Nakamura M, Negishi M, Altieri M, Chen Y T, Ikeda T, Tukey R H, Nebert D W
Eur J Biochem. 1983 Jul 15;134(1):19-25. doi: 10.1111/j.1432-1033.1983.tb07525.x.
Clone 46 was previously shown to represent mouse cytochrome P1-450 cDNA by both translation arrest experiments and segregation of induced P1-450 mRNA with induced aryl hydrocarbon hydroxylase activity among individual 3-methylcholanthrene-treated offspring of the (C57BL/6N)(DBA/2N)F1 X DBA/2N backcross. With clone 46 as a probe, a MOPC 41 mouse genomic-DNA library was screened. lambda 3NT12, a 16 X 10(3)-base-pair insert of genomic DNA grown in a recombinant Charon 4A lambda vector phage, was isolated and characterized. It was determined that clone 46 hybridizes to the extreme 5' end of lambda 3NT12. pMJE12, a 3.0 X 10(3)-base-pair fragment in the 5' region of lambda 3NT12, was subcloned in plasmid pBR322 and used as a probe to screen again the same mouse-DNA library; recombinant phages lambda 3NT13, lambda 3NT14, and lambda AhP-1 were isolated and characterized. The relative orientation of each of the four genomic clones on the mouse chromosome was determined. Only lambda AhP-1 contains the entire P1-450 genomic gene, which by R-loop analysis spans about 46 X 10(2) base pairs and contains at least five exons. Clone 46 is shown to be a 3' unique sequence of the genomic P1-450 gene. The lambda AhP-1 genomic-DNA clone from the MOPC 41 plasmocytoma is shown by a series of restriction enzymes to be the same as genomic DNA from normal mouse liver. With a subclone in the 5' portion of the P1-450 gene, two and three hybridizable fragments are found with mouse genomic DNA that has been digested with EcoRI and BamHI, respectively.
克隆46先前已通过翻译终止实验以及在（C57BL/6N）（DBA/2N）F1与DBA/2N回交的经3-甲基胆蒽处理的单个后代中诱导的P1-450 mRNA与诱导的芳烃羟化酶活性的分离，表明其代表小鼠细胞色素P1-450 cDNA。以克隆46为探针，筛选了MOPC 41小鼠基因组DNA文库。分离并鉴定了λ3NT12，它是在重组Charon 4A λ载体噬菌体中生长的16×10³碱基对的基因组DNA插入片段。已确定克隆46与λ3NT12的最末端5'端杂交。将λ3NT12 5'区域中的3.0×10³碱基对片段pMJE12亚克隆到质粒pBR322中，并用作探针再次筛选相同的小鼠DNA文库；分离并鉴定了重组噬菌体λ3NT13、λ3NT14和λAhP-1。确定了这四个基因组克隆在小鼠染色体上的相对方向。只有λAhP-1包含完整的P1-450基因组基因，通过R环分析，该基因跨度约为46×10²碱基对，并且至少包含五个外显子。克隆46被证明是基因组P1-450基因的3'独特序列。来自MOPC 41浆细胞瘤的λAhP-1基因组DNA克隆经一系列限制酶分析与正常小鼠肝脏的基因组DNA相同。使用P1-450基因5'部分的亚克隆，分别用EcoRI和BamHI消化的小鼠基因组DNA发现了两个和三个可杂交片段。
