Proc. Natl. Acad. Sci. USAVol. 82, pp. 5310-5314, August 1985Biochemistry

Cloning and characterization of cDNAs encoding2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible rabbit mRNAs forcytochrome P-450 isozymes 4 and 6

(monoclonal antibodies/mRNA precursors)

STEVE T. OKINO*, LINDA C. QUATTROCHI*, HENRY J. BARNESt, SUSAN OSANTO*, KEITH J. GRIFFINt,ERIC F. JOHNSONt, AND ROBERT H. TUKEY*t*Cancer Center, University of California, San Diego, La Jolla, CA 92039; and tDepartment of Biochemistry, Research Institute of Scripps Clinic, La Jolla,CA 92037

Communicated by Helen M. Ranney, April 24, 1985

ABSTRACT Monoclonal antibodies toward rabbit livercytochrome P-450 isozyme 6 (P-450 6) were used to identifyseveral recombinant clones from a pBR322 cDNA library thatexpress 13-lactamase-P-450 6 hybrid proteins. The nucleic acidsequence and predicted amino acid sequence of a rabbit P-4506 cDNA shows a high degree of homology with rat P-450c andmouse P1-450. When used as a probe to rescreen the library,the P-450 6 cDNA hybridized to several heterologous classes ofcDNAs. One such class was shown to encode. P-450 4 bycomparison of its predicted amino acid sequence to amino acidsequences of cysteine-containing tryptic peptides derived fromP-450 4. DNA sequence analysis of a cDNA clone belonging toa third class demonstrated that it contained a 131-base-pairintervening sequencing when compared to the cDNA coding forP-450 6. This sequence corresponds in location to intron E ofthe rat P-450c gene. Blot-hybridization analysis demonstratedthat 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramaticallyinduced P-450 4 and 6 mRNAs, which differ in size. The sizesof these mRNAs differ from their analogs in the mouse as aresult of divergence in the 3' untranslated portions of themRNAs.

The treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in the induction of two majorcytochrome P-450 isozymes, 4 and 6 (P-450 4 and 6) (1). Theinduction of P-450 6 occurs in several tissues (2) and atvarious ages (1). This P-450 is analogous to rat P-450c (3, 4)and mouse P1-450 (5) in both its pattern of expression andcatalytic function. In contrast, the induction by TCDD ofrabbit P-450 4, which is analogous to rat P-450d (3, 6, 7) andmouse P3-450 (5), is restricted to the liver of mature animals(1, 2). Sequence analysis ofcDNAs encoding the rat (8, 9) andmouse (10, 11)-cytochromes P-450 indicates extensive aminoacid sequence homology (>90%) between rat P-450c andmouse P1-450 and between rat P-450d and mouse P3-450 (10)as well as regions of substantial nucleic acid sequencehomology. In addition, seven exons comprise the geneencoding rat P-450c (12), and similar exogenic sequences areevident for the mouse P1-450 gene (11).We have extended the characterization of this P-450 gene

family to P-450 6 and P-450 4 of the rabbit. Using '25I-labeledmonoclonal antibodies directed toward P-450 6 to screen apBR322 expression library, we have identified a cDNA cloneencoding P-450 6. This cDNA was used in turn to detectadditional clones by colony hybridization permitting us toidentify cDNAs encoding P-450 4. We also have isolated andsequenced a cDNA to P-450 6 that appears to have retainedan unspliced intron, thus allowing a direct comparison

between the structure and location of this region and that ofthe rat P-450c gene.

MATERIALS AND METHODSProduction and Purification of Antibodies. Monoclonal

antibodies were generated toward a purified preparation ofP-450 6 in a fashion similar to that already described for P-4501 (13). Each antibody was purified from serum-free cellculture supernatants by using protein A-Sepharose and eitherlinked to Sepharose CL-4B activated with cyanogen bromidefor use in immunoprecipitation reactions or, where appro-priate, labeled with 125I as described (13).

