BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 48, 26-31 (1992)

Nucleotide Sequence of a cDNA Encoding Murine Fumarylacetoacetate Hydrolase

MARKUS GROMPE’ AND MUHSEN AL-DHALIMY

Department of Molecular and Medical Genetics, Oregon Health Sciences University, 3181 Sam Jackson Park Road, L 103, Portland, Oregon 97201

Received March 19, 1992

Hereditary tyrosinemia type I is caused by deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) (EC 3.7.1.2), the final step in tyrosine degradation. We report here the cloning and sequencing of a full length cDNA coding for murine FAH. This cDNA is highly homologous to the previously cloned human and rat genes. o 1992 Academic PESS, IIIC.

Fumarylacetoacetate hydrolase (FAH) (EC 3.7.1.2) catalyzes the last step in tyrosine catabolism, the degradation of fumarylacetoacetate into acetoacetate and fumarate. In man, deficiency of FAH causes tyrosinemia type I (1). Its main clinical features include progressive liver failure, renal tubular damage, neurologic crisis, and the development of liver cancer. Many aspects of the pathophysiology of tyrosinemia I are unclear at present, but unfortunately an animal model to study the disorder and its treatment are not available. Recently human (2,3) and rat (4) FAH cDNAs have been cloned by others. We here report the cloning of a full-length murine FAH cDNA as a first step toward isolating the whole gene and the creation of a murine model of FAH deficiency.

MATERIALS AND METHODS

Degenerate Uligonucleotide PCR

Total cellular RNA was isolated from adult mouse liver by the RNAzol method (5). First strand cDNA was generated from 10 pg of this RNA with random hexamers as primers (6,7). A pair of degenerate oligonucleotide primers (8) was designed to regions of known amino acid homology between human and rat FAH cDNA. Primer 1 covered codons 86-90 and primer 2 codons 213-218 according to the nomenclature of Phaneuf et al. (3). The primers were used to perform PCR amplification (9) of the mouse liver cDNA and a band of expected size (396 bp) was detected.

’ To whom correspondence should be addressed.

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0885-4505/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

cDNA ENCODING MURINE FUMARYLACETOACETATE HYDROLASE 27

Library Screening

A LambdaZAP mouse liver cDNA library (Stratagene No. 935302) was plated at a density of lo4 plaques/l35 mm dish. The 396-bp PCR product from above was gel purified (lo), radioactively labeled by random hexamer labeling (ll), and used to screen the cDNA library at high stringency (0.2 x SSC and 65°C for the final wash). Several positive clones were isolated and characterized by restriction mapping. The two largest clones were selected for DNA sequencing.

DNA Sequencing

Subclones containing overlapping partial cDNA fragments were created in vec- tor pTZ 19R (12) and sequenced by the dideoxy chain termination method (13).

RESULTS

A full-length open reading frame of 1257 bp coding for a protein of 419 amino acids was found in both cDNA clones that were sequenced (see Fig. 1). An “aataaa” polyadenylation signal was detected 20 bp upstream of a poly(A) tract in the 3’ untranslated region.

A comparison of the nucleotide and deduced amino acid sequences between the human and rat FAH cDNA sequences and our clone was performed with Intelligenetics software. In the protein coding region, nucleotide sequence ho- mology of our clone to the human FAH cDNA was 84.1% and to the rat FAH cDNA was 94.7%. The amino acid homology was 89% to human and 96.7% to the rat sequences (see Fig. 2) indicating that our cDNA indeed represents murine FAH. All three proteins have the same length of 419 amino acids and the high degree of amino acid identity is present along the entire sequence. When this murine FAH cDNA was used as a probe against Southern blots of human genomic DNA (14), bands identical to those detected with the human FAH cDNA were

-31 TAGGGGGCTCTGCTGCCCGGTGCTCGTCAGC

27 54 ATG XC TTT ATT CCA GTG GCC GAG GAC TCC GAC TTT CCC ATC CAA AAC CTG CCC MSFIPVAEDSDFPIQNLP

81 108 TATGGTGTP TTC TCCACTCAAAGCAAC CCAAAGCCACGGATTGGT GTA GCCATC YGVFSTQSNPKPRIGVAI

135 162 GGT GAC CAGATC TTG GACCTGAGT GTCATTAAACAC CTC TTTACC GGACCT GCC GDQILDLSVIKHLFTGPA

189 216 CTF TCCiVACATCAACATGlC ITCGATG?.GACAACTClCAATAAC TTCATGGGT LSKHQAVFDETTLNNFMG

243 270 CTGGGTCAACXTCX?.ATGGAAGGF+GGCAAGAGCATCCT~ATTAAA~ LGQAAWKEARASLQNLLS

FIG. 1. Nucleotide and amino acid sequence of murine FAH cDNA. The regions to which the PCR primers were designed are underlined. Amino acids are indicated in the one letter code.

