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Proc. Nall. Acad. Sci. USA Vol. 84, pp. 7473-7477, November 1987 Isolation and sequence of a eDNA clone for human tha maps at the mouse c-albino locus BYOUNG S. KWON*t, ASIFA K. HAQ*, SEYMOUR H. POMERANTZt, AND RUTH HALABAN§ *Molecular Laboratory, Guthrie Research Institute, Sayre, PA 18840; tDepartment of Biological Chemistry, University of Maryland School of Medicine, Baltimore, MD 21201; and §Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Aaron B. Lerner, July 16, 1987

ABSTRACT Screening of a Xgtll human on an oligo[d(T)I-cellulose column (11, 12). A cDNA li cDNA library with against hamster tyrosinase was prepared employing a Xgtll cloning vector (13-15) (monophenol, L-dopa: oxidoreductase, EC 1.14.18.1) Xgtll library contained 1.7 x 106 independent phages resulted in the isolation of 16 clones. The cDNA inserts from 13 immunobiological screening and analysis of the fusion of the 16 clones cross-hybridized with each other, indicating teins produced by Xgtll cDNA clones were carried c that they were from related mRNA . One of the cDNA described by Young and Davis (15). The rabbit anti-hal clones, Pmel34, detected one mRNA species with an approxi- tyrosinase antibodies and their application in the stun mate length of 2.4 kilobases that was expressed preferentially have been described in detail (10, 16). in normal and malignant but not in other RNA Blot Hybridization. Poly(A)+ RNA from n( types. The amino sequence deduced from the human melanocytes, melanoma cells, neuroblastoma sequence showed that the putative human tyrosinase is com- lines, HL-60 (human promyelocytic leukemia cell line) posed of 548 amino with a molecular weight of 62,610. HepG2 (human hepatocarcinoma cell line) was fractiol The deduced contains sites and - on a 1.2% (wt/vol) formaldehyde denaturing gel (17), t rich sites that could be used for binding. Southern blot ferred to a GeneScreenPlus membrane (New England analysis of DNA derived from newborn mice carrying lethal clear), and hybridized with 32P-labeled cDNA pr( albino deletion revealed that Pmel34 maps near or at Pmel14-2, a cDNA clone that was isolated from our hi the c-albino locus, the position of the structural for melanocyte cDNA library, was used as a control p tyrosinase. because the corresponding RNA was detectable in si5 amounts in all human and murine cells tested. Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, Genomic DNA Blot Analysis. Newborn mice of the g EC 1.14.18.1) is a copper-based oxidoreductase that cata- types C3H/C3H and cch/cH were provided by Sal lyzes the oxidation of to dopa and the oxidation of Gluecksohn-Waelsch (Albert Einstein College of Medic dopa to dopaquinone (1). It is a key in Newborn mice provided by M. Lynn Lamoreux (Texas A . Oculocutaneous albinism, a group of auto- somal-recessive diseases in humans (2) and animals, is University, College Station, TX) were littermates of characterized by reduced or absent melanin in skin, hair, and genotypes C14COs/C14Cos cCh/c14CWs, and cch/cch. These eyes. Tyrosinase-negative albino melanocytes have no were descended from three mice of the genotype C6H/C tyrosinase activity in vitro. that were given to M. L. Lamoreux by S. Gluecks Genetic control of pigmentation has been extensively Waelsch. These stocks were crossed twice onto JU/Ct/ studied in mice. There is evidence that the c-albino locus at +c/+c mice, then once onto C57BL/6J mice, then for e chromosome 7 for the structural gene for tyrosinase (3, generations onto a C57BL/6J-cch/cch stock descended I 4). Mutations at this locus affect both tyrosinase activity and mice provided by D. Townsend (University of Minnes coat color (5, 6). Minneapolis), and then sibling mated for six generation A probe for tyrosinase would be an invaluable High molecular weight of newborn mice of var tool for studies of the regulation of tyrosinase, of the genotypes were prepared as described (18). Restric molecular basis of human albinism, and of various mouse endonuclease digests ofDNA were electrophoresed in a 0 mutations affecting coat and eye color. We report here the agarose gel at 40C, transferred (19) to GeneScreenPlus, isolation and sequence ofa cDNA clone for human tyrosinase hybridized with the 32P-labeled cDNA probes. Pmell7- that maps at or near the mouse c-locus.