Immunological Identification of cDNAs That Encode P-4506. Liver RNA was isolated by the guanidine HCl method (14)from a rabbit 24 hr after administration (1) of 10 nmol ofTCDD per kg of body weight. Poly(A)+ RNA was enrichedby two cycles over a poly(dT)-cellulose column (15). Thepoly(A)+ RNA was then fractionated by sucrose densitygradient centrifugation (16), and fractions containing mRNAcoding for P-450 4 and P-450 6 were identified by in vitrotranslation and immunoprecipitation of the nascent proteinsby using monoclonal antibodies covalently linked to Sepha-rose. The immunoprecipitated products were analyzed byautoradiography following polyacrylamide gel electrophore-sis in the presence of sodium dodecyl sulfate (17). Fractionsenriched with mRNAs for P-450 4 and P-450 6 were used togenerate double-stranded sDNA (18), which was tailed withhomopolymer dCTP and hybridized into the Pst I site of the,8-lactamase gene in pBR322, which was tailed withhomopolymeric dGTP (19). E. coli HB101 were transformedwith the plasmid preparation (20) and plated onto nitrocel-lulose filters on LB plates containing tetracycline.Monoclonal antibodies 4B2 and 3A3, which recognize both

P-450 6 and P-450 4, and 1E4, which specifically recognizesP-450 6, were iodinated and used to screen approximately5000 recombinants on duplicate filters as described (21).To identify additional clones, a cDNA insert from an

antibody-positive clone, p6-34, was removed from the Pst Isite of pBR322, labeled with [32P]dCTP by nick-translation,and hybridized to a library of approximately 15,000 clones.Positive clones were then selected, replated to purify singlecolonies, and rescreened with the labeled cDNA.Other Methods. DNA sequencing was performed by the

dideoxy-termination method (22) after subcloning the restric-tion fragments into M13 mplO and mpll phage vectors (23).Blot-hybridization analysis of mRNA from control andTCDD-treated rabbits was performed as outlined (24).

Abbreviations: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; bp,base pairs; P-450, cytochrome P-450.tTo whom reprint requests should be addressed.

5310

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. NatL Acad Sci. USA 82 (1985) 5311

A3A3 1E4

B

6 6k\-

1E4

0*@

p6-34+ clones

P M P M

FIG. 1. Screening with monoclonal antibodies to purified pro-teins and cDNA clones. (A) A mixture of purified P-450 isozymes 4and 6 (5 pmol each) (lanes P) or microsomes (5 Attg of protein) fromTCDD-treated rabbit (lanes M) were subjected to- polyacrylamide gelelectrophoresis in the presence of sodium dodecyl sulfate (17), andthe proteins were transferred to nitrocellulose paper (25). Each filterwas treated with the lu5I-labeled antibodies (2 ,uCi, 1 gg/ml in 3%bovine serum albumin for 3 hr), and the filters were exposed to XARfilm. A Right shows binding of 1E4, while A Left shows binding with3A3. The antibody 1E4 reacts with only P-450 6, whereas antibody3A3 reacts with both P-450 4 and P450 6. (B) A cDNA librarygenerated by cloning the cDNA into the Pst I site of pBR322 wasscreened initially with '25I-labeled monoclonal antibodies. Oneimmunoreactive clone, p6-34, was identified and shown to bepositive with all three antibodies. The insert from p6-34 was labeledby nick translation and used as a probe to isolate 52 related cDNAclones. Each clone was grown on a nitrocellulose disc and screenedindividually with monoclonal antibodies'. B shows the positive clonesand a field of clones that were not antigenic when screened with theP-450 6-specific 1E4 monoclonal antibody.