28 GROMPE AND AL-DHALIMY

297 324 GCC AGC CAA GCC CGG CTC AGA GAT GAC AAG GAG Cl-l- CGG CAG CGT GCA TTC ACC ASQARLRDDKELRQRAFT

351 378 TCC CAG GCT TCT GCG ACA ATG CAC Cl-l- CCT GCT ACC ATA GGA GAC TAC ACG GAC s QASATMHLPATIGDYTD

405 432 TTC TAC CCTTTT CGGCAGCAT GCCACCAATGTT GGCATTATG lTCAGAGGCAAG FYPFRQHATNVGIMFRGK

459 486 GAGAAT GCG CTGTTGCCAAATTGG CTC CACTTACCTGTGGGATAC CAT GGCCGA ENALLPNWLHLPVGYHGR

513 540 GCT T-32 TCC ATT GTG GTA TCT GGA ACC CCG ATT CGA AGA CCC ATG GGG CAG ATG ASSIVVSGTPIRRPMGQM

567 594 ACX CCT GAT ARC TCA AAG CCT CCT GTG TAT GGT GCC TGC AGA CTC TTA GAC ATG APDNSKPPVYGACRLLDM

621 648 GAG TM; GAA ATG GCT TTC TTC GTA GGC CCT GGG AAC AGA TTC m CAG UXAlX ELEMAFFVGPGNRFGEPI

675 702 Ccc TCC AAAGCC CAT GAACACATT TTCGGGATG GTC CTCATGAAC GAC TGG PISKAHEHIFGMVLMNDW

729 756 AGC GCA CGA GAC ATC CAG CAA TGG GAG TAC GTC CCA CTT GGG CCA TTC CTG GGG SARDIQQWEYVPLGPFLG

783 810 AAA AGC TTT GGA ACC ACA ATC TCC CCG TGG GTG GTG CCT ATG GAT GCC CTC ATG KSFGTTISPWVVPMDALM

837 864 CCC TTT GTG GTG CCA AAC CCA AAG CAG GAC CCC AAG CCC TTG CCA TAT CTC 'XC PFVVPNPKQDPKPLPYLC

891 918 CACAGC CAGCCC TACACATTT GATATCAAC CTGTCTGTC TCT TTGAAAGGAGAA HSQPYTFDINLSVSLKGE

945 972 GGA ATG AGC CAG GCG GCT ACC Al!'2 TGC AGG TCT AX -ITT AAG CAC AI-G TAC 'KG GMSQAATICRSNFKHMYW

999 1026 ACC ATG Cl% CAG CAA CTC ACA CAC CAC TCT GTT AAT GGA TGC AAC CTG AGA CCT TMLQQLTHHSVNGCNLRP

1053 1080 GGGGAC CTC TlGGcpTcT GGAACCAXAGTGGATCAGACCCTGAAAGC TlTGGC GDLLASGTISGSDPESFG

1107 1134 TCC ATG CTG GAA CTG 'I-CC TGG AAG GGA ACA AAG GCC ATC GAT GTG GAG CAG GGG SMLELSWKGTKAIDVEQG

1161 1188 CAG ACC AGG ACC TTC CTG CTG GAC GGC GAT GAA GTC ATC ATA ACA GGT CAC TGC QTRTFLLDGDEVIITGHC

121s 1242 CAG GGG GAC G4X TAC CGT G T T GGC T T T GGC CAG TGT GCT GGG AAA GTG CTG CCT Q G D G Y R V G F G Q C A G K V L P

GCC CTT TCA CCA GCC TGA AGCTCCGGC~~C~A~~~~A A L S P A

MOUSE-FAH 1 ~FIpV~DSDFPIQNLPYGVFSTQSNpKPRI~AIG~ILDLSVIKHLFTGPaLSKHQHV

11111111111111111111111111111111111111111111111111111 lllllll RAT-FAJi 1 ~~FIPVAEDSDFPIQNLPYGVFSTQSNPKPRIGVAIGDQILDLSVIKHLFTGPVLSKHQHV

IIIlIIIIIIIIII llllIIllI I IIIIIIIIIIIIIII IIIIIIIIIIIIII I HUMAN-FAH 1 ~FIPV~DSDFPIhNLPYGVFSTrgdPrPRIGIGDQILDLSiIKHLFTGPVLSKHQdV

NUJSE-FAH 62 FDETTLNnFMGLGQAAWK LQNLLSASQArLRDDKELRQRAFTSQASATMHLPATIG lllllll 1111111111111111111111111 llllllllllllllllllllIIIIIII

RAT-FAH 62 FDETTLNSFMGLGQAAWKEARAS LQNLLSASQAqLRDDKELRQRAFTSQASATMHLPATIG I IIIIIIIIIIIIIIIII llllll III III1 III II IIIIIIIIIIIIII

HUMAN-FAH 62 FnqpTLNSFMGLGQ AAWKEARvfIQNLLSvSQArLRDDtELRkcAFiSQASATMHLPATIG

consensus FdetTLNsFMGL sLQNLLSaSQArLRDDkELRqrAFtSQASATIG

HOUSE-FAH 123 DYTDFYpfrQHATNVGIl4FRGKENALLpNWIJiLpVGYEGRASSiWSGTPIRRPM~D llllll IIIIIIIIIIIIIIIIIIllllllllllllllll IIIIIIIIIIIIIIIII