¶ cDNA clone that was isolated from our human melanoi cDNA library, was utilized as a control probe for the purr MATERIALS AND METHODS of estimating the amounts of DNA in each lane bect Pmell7-1 detected a single EcoRI fragment in all mouse D . Normal human melanocytes, melanotic mel- tested. The intensity of the hybridizing bands was meast anoma cells (LG), and neuroblastoma cells (SK-N-SH) were by a densitometer at 500 nm (Beckman DU-8BUV/ cultured as described (7-9). The murine neuroblastoma cell Spectrophotometer). line NIE115 was obtained from X. 0. Breakefield (E. K. DNA . DNA restriction fragments subclone Shriver Center, Waltham, MA). of normal melano- M13 vectors (20) were sequenced by the dideoxy ch cytes were radiolabeled with [355] (Amersham) termination technique (21), with modifications made to (100 ,XCi/ml, 1390 Ci/mmol; 1 Ci = 37 GBq) as described commodate 2'-deoxyadenosine 5'-[a-[35S]thio]triphospt (10). (22). A forward primer (New England Biolabs) compleir cDNA Libraries and Screening. RNA from normal human tary to the lacZ sequence adjacent to the 5'-side of the Ec melanocytes was prepared, and poly(A)+ RNA was purified tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge 9This sequence is being deposited in the EMBL/GenBank data t payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03581). Downloaded by guest on October 5, 2021 7474 Biochemistry: Kwon et al. Proc. Natl. Acad. Sci. USA 84 (1987) site in Xgtll was used for the direct sequencing of cDNA of the cDNA clones. A lysogen of Xmell6 was prepared, and insert end points in Xgtll (23). the bacterial lysate was analyzed by 6% NaDodSO4/poly- acrylamide gel electrophoresis and by competitive immuno- RESULTS precipitation assay. Xmell6 was used for this experiment because the Xmell6 plaques produced the strongest signal in Initial screening of 500,000 recombinant phage plaques with plaque screening with anti-tyrosinase antibodies even though the rabbit anti-tyrosinase antibodies identified 16 indepen- Xmel6 contained an incomplete cDNA of -0.7 kilobase dent clones. The cDNA inserts varied in size from 0.2 to 1.6 pairs. A fusion protein was produced in Y1089/Xmell6 that kilobase pairs. The longest cDNA insert (-1.6 kilobase pairs) had a relative size of 140 kDa (Fig. lb). The fusion protein from Xme134 hybridized to 12 other cDNA inserts and shared was 25 kDa larger than 8-galactosidase, an overlapping restriction enzyme pattern (Fig. la). The indicating that the incomplete cDNA in Xmell6 was fused to other three cDNA inserts (not included in the figure) were not .3-galactosidase gene in frame. Synthesis of both /3-galacto- related to the 13 cDNA clones. A cDNA insert contained in sidase and the fusion protein was dependent on induction one of these, Xmell7-1, was utilized as a control probe. The with isopropyl 13-D-thiogalactopyranoside. The production of cDNA inserts of Xmell6, Xmel34, Xmel4O, and Xmell7-1 were immunologically reactive tyrosinase protein in the lysate of subcloned into pBR322 to yield Pmell6, Pmel34, Pmel4O, and Xmell6 lysogen was tested by competition assay using Pmell7-1, respectively, and used for further characterization metabolically labeled melanocyte cell extracts as a source for tyrosinase. Bacterial lysates of the Xmell6 lysogen (2 x 107 a 0 200 400 600 800 1000 1200 1400 1600 180 2000 2200 2400 bacterial cells in 100 pil) competed with roughly 70% of the anti-tyrosinase antibodies as judged from the intensity of the tyrosinase bands and the radioactivity