RESULTS AND DISCUSSION

Characterization of the Monoclonal Antibodies. Monoclonalantibodies were selected from hybridomas that displayeddistinct reactivity toward purified P450 isozymes 4 and 6 asjudged by an ELISA (13). When the antibodies were testedfor their ability to react with microsomal proteins separatedby polyacrylamide gel electrophoresis in the presence of

sodium dodecyl sulfate and transferred to nitrocellulosepaper (25) (Fig. LA), antibody 1E4 reacted with purified P-4506 and to a single electrophoretic class corresponding to P-4506 from TCDD-treated microsomes. In contrast, antibody 3A3reacted with purified P-450 4 and P-450 6 and to similarelectrophoretic forms present in liver microsomes preparedfrom TCDD-treated rabbits. Both antibodies were used asprobes to screen the cDNA library.

Identification of P-450 6 and Related cDNAs. From an initialscreen of 5,000 recombinants, one positive colony, termedp6-34 was selected. To obtain additional clones, the cDNAinsert from p6-34 was nick-translated and used as a probe toscreen approximately 15,000 clones. From 52 recombinantsthat hybridized with the p6-34 cDNA, six generated f3-lactamase gene fusion products that reacted with the I-labeled antibodies. After colony purification and rescreeningof the six immunopositive clones, each was tested for theirability to react with the 1E4 monoclonal antibody, whichrecognizes only P-450 6. As shown in Fig. 1B, all sixrecombinants produced fusion proteins reactive with 1E4.The fusion proteins were also reactive with antibody 3A3,and all of the inserts from the six plasmids contained similarrestriction patterns. Because 1E4 is specific for P450 6, thisinitial screen suggested that each of the recombinants con-tained an insert encoding this cytochrome.The plasmids from all of the antibody- and hybridization-

positive clones were characterized by restriction enzymemapping and assigned to three classes that exhibit distinctpatterns ofrestriction fragments as summarized in Fig. 2. Thefirst group of clones was similar to the immunopositive P-4506 clones, with p6-34-1 containing the largest cDNA insert.From the second class of clones, p6-34-2 contained thelargest insert. A single clone representing a third type,p6-34-3, exhibited an identical restriction map to p6-34-1 inboth its 3' and 5' portions but contained several additionalrestriction sites within a short region (underlined in Fig. 2)that were not found in p6-34-1.

Nucleotide Sequence of P450 6 cDNA p6-34-1 and P450 4cDNA Clone p6-34-2. The strategy for determining the nucle-otide sequence of the clones is shown in Fig. 2. Fig. 3 showsthe nucleotide sequence of the P-450 6 cDNA clone p6-34-1.It contains an open reading frame of 1338 nucleotides,encoding 446 amino acids, followed by a 686-bp noncodingregion. Alignment of the predicted amino acid sequence fromthe P450 6 cDNA with that of rat P-450c (8) revealed that theP-450 6 sequence starts at the tyrosine residue equivalent toresidue 73 of rat P-450c (Fig. 4). This alignment elicits 75%homology with the rat sequence.

4%SS -.,, SQ. s~4

Ql -4

4e T T~~~~'I nAJ-JAA- II_______ I I~-4 olI .

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p6-34-2

AMI

-*4%

**L-ve~ra.

pT

i . .*__ o- .._yS'. top6-34-3

FIG. 2. Restriction maps and strategy for sequencing p6-34-1, p6-34-2, and p6-34-3. Restriction sites are shown for the longest representativesof each of three classes of P450 6-related cDNA clones. The arrows indicate restriction fragments that were subcloned into M13 mplO or M13mpll as well as the direction and distance of the subsequent sequence analysis. The underline in p6-34-3 represents the location of the intron,shown in Fig. 5.

4%>

200 bpi --

4e4%VVo.

