Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes.

Filter-hybridization studies show that major phenobarbital and pregnenolone-16alpha-carbonitrile-inducible cytochrome P-450 mRNAs in rats were encoded by members of separate, distinct gene families. These gene families are genetically divergent from each and show no cross-hybridization, even under low-stringency conditions. Furthermore, sequences contained in the P-450PB and P-450PCN gene families map to separate chromosomes of the mouse genome. Using mouse X Chinese hamster somatic cell hybrids (EBS cell lines), all distinguishable P-450PCN sequences were found to map to chromosome 6, whereas all P-450PB sequences were located on chromosome 7. Our data support the proposition that the region of the Coh locus on chromosome 7 is the site of the cytochrome P-450PB gene family. The presence of gene families for the cytochromes P-450 occurs in many mammalian species and is likely an important part of the mechanism by which the mixed-function oxidase system is capable of recognizing and metabolizing such a wide array of endogenous and foreign compounds. Conversely, NADPH-cytochrome P-450 oxidoreductase appears to be encoded in many vertebrate species by a single gene and is located on chromosome 6 of the mouse. Corroboratory data are presented to show that the Eph-1 locus on chromosome 1 is the site of at least one microsomal epoxide hydratase gene.

The mixed-function oxidase system, comprising the cytochromes P-450 and the electron-donating NADPH-cytochrome P-450 oxidoreductase, has been the focus of intensive research by many laboratories. The ability of these proteins to catalyze the oxidation of a broad spectrum of xenobiotics, as well as to participate in the metabolism of various endogenous substrates such as steroids and fatty acids, makes them enzymatically important. Recent studies (1-7) have demonstrated that the diverse substrate specificities are attributable in large measure to the existence of numerous isozymic forms of cytochrome P-450. Characterization of individual cyto-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  chromes P-450, such as the major forms induced by phenobarbital, 3-methylcholanthrene, and pregnenolone-16a-carbonitrile, indicates differences in antigenic and catalytic properties as well as amino-terminal sequences and peptide maps (1)(2)(3)(4)(5)(6)(7)(8)(9). On the other hand, research directed at both the protein and nucleic acid levels clearly demonstrates the existence of other closely related isozymes which show extensive homology to previously known forms (3,5,10,11). The basis for this microheterogeneity is thought to be primarily genetic, with each slightly variant cytochrome P-450 being encoded by a separate gene, or an allelic form of a gene, which is itself a member of a gene family.
This study presents data regarding the genetic localization of two cytochrome P-450 gene families in mice, one detected by a cDNA to a major phenobarbital-induced cytochrome P-450 mRNA in rats (pP-450b-5) and the other detected by a cDNA to a major pregnenolone-16a-carbonitrile-induced species also in rats (pP-450PCN-10). The chromosomal assignments of the NADPH-cytochrome P-450 oxidoreductase and epoxide hydratase genes are also reported, and the significance of the genetic arrangement of the mixed-function oxidase system to its function is discussed.

EXPERIMENTAL PROCEDURES
Somatic Cell Hybrids-Cell hybrids (EBS) between Chinese hamster cells (clone E36) deficient in hypoxanthine phosphoribosyltransferase (HPRT-) and mouse spleen cells from BALB/c mice were generated and maintained as described (12). Under the conditions employed, hybrid clones retain all the Chinese hamster chromosomes but randomly segregate mouse chromosomes, resulting in the isolation of Chinese hamster X mouse cell hybrids that contain different numbers and combinations of mouse chromosomes. The mouse chromosome content of the hybrid clones was determined enzymatically and cytogenetically. Each hybrid clone was analyzed for the expression of 25 enzymes representing linkage groups assigned to 16 of the 19 autosomes and the X chromosome as described (13-15). Trypsin-Giemsa banding was used to identify each of the mouse chromosomes as described (15). The enzymatic, cytogenetic, and Southern blotting analyses were carried out on parallel cultures of each hybrid clone so that all the data were correlated. Individual clones were considered positive for a given chromosome if greater than 15% of the metaphases examined contained the chromosome; the clones were scored negative for that chromosome if less than 5% of the metaphases contained the chromosome. If chromosomes were retained with a frequency between 5 and 15%, the score was considered indeterminant and not included in the segregation analysis of that chromosome.
Animals-Outbred Holtzmann rats and New Zealand White rabbits were used in this study. Recombinant inbred mouse strains (RI strains) as well as inbred strains came from the Jackson Laboratory (Bar Harbor, ME).