in the relevant gel slices (Fig. lc). A"34 To examine whether mRNA homologous to Pmel34 is ex- A me 33 Xmel44 pressed preferentially in melanocytes, RNA gel blot analysis of A me 16 poly(A)+ mRNA from cells ofmelanocytic and nonmelanocytic Xmel 4 lineage was performed. Pmel34 hybridized to 21S (=2.4 A mM 52 Xmel 16 kilobase) mRNA species from normal human melanocytes and me 106 human melanotic melanoma cells (LG) but not from HepG2, mel? HL-60, human or murine neuroblastoma (Fig. 2), human and A me 20 mouse fibroblasts, or lymphocytes (data not shown). The skin of mice carrying the radiation-induced albino b C alleles, such as c3Hlc3H, had no tyrosinase activity (4). The Mr tyrosinase activity levels in the skin of mice heterozygous 1 2 3 4 IKdI 1 2 between the lethal albino deletion and chinchilla were shown Mr IKdl to be intermediate between the normal and mutant homo- zygotes, consistent with the murine albino locus encoding the structural gene of tyrosinase (unpublished observation of S. 200 - Gluecksohn-Waelsch, reviewed in ref. 4). If the c-albino 94- locus codes for the structural gene for tyrosinase, tyrosinase 116 - cDNA should not detect any hybridizing band in DNA 67-b4 - 66.2 - extracted from c3H/c3H or c14Cos/c14Cos mice but should detect 40 -- hybridizing bands of normal and half intensity in DNA from 43- homozygote (cch/cch) and heterozygotes (cch/c3H and cch/ c)4Cos) respectively. 1 2 3 4 5 6 7 8 20- 28S-

FIG. 1. Isolation of human tyrosinase cDNA by screen- ing. (a) Alignment of Xmel34-related cDNA inserts. Inserts from 13 pmel 34 cDNA clones whose gene products bound to anti-tyrosinase anti- 18S bodies were aligned, based upon restriction mapping and partial nucleotide sequencing. (b) Analysis of lacZ-cDNA fusion proteins. Bacterial lysates were prepared from the lysogens of Y1089/Xgt1l (lanes 1 and 2) and Y1089/Xmell6 (lanes 3 and 4) that were cultured in the absence (lanes 1 and 4) or presence (lanes 2 and 3) of isopropyl pmel 14-2----e- f3D-thiogalactopyranoside. Samples were electrophoresed on a 6% NaDodSO4/polyacrylamide gel and stained with . The isopropyl /3-D-thiogalactopyranoside-dependent produc- tion of 3-galactosidase (lane 2) and a - 140-kDa Xmell6 fusion protein (lane 3, arrow) was noted. Protein sizes in kDa (Kd) are at the left. (c) Competition immunoprecipitation assay. The lysate of Xmell6 or FIG. 2. Blot analysis of poly(A)+ RNA derived from cultures of Xgtll lysogen was used to compete with metabolically labeled human melanocytic and nonmelanocytic lineage. Four micrograms of melanocyte cell extract for anti-tyrosinase antibodies. Equal poly(A)+ RNA of normal human melanocyte (lane 1), LG (lane 2), amounts of lysates of Xgtll lysogen and Xmell6 lysogen (-2 x 107 HepG2 (lane 3), and HL-60 (lane 4) cells, and 10 ,ug of poly(A)+ RNA bacterial cells in 100-1,l volume) were incubated with 5 ,ul of a 1:100 of normal human melanocytes (lane 5), LG (lane 6), human neuro- dilution of anti-tyrosinase antibodies. The respective supernatants blastoma (lane 7), and murine neuroblastoma cells (lane 8) were were used to immunoprecipitate [35S]methionine-labeled human fractionated on a 1.2% (wt/vol) formaldehyde denaturing agarose melanocyte extract (6 x 106 cpm in protein per tube). The eluted gel, blotted, and hybridized with 32P-labeled Pmel34. The same filter immune complexes were separated on 8.5% NaDodSO4/polyacryl- was used to hybridize to 32P-labeled Pmel14-2 to show that each lane gel. In lanes 1 and 2, the antibody preparation was contained RNA as indicated above and that the RNA was relatively preabsorbed with lysogens Xgtll and Xmell6, respectively. intact. Downloaded by guest on October 5, 2021 Biochemistry: Kwon et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7475

