Ian R . Phillips , Elizabeth a . Shephard , Alan Ashworth , and Brian

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family. The cytochrome P-450-mediated mixed-function monooxygenases of liver microsomal membranes are of central importance in the metabolism of steroid hormones and xenobiotics, including many drugs, carcinogens, and environmental pollutants (1, 2). These enzyme systems are also present in many other tissues and in a wide range of organisms (3). Each individual organism usually possesses a number of different cytochromes P-450 (4, 5), many of which are selectively inducible by a variety of foreign compounds. The spectrum of isozymes induced varies with the nature of the xenobiotic to which an organism is exposed (6, 7). In previous studies, we isolated and sequenced a cDNA clone [pP450(1)]* that codes for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.) (8, 9). cDNA clones coding for several cytochromes P-450 of rat, rabbit, mouse, cow, and chicken have been isolated by ourselves and others (10-18). They have already provided valuable information on the amino acid sequences of some cytochromes P-450 from different species and on the molecular biology of the induction processes. Individual human variations are well known in the metabolism of some foreign compounds such as therapeutic drugs (19-22). This metabolic variability may be due, in part, to genetically determined factors relating to the structure and expression of cytochrome P-450 isozymes. The existence of multiple forms of cytochrome P-450 in humans has been directly demonstrated by the isolation, from human liver microsomes, of several cytochrome P-450 isozymes that vary in their catalytic activity toward drugs and carcinogens (23). The amounts of some of the isozymes were found to vary greatly between individuals. Virtually nothing is known about the structure of human cytochromes P-450 nor is there any information available on the organization, expression, and control of the genes that code for these proteins. The isolation and characterization of DNA clones coding for human cytochrome P-450 isozymes will facilitate elucidation of the structure of these proteins and is essential for investigation of the molecular biological basis of genetically determined variation in the metabolism of foreign compounds. In this paper, we describe the use of a cloned sequence coding for a rat liver cytochrome P-450 as a molecular hybridization probe for the isolation of a cDNA clone coding for a human cytochrome P-450. The nucleotide sequence of the human cDNA and the amino acid sequence deduced from it is reported and compared with those of cytochrome P-450 variants from other organisms. MATERIALS AND METHODS Screening of the Adult Human Liver cDNA Library. The cDNA library used was provided by Derek Woods of the Harvard University Hospital Medical School, and its construction has been described (24). Bacteria were plated on two 22 x 22 cm nitrocellulose filters (Millipore Triton Free HATF) at a density of -50,000 colonies per filter and grown at 37°C on L-agar plates containing tetracycline at 12.5 ,ug/ml. When the colonies had reached a diameter of -0.5 mm, they were replica-plated twice. The colonies on the master filters were grown to a diameter of 2 mm on plates containing 25% glycerol (25) and stored at -20°C. After growth of the colonies on the duplicate replica filters to a diameter of =1 mm plasmids were amplified by chloramphenicol treatment. Colony DNA was fixed to the filters (26) and bacterial debris was removed (27). The filters were prehybridized overnight at 30°C in 50% (vol/vol) formamide/0.9 M NaCI/50 mM sodium phosphate, pH 7.7/5 mM EDTA/5 x standard Denhardt's solution (28)/0.1% NaDodSO4/denatured salmon sperm DNA (200 ,ug/ml)/ poly(A) (10 ,g/ml). The buffer was then replaced with 50% formamide/0.9 M NaCl/50 mM sodium phosphate, pH 7.7/5 mM EDTA/2 x standard Denhardt's solution/0.1% NaDodS04/denatured salmon sperm DNA (100 ,g/ml)/Escherichia coli DNA (5 ,ug/ml)/pAT153 (10 ,g/ml)/poly(A) (10 ,ug/ml)/9% (wt/vol) dextran sulfate (50 1td/cm2 of filter) and incubation was continued for 5 hr. The 32P-labeled insert of pP450(1) (ref. 9) was added to a final concentration of 5 ng/ml and the filters were hybridized for 40 hr at 30°C. The filters were washed with gentle agitation for four 15-min periods with 1 M NaCl at room temperature then for two 1-hr periods with 1 M NaCl/0.1% NaDodSO4 at 50°C (29, 30). *pP450(1) refers to the first recombinant plasmid that our group identified as containing a cDNA insert coding for a rat cytochrome P-450. pHP450(1) and pHP450(2) refer to the first and second recombinant plasmids, respectively, that were identified as containing a cDNA insert coding for a human cytochrome P-450. 983 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 984 Biochemistry: Phillips et al. Bacterial colonies in areas corresponding to strong positive signals were picked from the master filters and screened as above at a colony density of -300 per 88-mm-diameter nitrocellulose filter. Positive colonies were picked into microtiter trays, grown at 370C, transferred in an ordered array to nitrocellulose filters (9), and rescreened as above. Preparation of Radiolabeled Nucleic Acid Probes. Plasmids pP450(1) and pHP450(1) were isolated by alkaline lysis (31) and purified by centrifugation to equilibrium in a CsCl gradient (26). The cDNA insert of pP450(1) was excised by digestion with BamHI and isolated by electrophoresis in a gel of 0.8% agarose (low-melting; Seakem). The insert was recovered by chromatography on an NACS-52 column (Bethesda Research Laboratories) according to the manufacturer's recommendations and precipitated by addition of ethanol. DNAs were labeled by nick-translation (26) to a specific radioactivity of =108 dpm/,ug with [a-32P]dATP (specific radioactivity, 400-800 Ci/mmol; 1 Ci = 37 GBq; Amersham International). Southern Blotting. Plasmid DNA. Plasmid DNA was isolated (31) and cDNA inserts were excised by digestion with the appropriate restriction endonuclease. DNA was electrophoresed in a 1.5% agarose gel and blotted onto a nitrocellulose filter (0.45 ,Am; Schleicher & Schuell) (32). The filter was prehybridized at 30'C for 2 hr. The solution was replaced with hybridization buffer and incubation was continued at 30'C for 1 hr. Prehybridization and hybridization buffers were as described above for colony hybridizations. The radioactive probe was added to a final concentration of 1 ng/ml and the filter was hybridized at 30'C for 40 hr. The filter was washed under low-stringency conditions (see below). Genomic DNA. High molecular weight genomic DNA was isolated from rat liver or human leukocytes (33) and digested for 5 hr with EcoRI (New England Biolabs) (4 units/,g of DNA) under conditions recommended by the supplier. Digested DNA was electrophoresed in an 0.8% agarose gel at 1.5 V/cm for 16 hr. DNA was denatured and blotted onto a nitrocellulose filter (32). Filters were prehybridized and hybridized as described for plasmid DNA Southern blots but with the following modifications. E. coli DNA and pAT153