I,"

~~~~~O

Biochemistry: Okino et aL

pvU-o.-,-

I

I-

5312 Biochemistry: Okino et aL

P450 6

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6P450 4

P450 6

P450 6

ATGAGGA C C G AA AG A G GC

Proc. Natl. Acad Sci. USA 82 (1985)

p6-34-3

ATGCTGACGCTGGGCAAGAACCCACATGTGGCGCTGGCGGCGCTGAGCCGGCp6-34-1 .

CGTACGGGGACGTGTTCCAGATCCGCCTGGGCTCCACGCCC [ACA A

'GATCAAGCACTACCGGGTGGACAGGTTGGACGAGAATGCCAACGTCCAGGTC CA CGAAA G CTCCA G C ACGG G CC C CCCCA

GTCGGACGAGAAGACCGTTGGTATTGTCTTGGACCTCTTCGGAGCTGGGTTTGACACGGTCACAACTGCCATCTCGTGGAGCCTCATGTACCTGGTGACC------ T CACCC C AAC A T CA C A AC A

AAGCGCCAGCATACACGAGAGAAGATCCAGGAGGAGCTGGACGCTGTGGTTGGCAGGGCGCGGCGGCCGCGGTTCTCCGACAGACCCCAGCTGCCCTACCC C AG AGA--- AGAGA A C TCC

TGGAGGCCGTCATCATGGAGACCTTCCGACACACCTCTTTCCTCCCCTTCACCATCCCCCACAGCACCACAAGGGACACCAGTCTGGGTGGCTTTrTACATT C CT G C CA AAC CC

CCCCAAGGGACGCTGTGTCTTCGTGAACCAGTGGCAGAACAACCACGACCGGGAGCTGTGGGGTGTACCCCGAAGCTTCCGGCCCGAGCGATTTCTCACCT A T CA T A A A T C C G AC GGAGGAG C G

CCCAGCGGCACG---CTGGACAAGGGCCCTGACGACAAGGTGCTGCTCTTTGGGCTGGGCAAACGCAAGTGCATCGCGGAGACCATCGGCCGCCTGGAGGG GGA G GCCA CA CC TGAGT G CAC C C C G G G T C G C A GTG A

TCTTTCTCTTCCTGGCCACGCTGCTGCAGCAGGTGGAATTCAGCGTGTCGCCCGGCACGA~CGGTGGACATGACCCCCATCTACGGGCTGACCATGAAGCAC TC C G C G C A GT C C C

CGCCCGCTGTGAACACTTCCAAGCAAAGCTGCGCTTCGAGGCC---TAGACTGTGCCCGGGCCTAATGGGCAGCCTGACCTGGGGACCTGTGCAGGGGGCC G C G TG A TCGC CA TCC A CAG GAGGGCG G GGGGC T G G CG G C T GGGCTC GCC CCC

TGTATTCTGCCTGAAGCCTCACAGCCGCTGGTCAGGCTGATTCTGTGCTCTCTGGGCCGTGGGCAAAGCCAGAGGAACCCAGCCTGCCCCCTCCTCTGGAC CC GCT T CTTCA AGTA CTTC CCG ATGCCCCG CCC TC C A AC CCACC TCCC TGCGCC G T GGCAG GG GG ACT

CTTGTCCCTCTGCGTGCTGACCGCGGTGGGGGCCCACAGGAGCTGCTGGAGGGAGGAGG~CCCTGAGGGCCTCTGATGAACCTCTCTGGAAGCCCCCACGCT CC GTGCGGCTTCCT C TGC CCCACC TGT ATG TCTTGT GCT GCT CTTT CC A GCAAAGCTCC AGG CC GG TA GTC CA

CCGCGGGTGGCAGGCAGCATGTGGGTGCTGACTGGCTGGGGAAACACGAGAGCGGGCCCcATCAGGGCCTGTGTAGTCTZGCTGCACAGCCCACGATCAGGGCTA TG C G CTCT TCTGCTC GCCCAA T T AAC GTGAAGCGC T GGAGGAAG CAACCCAGA GAAGCTC T G TTCTG CC

GCCAGGAGTGAAGGGTGAAAACAGCCCAGGTGCTGGTCCCCAGTGGGTC~CACTGCGGGCGcGGGCTGAGCTGTGTGCCCTGGGGTCTGGGACACCTGGTG CC GCCCTG CTGTT GT CATTTG GAG AA AG AGAT GAAG TCT TCTCT TCTCTCTCT C T TG CT C TCTCTGT A TCT

GCCTATGGGACAAACAGGGCCGTCTCCTGGGAGCAGAGA6CAGAGCGGG;GCCGGCACAGGGGCTGGTGT~CCCAGTGCCTTTTCCACATCCCGTGTTGiT CAA T T AAT]

AATGTCTGTCCTTTAACATTAACGCCCTGTGATGCGCTTGCCTGCTTGCTTAAGAGCCATTTATTGAGTGCAACAGAGAGGGTTCTAACAGCTGCGTGGC

GGTGTGAATAAACACACCTACCAGACA

FIG. 3. The nucleotide sequence for P-450 6 and P-450 4 cDNAs. The sequence of the P-450 6 cDNA p6-34-1 starts at nucleotide 52, whilethe 5' portion of p6-34-3 is 51 base pairs (bp) longer and is shown as the top line of the figure. The sequence of the intervening sequence locatedin p6-34-3 at nucleotide 1015 is shown in Fig. 5. The alignment of the P-450 4 cDNA p-34-2 is based on the alignment of predicted amino acid

