Open AccessStructure of the Mouse Cytochrome P 1 ‐450 Genomic Gene
Author(s) -
NAKAMURA Michitoshi,
NEGISHI Masahiko,
ALTIERI Mario,
CHEN YuanTsong,
IKEDA Toshihiko,
TUKEY Robert H.,
NEBERT Daniel W.
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07525.x
Subject(s) - biology , genomic dna , microbiology and biotechnology , genomic library , restriction enzyme , genetics , complementary dna , gene , clone (java method) , dna , base pair , restriction map , plasmid , peptide sequence
Clone 46 was previously shown to represent mouse cytochrome P 1 ‐450 cDNA by both translation arrest experiments and segregation of induced P 1 ‐450 mRNA with induced aryl hydrocarbon hydroxylase activity among individual 3‐methylcholanthrene‐treated offspring of the (C57BL/6N)(DBA/2N)F 1 × DBA/2N backcross. With clone 46 as a probe, a MOPC 41 mouse genomic‐DNA library was screened. λ3NT12, a 16 × 10 3 ‐base‐pair insert of genomic DNA grown in a recombinant Charon 4A λ vector phage, was isolated and characterized. It was determined that clone 46 hybridizes to the extreme 5′ end of λ3NT12. pMJE12, a 3.0 × 10 3 ‐base‐pair fragment in the 5′ region of λ3NT12, was subcloned in plasmid pBR322 and used as a probe to screen again the same mouse‐DNA library; recombinant phages λ3NT13, and λ3NT14, and λAhP‐1 were isolated and characterized. The relative orientation of each of the four genomic clones on the mouse chromosome was determined. Only λAhP‐1 contains the entire P 1 ‐450 genomic gene, which by R‐loop analysis spans about 46 × 10 2 base pairs and contains at least five exons. Clone 46 is shown to be a 3′ unique sequence of the genomic P 1 ‐450 gene. The λAhP‐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 P 1 ‐450 gene, two and three hybridizable fragments are found with mouse genomic DNA that has been digested with Eco RI and Bam HI, respectively.