RAT-FAH 123 DYTDFYSSlQHATNVGIXFRGKENALLpNWLELP VGYliGRASSVWSGTPIRRPI4GQMRpD IIIIIIII llIIIllllll lllll IIIIIl1llllllllllllllll1lIIIIII II

BOIIAN-FAH 123 DYTDFYSSrQHATNVGIMFRdICENAbnpNWLBLpVGyHGRASSVWSGTPIRRPMGQMkPD

consensus DYTDFYssrQHATNVGIKFRgKENALlPNWLHLPVGYHGR&SSvWSGTPIRRpMGQMrPD

MOUSE-FAH 184 NSKPPVYGAcrlLDMELEMAFFVGPGNRFGEPIPISKAhEHIFGMVLMND WSARDIQQWEY lllllllll 11111111111111111111111111 1111111111111111111111

RAT-FAH 184 NSKPPVYGAsKrLDMELEMAFFVGPGNRFGEPIPISKAqEHIFGMvLMNDWSARDIQQWEY

llIllllI I IIIIIIIIIIIIIIII llllllllI 111111111111111111 III aw-Fm 184 ~SKP~VYGACK~LDMELEMAFFVGPGWGEPIPISKA~EHIFGMVLKN DWSARDIQkWEY

consensus nSKPPVYGAcklLDMELEMAFFVGPGNRfGEPIPISKAhEHIFGMVLNNDWSARDIQqWEY

MOUSE-FAH 245 VPLGPFLGKSFGTTISPWWPMDALM~FVV~N~KQDPKPL~YLCHSQPYTFDINLSVSLKG 111111111111111111111111111111111111111111111111111111111 III

RAT-FAH 245 VPLGPFLGKSFGTTISPWVVPMDALMPEVVPNPKQDPKPLPYLCHSQPYTFDINLSVaLKG

IIIIIIIIIIIIII IIIIIIIIIIIII llllllll lllllll lllllllIll III HUNAN-FAH 245 VPLGPFLGKSFGTTvSPWWPMDALMpFaVPNpKQDPrPLPYLCHdePYTFDINLSVnLKG

consensus VPU;PFLGKSFGTTiSPWVVPMDALMPFvVPNPKQDPkPLPYLCHsqPYTFDINLSV-LKG

mMSE-FAH 306 EGMSQAATICRSNFKHMYWRLTHHSVNGCNLRPGDLLASGTISGSDPESFGSMLELS IIIIIIIIIIIIIIIIIIII 1111111111111111111111111111111111111111

RAT-FAH 306 EGMSQAATICRSNFKHMYWTiLQQLTHHSVNGCNLRpGDLLASGTISGSDPESFGSMLELS llllllllll IIII IIII IIIIIIIIIIIIIIIIIIIIIIIIIII II IIllIIII

HUMAN-FAH 306 EGMSQAATICkSNFKyMYWTmLQQLTHHSVNGCNLRPGDLLASGTISGpePEnFGSMLELS

consensus EGMSQAATICrSNFKhMYWTmLQQLTHHSV?%CNLRPGDLIA.SGTISGsdPEsFGSMLELS

MOUSE-FAH 361 WKGTKAIDVeQGQTRTFLLDGDEVIITGHCQGDGYRVGFGQCAGKVLPALSPA lllllllll lllllllllllllllllllllllllllllllllllllllllll

RAT-FAH 367 WKGTKAIDVGQGQTRTFLLDGDEVIITGHCQGDGYRVGFGQCAGKVLPALSPA lllll II I III1 IIIIIIIIlIII IIIIIII IIIIIIIIIIIII I

HUMAN-FAH 361 WKGTKpIDlGnGQTRkFLLDGDEVIITGyCQGDGYRiGFGQCAGKPALlPs

consensus WKGTKaIDvgqGQTRtFLLDGDEVIITGhCQGDGYRvGFGQCAGKVP~sPa

FIG. 2. Alignment of the amino acid sequences of human, rat, and murine FAF.

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30 GROMPE AND AL-DHALIMY

detected again confirming the high degree of sequence conservation between species (data not shown). The EMBL Accession Number is 211774.

DISCUSSION

We here report the cloning and sequencing of the murine homologue of the gene deficient in human tyrosinemia I. The human, rat, and murine proteins are very similar at both the nucleotide sequence and amino acid levels, indicating tight functional constraints on sequence evolution in all parts of the protein. The degree of homology between the murine and human FAH proteins reaches 98%, when amino acid similarity rather than identity are compared. The cloning of the murine FAH cDNA serves as a first step in the isolation of the chromosomal mouse gene and the creation of an animal model via homologous recombination in embryonic stem cells (15). In addition, the sequence comparison between different species will facilitate the interpretation of the functional significance of sequence alterations detected in human tyrosinemia I patients.

ACKNOWLEDGMENT This work was supported by NICHHD Grant HD28585-01.

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