sequences as shown in Fig. 4. Only the nucleotides of p6-34-2 that differ from those of p6-34-1 are shown. The termination codons of each are

overlined.

The DNA sequence of clone p6-34-2 also is shown in Fig.3. The clone contains a 1743-bp insert, with an open readingframe of 1272 bp encoding 424 amino acids. Alignment of thepredicted amino acid sequence of p6-34-2 with that of ratP-450d (9) demonstrated that the NH2-terminal histidinecorresponded to Thr-92 of rat P-450d, and the sequence

exhibits 76% homology with the rat enzyme (Fig. 4). Addi-tional evidence that p6-34-2 encodes rabbit P-450 4 is pro-

vided by a comparison of the amino acid sequences reportedfor cysteinyl peptides derived from rabbit P-450 4 (26). Asshown in Fig. 4, they differ at only 6 of 302 residues. Thesesix differences could reflect the occurrence of two or more,

possibly allelic, P-450 4-like genes.A comparison of the available sequence indicates an amino

acid homology between P-450 4 and P-450 6 of 64% (Fig. 4),

which is similar in extent to the 69% homology observedbetween rat P-450d (9) and P-450c (8) and mouse P3-450 andP1-450 (10) for this same region of sequence. Regions ofdivergence between the two cytochromes within species andbetween species occur in similar portions of the sequence,

suggesting that functional domains are conserved. However,it is interesting to note that a comparison of the 3' noncodingregions of the mRNAs for rat P-450d and mouse P3-450 andfor rat P-450c and mouse P1-450 indicates that they are very

similar, whereas those of rabbit P-450 isozymes 4 and 6 are

considerably different from their mouse and rat analogs inboth size and sequence.DNA Sequence Analysis of p6-34-3. Ofthe 52 positive clones

that hybridized to p6-34, p6-34-3 was unique in its restrictionpattern, suggesting that it was present in very low abundance.

CTTCAAGGGCCGGCCTGACCTCTACAGCTTCTCCTTCGTCACTAAGGGCCAGAGCATGATCTTCGGCTCAGACTCGGGGCCCGTGTGGGCCGCCCGCCGGC A CG C A C

CGCCTGGCCCAGAACGCCCGTAACAGCTTCTCCGTGGCCTCCGACCCCGCCTCCTCCAGCTCCTGCTACCTGGAGGAGCACG;TGAGCCAGGAGGCCGAGAiG T TG G A C A

ACCTCATCGGCAGGTTCCAGGAGCTGATGGCGGCGGTGGGGCACTTCGACCCCTACAGGTACGTGGTGATGTCCGTGGCCAATGTCATCTGTGCCATGTGT G CC GC G G C GG G G

CTTGGCCGGCGCTATGACCACGACGACCAGGAGCTGCTGAGCCTCGTCAACCTGAACCGATGAGTTCGC.GAAGGTGGCGGCCTCCGGAAGCCCCGCCGACG A TCCCG G G AGTG A GA G G GGAAC G AGCA T G ACC CT T C TG

TTCTTCCTCiTCCTCCGCTACCTGCCCAACCCTGCCCTGGACACCTTCAAGGACCTGAATGAGAGGTTCTACAGTTTCACCCAGGAGAGGGTCAAGGAGCC G GCC C G GG G T C CC CTGC G CTG A A CC CG

ACTGCAGAAGCTTTGAGAAGGGCCACATCCGGGACATCACGGACAGCCT

-.GTGGTGGTGCTCAGCGGCCTGGACACCATCAAGCAGGCCTTGUTUCtiucAtitiwuAt;tij%

Proc. Natl. Acad. Sci. USA 82 (1985) 5313Biochemistry: Okino et al.

56 60 80V--- -----LS-TK --QQ---LL- --I-------MLTL GKNPHVALAA LSRPYGDVFQ IRLGSTPVVV

----N-----LSGLDTIKQA

HIKQAT----

100--K-----NPLVRQGDDFKG

LVRQGDDFKG--K-------

