 1983 Sep 25;258(18):11285-95.
The capacity of the liver microsomal mixed function oxidase system to metabolize a great variety of exogenous as well as endogenous compounds is thought to reflect the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. In order to approach the questions of the genetic basis for the existence of multiple cytochrome P-450 isozymes and the molecular mechanisms of the induction process, we have used a cloned cDNA for a major phenobarbital-induced form of cytochrome P-450 to identify and characterize thirteen distinct rat genomic clones containing segments of six different genes. Only three clones, representing overlapping segments of a single gene, hybridized to the cloned cDNA or to phenobarbital-induced mRNA under conditions of high stringency. In vitro transcription studies with isolated rat liver nuclei indicated that only this gene, but not the genes represented by the other genomic clones, appears to be markedly activated by phenobarbital treatment of rats. Although there are a small number of differences, DNA sequence analysis of the eight exons of the gene present in these genomic clones indicates that they encode residues 58 to 491 (the COOH terminus) of cytochrome P-450e, a major phenobarbital-induced isozyme. It appears that the other cloned genes may contain only a small region of very strong homology to the cytochrome P-450e gene, a region which includes the exon encoding a tridecapeptide which is also present in two dissimilar forms of rabbit liver cytochrome P-450, one constitutive and one induced by phenobarbital. Southern blotting analysis of rat liver DNA indicates that the rat genome may contain two additional genes which are very closely related to the cytochrome P-450e gene but which we have not yet isolated from the genomic library. One of these genes is likely to encode cytochrome P-450b, the major phenobarbital induced form of cytochrome P-450 whose mRNA is greater than 95% homologous to that encoding cytochrome P-450e.
肝脏微粒体混合功能氧化酶系统代谢多种外源性和内源性化合物的能力，被认为反映了末端氧化酶细胞色素P - 450多种形式的参与，这些形式具有不同但重叠的底物特异性。动物接触特定诱导剂后，其中几种同工酶会在肝脏中积累。为了探讨多种细胞色素P - 450同工酶存在的遗传基础以及诱导过程的分子机制问题，我们使用了一种克隆的cDNA，它对应一种主要由苯巴比妥诱导产生的细胞色素P - 450形式，来鉴定和表征13个不同的大鼠基因组克隆，这些克隆包含六个不同基因的片段。在高严格度条件下，只有三个代表单个基因重叠片段的克隆与克隆的cDNA或苯巴比妥诱导的mRNA杂交。用分离的大鼠肝细胞核进行的体外转录研究表明，只有这个基因，而不是其他基因组克隆所代表的基因，在苯巴比妥处理大鼠后似乎被显著激活。尽管存在少量差异，但对这些基因组克隆中存在的该基因的八个外显子进行DNA序列分析表明，它们编码细胞色素P - 450e（一种主要由苯巴比妥诱导产生的同工酶）的58至491位残基（COOH末端）。其他克隆基因似乎可能只包含与细胞色素P - 450e基因具有非常强同源性的一个小区域，该区域包括编码一个十三肽的外显子，这个十三肽也存在于兔肝脏细胞色素P - 450的两种不同形式中，一种是组成型的，另一种是由苯巴比妥诱导产生的。大鼠肝脏DNA的Southern印迹分析表明，大鼠基因组可能还包含另外两个与细胞色素P - 450e基因非常密切相关的基因，但我们尚未从基因组文库中分离出来。其中一个基因可能编码细胞色素P - 450b，它是细胞色素P - 450主要的苯巴比妥诱导形式，其mRNA与编码细胞色素P - 450e的mRNA有超过95%的同源性。