RESlII.TS A N D DlSClJSSION
('hromosomnl Assignmant of thc P-450PCN Gana Fnmi1.v-Although only a single pregnenolone-16w-carhonitrile-induced cytochrome P-450 has so far heen purified (61, Sout.hern hlots ( Fig. 1) suggest that like the phenoharhit,al-induced fnrms, other related isozymes exist. This conclusion is hased on the ohservation that the cytochrome P-450PCN prohe hvhridizes with approximately 50-60 kh of rat genomic DNA. Hy comparison, cytochrome P-450h cDNA, which is known to recognize approximately eight genes (26,27), detects in the range of 90-120 kh of DNA. These data are taken as being a strong indication that multiple genes do exist for cytochrome P-450PCN rather than a single large gene. We have termed the genes for these putative proteins the P-450PCN gene familv hecause of the homology of these gene sequences to the pP-45OPCN-10 prohe. However, it is important to note that all these genes may not he functional and, if functional, may not he induced hy pregnenolone-16n-carhonitrile. may he induced hv pregnenolone-lftr-cart~onit rile. Hence, the nomenclature used is hased on sequence similarity and not inducihility.
Digestion of DNA from the ERS somatic cell hyhrids with the restriction enzyme PouII generates seven fragments, of which four can he discriminated from hamster sequences on Southern hlots with the P-4:iOPCN-10 prohe (Fig. 2 ) . Comparison of the autoradiographic pattern with the chromosomal complement of each hybrid (Tahle I ) indicates that a11 four fragments map to chromosome 6 of the mouse. An identical chromosomal assignment has also been ohtained for a11 discernihle fragments of DNA from these cell lines digested with  with pP-450PCN-10. Aliquots (10 wg) of DNA from each cell line were digested with PuuII and electrophoresed on a 0.9% agarose gel. Following transfer to nitrocellulose, the filter was hybridized to nick-translated pP-450PCN-10 (2 X lo6 cpm/ml) for 48 h at 50 "C under high-stringency formamide conditions. Standards are HindIII-digested X DNA. E36 is the hamster cell line and BALR/cByJ is the mouse strain closely related to the BALB/c strain from which the cell hybrids were originally derived. The black dots bracket mouse restriction fragments used to map the cytochrome P-450PCN gene(s) to chromosome 6. some 6. We have named the locus for this gene complex the Pcn locus.
Linkage of the Cytochrome P-45OPB Gene Family on Chro-mosome 7-We have previously reported the mapping of five restriction fragment-length polymorphisms, detected by hybridization to the pP-450b-5 probe, to the Coh locus on chromosome 7 in mice (18). This locus was shown by Wood and Taylor (28) to code for coumarin hydroxylase, a phenobarbital-inducible, cytochrome P-450-dependent, enzyme activity. When the pP-450b-5 restriction fragment-length and coumarin hydroxylase polymorphisms were compared among 24 recombinant inbred and 15 inbred mouse strains, a single discordance (in the CBA/CaJ mouse) was noted. From these observations, we postulated that the coumarin hydroxylase gene was very closely linked to other similar cytochrome P-450 gene sequences which collectively were designated the cytochrome P-450PB gene family. The autoradiographic pattern, observed among DNAs obtained from the EBS cell lines digested with MspI and probed with pP-450b-5, demonstrates that 12 of 19 mouse MspI restriction fragments can be discriminated (Fig. 3). All 12 fragments map to chromosome 7 , and similar results have been obtained with PuuII and EcoRI digests of genomic DNA. Therefore, in conjunction with the previously reported data, these results strongly suggest clustering of the P-450PB gene family on chromosome 7, probably at the Coh locus (complex). Anot.her factor which substantially increases the complexity of this gene complex is the relatively high degree of genetic polymorphism evidenced by the cytochromes P-450 in such species as mice (18,28), rabbits (29), rats (30,311, and humans (32-34). Vlasuk et al. (35) demonstrat,ed using t.wo-dimensional gel electrophoresis that at least four variant cytochromes P-450 were induced by phenobarbit,al in rats. Outbred rat strains and even individual colonies of the same strain exhibited different combinations of the variant forms. Recently, Rampersaud and Walz (30), using the same electrophoretic technique, showed that at least six variants were detectable, and appropriate crosses between outbred rats having different phenotypes suggested that t,hese six forms were encoded by two very closely linked autosomal loci. These loci, designated P-450b and P-450e, have multiple alleles, with the P-450b locus having four allelic forms and the P-450e locus having two. The genetic mechanism for this relatively large number of polymorphisms has yet to be explored. Certainly, TABLE I Segregation of epoxide hydratase, oxidoreductase, P-450PB, P-450PCN, and mouse chromosomes in mouse x hamster cell hybrids Restriction-endonuclease digestion was performed on DNA extracts of mouse X Chinese hamster hybrid clones; duplicate cultures of' the same passage were used for enzyme and karyotype analyses. Whether a particular chromosome was scored, + orwas determined as described under "Experimental Procedures."