DSGPLWATLIRPDLYSFSFV****** **

RPDLYSSSFI------FTL-__________

120 140AN----T-NP ----L----- ------LK--TKGQSMIFGS DSGPVWAARR RLAQNARNSF* *** * ********** **** **

TEGQSMTFSP DSGPVWAARR RLAQDSLKSF-N-X----N- ----1----- -----A----_1 I-A----------

i80

---Y--SK-- K---E----- -FK-L-V---EAENLIGRFQ ELMAAVGHFD PYRYVVMSVA* ******* ******* ** ** * * *

EAENLIGRFQ ELMAAVGRFD PYSQLVVSAA------SK-- K---E--H-E -VN-V-E-V-

---S ---------.

240E-TG--Y--- -IP-------KVAASGSPAD FFLILRYLPN

* **** * ** *******

ETASSGSPVD FFPILRYLPN-NVT--NA-- ---V------S- V]

-E-CQDR---IKHYRVDRLD

FKHSEKNSKA------Y-D

----T-I--DEELDAVVGRA

EELDAVVGRA----T-I--D

160-I----TLA- ---------KSVASDPASSS SCYLEEHVSQ* ** ***** **********

SIASNPASSS SCYLEEHVSQ------T-V- ---------K_ _ _ _ _ _ _ _ _ _

- - - - - - - - - -

200 220-----I---- ---------- -----SN---NVICAMCFGR RYDHDDQELL SLVNLNDEFG** ****** * * * *

RVIGAMCFGR HFPQGSEEML DVVRNSSKFVN--------K N--RK----- NL-KS-KD--x- ] I-

260 280SS--A----- KK----MKKL I---Y-T--- ----------

PALDTFKDLN ERFYSFTQER VKEHCRSFEK GHIRDITDSL* *** * ** * * **** * * *** *

RPLQRFKDFN QRFLRFLQKT VREHYEDFDR NSIQDITGALPA-K---N-- DN-VLS---- -Q---Q--NK ----------

I-- ---.---] -E

300 320------L--D -VIT--F--- ------I---ENANVQVSDE KTVGIVLDLF GAGFDTVTTA

* * * * * * ****** ***

NGGLIPQ..E KIVNLVNDIF GAGFDTITTA---------- ---------- ----E-V---**-

360-Q--L----- ------F-L-RRPRFSDRPQ LPYLEAVIME* * ***** ****** *

RQPLLSDRPQ LPYLEAFILE---R------ ---L------

420H--------V ---Q----D- NE--------RCVFVNQWQN NHDRELWGVP RSFRPERFLT* * **** *** *** * ********

CCIFINQWQI NHDPQLWGDP EEFRPERFLT---------V ---EKQ-K-- FV--------__________ ---------- ----------

340---------- NPRI.QR---ISWSLMYLVT KRQHTREKIQ

********** ***

LSWSLMYLVT NPRRQR.KIQIF--ILL--- E-KV--.--H.)_- ]

380 400-- -S--V---------- --N-------TFRHTSFLPF TIPHSTTRDT SLGGFYIPKG****** ** ********** * ** ***

LFRHTSFVPF TIPHSTTRDT TLNGFHIPKEIY-Y------ ---------- S-I---------xx---) ------

440 460S------HLS E--I------ ----------

PSGTLDKGPD DKVLL.FGLG KRKCIAETIG* * **** ** ** *

ADGAAINKPL SEKVTLFGLG KRRCIGEILAN-NT--D-T- ----M----- --------P----------- A--------- -------T--

480 500 520P-450c ----------I ----M--N-- --EK-----A ----L----- ----VQM-SS GPQHLQAP-450 6 RLEVFLFLAT LLQQVEFSVS PGTTVDMTPI YGLTMKHARC EHFQAKLRFE A

* ******* *** *** ** ** *** ******* * ** ** **

P-450 4 RWEVFLFLAI LLQRLEFSVP PGVPVDLTPI YGLTMKHPRC EHVQARPRFS DQP-450d K--------- --HQ---T-- ---K-----S------PRT- -----W---- KCYS-4 --- X .- --] ---- ----- )-

FIG. 4. Comparison of the amino acid sequences of rabbit P-450 6 with rat P-450c and rabbit P-450 4 with both rat P-450d and cysteinylpeptides of rabbit P-450 4. The predicted amino acid sequence of P-450 6 and P-450 4 are shown. The amino acid sequence for rat P-450c (8)is aligned above the sequence for rabbit P-450 6, starting at residue 56 of the rat P-450. The rat P-450d sequence (9) and the rabbit cysteinylpeptides (cys-4) characterized by Fujita et al. (26) have been aligned with the sequence of P-450 4. The asterisks represent regions that are

