Negishi M, Swan D C, Enquist L W, Nebert D W
Proc Natl Acad Sci U S A. 1981 Feb;78(2):800-4. doi: 10.1073/pnas.78.2.800.
Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P(1)-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric "tailing" and cloned in E. coli LE392. Clone 46 hybridized with [(32)P]cDNA made from 23S mRNA from "Ah-responsive" C57BL/6N mice but did not hybridize with similarly prepared [(32)P]cDNA from "Ah-nonresponsive" DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [(32)P]cDNA probes; these two clones were therefore used as "positive" and "negative" control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-P(1)-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 (32)P-labeled nick-translated DNA to EcoRI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P(1)-450.
利用已知与Ah位点及3 - 甲基胆蒽诱导的细胞色素P(1)-450相关的部分纯化的小鼠肝脏23S mRNA，我们通过逆转录酶（RNA指导的DNA核苷酸转移酶）和大肠杆菌DNA聚合酶I的Klenow A片段的连续作用合成了双链cDNA。双链cDNA通过Pst I切割和同聚物“加尾”插入pBR322质粒DNA，并克隆于大肠杆菌LE392中。克隆46与由“Ah反应性”C57BL/6N小鼠的23S mRNA制备的[(32)P]cDNA杂交，但不与由“Ah无反应性”DBA/2N小鼠类似制备的[(32)P]cDNA杂交。克隆30对C57BL/6N和DBA/2N的[(32)P]cDNA探针均呈阳性，克隆7均呈阴性；因此这两个克隆分别用作“阳性”和“阴性”对照克隆。通过翻译抑制实验表明，克隆46 DNA和克隆30 DNA分别与抗P(1)-450和抗白蛋白可沉淀物质相关。通过对Pst I消化产物进行琼脂糖凝胶电泳，显示克隆46 DNA插入片段全长为1100个碱基对，并含有一个内部Pst I位点。由3 - 甲基胆蒽处理的C57BL/6N小鼠分离的总mRNA制备的cDNA与克隆46经Pst I消化的DNA的两个片段杂交，而由3 - 甲基胆蒽处理的DBA/2N以及对照C57BL/6N和DBA/2N小鼠类似制备的cDNA则不杂交。在所使用的11种限制性内切酶中，两种（Pst I和Xba I）在克隆46 DNA插入片段内有切点。将克隆46经(32)P标记的切口平移DNA与A/HeJ小鼠基因组DNA的EcoRI片段杂交，经RPC - 5柱层析和凝胶电泳分离后，仅出现一条阳性带（3 - 4千碱基对）。这些数据确凿地证明，pBR322克隆46 DNA与受小鼠Ah位点控制的mRNA相关，推测该mRNA为编码3 - 甲基胆蒽诱导的P(1)-450的结构基因。