Markers
Chromosomes Hybrid  Rampersaud and Walz (30) are in agreement with our conclusions that memhers of t,he P-450PR gene family are tight,ly linked, with the Coh locus (complex) being the site of the clustered genes in mice and the P-450b and P-450e loci representing the homologous complex in rats.

4.5f)l'CN-Major differences have been noted
at the cDNA and mRNA levels for cytochromes P-450h and P-450PCN (21). Comparison of the respective cDNAs by restriction mapping revealed no similarities (21), and Northern hlot analysis demonstrated that P-450b and P-450PCN mRNAs were 2150 and 2500 nucleotides in length, respectively (21). Furthermore, Southern blot experiments (Fig. 1) indicate that the genes encoding the P-450b-and P-450PCN-like cytochromes are either unrelated or possess a very low level of homology.
Direct hybridization between pP-4.50b-5 and pP-450PCN-1 0 under conditions of low stringency (30% formamide, 6 X SSC, 42 "C) failed to detect any sequence homology (Fig. 4). No hybridization of nick-translated pP-45OPCN-10 probe to the filter-hound 1.8-kh pP-450b-5 insert was observed (Fig.   4C), even on long exposures of the autoradiogram. Similarly, the isotopically labeled pP-450b-5 probe did not hybridize to the Pstl restriction fragments (1.1 and 0.9 kb) derived from pP-450PCN-10 (Fig. 4H). Furthermore, no hybridization was noted between the respective probes and epoxide hydratase, oxidoreductase, and serum albumin cDNAs. Taken in total, the data indicate that marked genetic divergence has occurred to produce these significantly different c-ytochrome P-450 species. Physicochemical and immunological studies have also illustrated clear differences in the properties of cytochromes P-450b and P-450PCN (6).
Chromosomal Assignment of the NADPH Oxidorrductnsr Gene (Par)-The pattern ohtained on a Southern hlot analysis of restricted genomic DNA using pOR-7 cDNA as a prohe is illustrated in Fig. 5 . A single fragment in hoth mouse (15 kt)) and hamster (9.4 kh) DNA was generated upon EcoRI digestion. Comparison of the electrophoretic pattern with the chromosomal complement of each hybrid clone indicates that the locus for the NADPH-cytochrome P-4-50 oxidoreductase gene, designated Por (NADPH-c-ytochrome _P-450 oxidoreductase), maps to chromosome 6 of the mouse (Table  I). Identical results have heen obtained with I'uuII digests of these DNAs.
Chromosomal Assignment of thr Epoxidc Hydratnsr Crnr (Eph-l)-The autoradiographic pattern resulting from the hyhridization of epoxide hydratase cDNA (pF:H-I) and EcoRI-digested ERS somatic cell hybrid and control DNA is presented in Fig. 6. Several hybridizahle fragments are noted in hamster (EX<), while a single restriction fragment (>23 kh) capable of hyhridizing with epoxide hydratase cDNA was found in the RALR/cHyJ mouse genome. The mouse specific epoxide hydratase gene fragment mapped to chromosome 1 (Table I). This result is in agreement with the assignment made hy Lyman at al. (36) who used an enzyme-activity polymorphism in C57RL/Gd and DRA/P.J mice to map epoxide hydratase using RXD recombinant inbred mice. Recent evidence suggests that in hamsters at least two related microsomal epoxide hydratase-like genes are expressed ( X ) . This is consistent with the multiple hyhridizing sequences observed on Southern hlots for this species (Fig.  6). Mice, however, consistently show fewer hybridizing DNA fragments in genomic blots and may possess fewer genes coding for this protein.