homologous between rabbit P-450 isozymes 4 and 6, while the hyphens represent homologous residues of the other sequences compared witheither P-450 4 or 6. The dots reflect gaps that have been inserted to maximize homology.

The clone was identical to the P-450 6 cDNA, p6-34-1, innucleic acid sequence with two exceptions. First, the se-

quence extended 51 bp beyond the 5' terminus of p6-34-1, as

shown in Fig. 3, with the first amino acid aligned with residue56 of rat P-450c. Second, the clone exhibits an interveningsequence of 131 bp located at nucleotide 1015 in the sequence

393Phe Val Pro Phe Thr Ile Pro His Se

P450c TTT GTC CCA TTC ACC ATC CCC CAC AG GTCAGGCATAGATCCATGACATCTGATACTAAP450 6 TTC CTC CCC TTC ACC ATC CCC CAC AG

Phe Leu Pro Phe Thr Ile Pro His Se

P450c CCCccATG&¶C&TGG&AGGACATGGCA ,ZG.r-OsCn' E- B::: pT G A& TT CCP450 6 CCTCTGCA.-----

r Thr Ile Arg Asp Thr Ser LeuP450c AG C ACC ATA AGA GAT ACA AGT CTGP450 6 -- C ACC ACA AGG GAC ACC AGT CTG

r Thr Thr Arg Asp Thr Ser Leu

FIG. 5. A comparison of the putative intron of rabbit p6-34-3 with intron E of rat P-450c. The entire p6-34-3 clone was sequenced. Shownin the figure are codons flanking the codon equivalent to that encoding rat P-450c Ser-393. The sequence of p6-34-3 is identical to that of p6-34-1with the exception of sequence shown in the upper portion of the figure, which intervenes at a site equivalent to the splicing junction of twoexons of the rat P-450c gene. Intron E of the gene encoding rat P-450c occurs at this site, and its sequence is shown above that of the interveningsequence found in p6-34-3.

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

P-450cP-450 6

P-450 4P-450dCYS-4

Proc. NatL Acad Sci. USA 82 (1985)

A B

I-~r -9ir,:1..4-, 0

Q)H

kb

f-- -2.4a_~~ ~~22.0

P-450 6 P-450 4 3'-P-450 6 P-450 6 3Y-P-450 4

FIG. 6. Blot-hybridization analysis. Preparations of rabbit ormouse liver mRNA (30 ,ug) from either control or TCDD-treatedanimals were electrophoresed in 0.8% agarose gels containing 10 mMmethylmercury(II) hydroxide and transferred to diazobenzyloxy-methyl paper (24). Samples were run in duplicate and hybridized witha 32P-labeled insert from either the P-450 4 or P-450 6 cDNA. (A)Filters were hybridized with the complete cDNA inserts. The livermRNA from the mouse and rabbit were isolated 24 hr after a singleinjection of TCDD (10 nmol/kg). (B) The 3' P-450 6 cDNA is the 3'Pst I fragment from clone p6-34-1, while the 3' P450 4 cDNA is the3'-most Ava I fragment derived from clone p6-34-2. Molecularweights were estimated by comparison to mobilities of fragmentsderived by HindIl digestion of X phage DNA.

of p6-34-3 with a stop codon in the reading frame. Thesequence before and after this segment is identical to p6-34-1(Fig. 5). This intervening sequence is within the codoncorresponding to Ser-393 of rat P-450c, the location of thesplice junction for intron E of the gene encoding rat P-450c(12). Since the rabbit cDNA library was generated frompoly(A)+ RNA derived from total liver homogenates, whichmay include nuclear RNAs, these results suggest that P6-34-3represents a form of P-450 6 mRNA that has not undergonecomplete removal of introns. However, there is little homol-ogy between the 149-bp rat P-450c intron E and the 131-bpintervening sequence of p6-34-3.

Expression of P-450 4 and P-450 6 after Treatment withTCDD. When adult rabbits are treated with TCDD, bothP-450 4 and P-450 6 are induced (1). To determine if P-450 4and 6 mRNAs are elevated, total liver mRNA from untreatedand TCDD-treated rabbits was hybridized with the P-450 4and P-450 6 cDNAs by blot-hybridization analysis. Thehybridization of the two cDNAs to liver mRNA from aC57/B6 mouse treated with TCDD was examined also tocompare the approximate sizes of the rabbit mRNAs to thoseof mouse P1-450 and P3-450 mRNAs. Both P-450 4 and P-4506 cDNAs hybridized to two inducible species of rabbitmRNA, one of approximately 2000 bp and the other of 2300bp (Fig. 6). When the 3' regions were used, the probe derivedfrom the P-450 4 cDNA hybridized exclusively to the 2000-bpspecies, while the P-450 6 cDNA probe hybridized to the2300-bp form (Fig. 6). Both rabbit cDNAs hybridized to2600-bp and 1900-bp mouse mRNAs, corresponding to P1-450and P3-450 (16). The difference in size between rabbit P-4506 and mouse P1-450 mRNAs is the result of a difference of>300 bp in the 3' noncoding regions.

CONCLUSION

The conservation of sequence between analogous forms ofP450-i.e., rabbit P-450 6 and rat P-450c, rabbit P450 4 and ratP450d-exceeds that observed between distinct isotypes ofP4S0 within the same species. The extent of homology seen

between the analogous proteins (about 75%) is similar to thatobserved between the major phenobarbital-inducible forms ofthe two species (27, 28). The differences (about 35%) evident

between P450 isozymes 6 and 4 are similar to those exhibitedby three related forms ofrabbit P-450 characterized by Leightonet al. (29). A putative nuclear precursor ofthe mRNA encodingP-450 6 containing an unspliced intron to what is equivalent inlocation to the rat P-450c intron E suggests that this intronlocation may be conserved across species. The structure ofthisintron may be of little importance in the regulation of P-450 6expression because the rat P-450c E intron and its equivalent inthe rabbit are of different lengths and exhibit little homology. Itis more likely that regulatory regions controlling the expressionof P450 6 occur upstream of the structural gene, as has beenshown for mouse P1-450 (30).

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