Genetic Arrangement of the Mixrd-function Oxidasr Sys-
tem-Somatic cell hybrids in combination with cloned cDNA prohes are powerful tools to analyze the genetic arrangement of the mixed-function oxidase system and to clarify the relationship of individual c-ytochromes P-450 to one another. Our studies have shown that the major phenoharhital-and pregnenolone-16n-carbonitrile-induced forms of cytochrome 1'-450 arise from distinct gene families, which are not syntenic, located on chromosomes 7 and 6 respectively. In addition, it is likely that these gene families are clustered on these chromosomes, since all restriction fragments, regardless of the enzyme utilized, map to chromosome 7 in the case of pP-450b-5 or chromosome 6 in the case of pP-4501'CN-10. Hased on earlier mapping studies (18), we identified the C'oh locus as the site of the P-450PR gene family, and we now recommend that the locus on chromosome 6 representing the site of the P-450PCN gene family be termed the I'm locus.
From the available data, it may be hypothesized that mammalian c-ytochrome P-450 gene families arose from R primordial gene through a series of duplications. These events could have given rise by genetic divergence to generally nonsvntenic, distantly related clusters of c.ytochrome P-4.50 genes of which the P-450PR and P-450PCN gene families are representative. A similar scenario is thought to have created such nonsvntenic, but homologous, gene families as the O -and /j-glot)ins (38) as well as the genetic superfamily comprising the class I and I1 histocompatibility, IgG, &microglohulin, and Thy 1 genes (39). Hybridization of both the PI'-45011-5 and pP-450PCN-10 probes to filter hound DNA from diverse vertebrate species (Table  11) is intense in mammals which are evolutionarily closely related to the rat, such as mouse and hamster. In contrast, much weaker hyhridization signals are of the Mixed-function Oxidase Systcm 5 19 pOlt-7, and pl'-4.5Oh-5, and pHIU22 were digested with I'stl to liberate the insert from the vector. IfindlII-digested X DNA (rnnr/wr I ) and Hinfl-digested pHIt322 (mnrhrr 2) were used as size stnndnrds. 1)igests were electrophoresed in 0.9"; agarose gels, hlotted, and hyhridized under low-stringency ronditions (see "Experimental Procedures"). A is the ethidium bromide-stained gel, and H and C are autoradiographs o l filter lilts hyhridized with nick-translated pP-45Oh-5 and p1'-4501'CN-10, respectively. No isotopically lt1t)eIrd A was inclr~ded in the hyhridization solution; therefore. only the Hinll-digested pHKS22 marker appears. detected in more distantly related species such as rabbit (18) and human, and hybridization has not been detected to nonmammalian DNAs such as from turkey, yeast, or slime mold even under low-stringency conditions (Table 11). These data indicate that. the cytochrome P-450 genes are not conserved among species to the same extent as genes coding for such highly conserved proteins as tubulins (40, 41), actin (41, 42), and cert,ain oncogenes (43, 44) which are conserved not only among mammals, but among other vertebrate classes and nonvert,ebrates as well.
In contrast to the cytochromes P-450, NADPH-cytochrome P-450 oxidoreductase is encoded by a single gene (Por) located on chromosome 6. Fig. 7 shows the hybridization of pOR-7 2.0 -2.3 -

'
cDNA with EcoRI-digested DNA from a collection of vertebrate and nonvertebrate species. Two salient features differentiate the patterns observed for the cytochromes P-450 and that seen for NADPH-cytochrome P-450 oxidoreductase: 1 oxidoreductase cDNA detects one to two fragments as opposed to multiple fragments detected bv the cvtochrome 1)-450 cDNAs, and 2) homologous sequences are detected in a nonmammalian species (turkey) by pOR-7. These observations are consistent with oxidoreductase being encoded bv a single

TARIX II E:idunlirrn nf inlcrsprcics rfhlionships of the mixrd-function oxidnsr systrm by Southcrn h/nt nnn/ysis
All hvhridizations were performed at hoth high and low stringencies. +, sequences were detectable under high or low stringency: -, n o detectahle hyhridization; f, data were inconclusive. Multiple genes were suggested hv Southern hlots (;,e. at least five fragments were visualized, with a t o t a l size of greater than 30 kilnhases of hyhridizing IINA).

Q, v)
Flc:. 7. Hybridization of pOR-7 to EcoRI-digested DNA from diverse vertebratespecies. Nitrocellulose-t)lottrd I)NA from w c h vertet)rIlte species was hyhridizcd t o pOH-7 under either lowst ringcwcy formnmitle conditions (rat, mouse. rahhit. human, yeast, and /'hjsnrurn) or aqueous high-stringency conditions (turkey). The tlil'l'llsc. hnntling pattern seen upon hybridization t o I'h\'sorum DNA is charncteristir 01' nonspecific hinding of the prohe and, unlike the patterns s e m lor the o t her organisms. disappears upon more stringent hyhridiztlt ion condit inns. gene which is more highly evolutionarily conserved than the cytochromes P-450. Physical and functional similarity of microsomal NADPH-cytochrome P-450 oxidoreductase in such distantlv related vertebrates as rahbit,s (451, elasmohranchs (46), and teleosts (47, 48) further indicates evolutionary preservation of' this protein. This high degree of conservation is important in light of the diversity of forms of cytochrome P-450, since it is thought that all microsomal cytochrome P-4.50s directly interact with NADPH-cytochrome P-450 oxidoreductase as terminal electron acceptors. I t appears that oxidoreductase has been structurally conserved, whereas the cytochromes P-450 have retained only those structural regions essential for heme binding and correct interact ion with the oxidoreductase, thus preserving the functional relationship in the electron-transport chain. The relatively high degree of evolutionary conservation of the oxidoreductase gene presumably reflects the essential nature of this protein.
In conclusion, the evidence suggests that gene duplication, possibly in conjunction with a high degree of genetic polymorphism (18, 301, is a critical part of the mechanism hy which t h e c-ytochromes P-450 as a group manifest such a broad range of catalytic ahilities.