9h k a U , strategy shown in Fig. 4. Most of the other 10 clones were also sequenced. The nucleotide sequence of tyrosinase cDNA re- vealed a single long open , beginning with the first nucleotide after the EcoRI linker. Direct sequence analysis with a Xgtll forward primer (23) revealed that this open reading frame is in frame with the lacZ gene of the Xgtll vector. We SiizeMarker_ found that other clones were also fused in frame with the IacZ IKbi gene. This property was helpful in assigning the open reading 21.7 frame even though the cDNAs did not start with the first ATG codon. The open reading frame coded for a polypeptide of 566 amino acids with a molecular weight of 63,549 (Fig. 5). The codon specifying carboxyl-terminal was followed by the termination codon TAA (nucleotide residues 1645-1647). No nucleotide differences were observed among 5.2- _ the three cDNA clones except that they differed in length. The 3'-untranslated sequence determined from PmeI34, Pmell6, and Pmel4O did not extend as far as the poly(A)+ tail. However, Pmel4O contained a potential polyadenylylation signal of AT- TAAA (24, 25) (underlined nucleotide residues 1822-1827) that appears upstream of the consensus polyadenylylation signal AATAAA. The deduced sequence of the first 12 residues 1.6- of the tyrosinase has characteristics of the signal of secretory and membrane-associated proteins (26), which mainly contains hydrophobic amino acids (10 out of 12 residues) and terminates with (27) (Fig. 5). We puta- tively assign the first 12 amino acids as a part of a . A possible site cleavage of the signal peptide of the FIG. 3. Southern blot analysis of genomic DNA of murine lethal tyrosinase precursor is after the serine at phenylal- albino deletion mutants. Genomic DNA from newborn mice of anine-1 (Fig. 5). Thus, the protein backbone of processed strains cch/cch, cc /c3H, c3H/c3H, c/C14Cos, and c)4Cos/cI4Cos was digested with EcoRI, electrophoresed on a 0.8% agarose gel, trans- tyrosinase is composed of 548 amino acids with a molecular ferred to GeneScreenPlus, and hybridized to Pmel34 (a). The same weight of 62,160. filter was probed with Pmell7-1 after removal of the Pmel34 probe As tyrosinase is a , we have looked for (b). Spectrophotometric analysis of the intensity of Pmell7-1 bands possible N-glycosylation sites and found five potential as- revealed that there was -20% more cch/cch DNA compared with that paragine-linked glycosylation signals (28, 29) at positions 73, in the other four lanes. Kb, kilobases. 98, 148, 217, and 324 as underlined in Fig. 5. The protein contains two -rich domains (Fig. 6a, As shown in Fig. 3a, the Pmel34 probe detected three Pmel34P). There are 17 cysteine residues; 10 residues are hybridizing cch/cch genomic DNA fragments whose sizes clustered within the first 100 amino acids, and 5 residues are were 4.5, 12.0, and 14.5 kb. The different extent of hybrid- clustered between amino acids 231 and 308. ization of the three bands appears to be due to the hetero- The tyrosinase molecule contains two copper atoms (30, geneity between the human probe and the mouse . The 31), and histidine residues serve as copper binding sites (31, same three bands were detected at half intensity in cch/c3H 32). There are 15 histidine residues in the deduced amino acid and cch/c14Cos DNA when normalized with respect to the sequence of human tyrosinase. Four of these appeared Pmell7-1 band (Fig. 3b). No hybridizing fragments were between the two cysteine-rich domains (histidine-130, -167, detected in c3H/C3H or c14CoS/c14C0s DNA even after pro- -189, and -198), and 6 residues appeared after the second longed exposure. Therefore, the murine genes whose se- cysteine-rich domain (histidine-350, -354, -360, -377, -390, quences are homologous to cDNA contained in Pmel34 are and -406). Histidine residues at positions 350, 354, 360, and located at or near the albino locus. 377 had an arrangement similar to that of bovine superoxide The nucleotide sequence of three overlapping cDNA clones dismutase, another copper-containing enzyme (32). There is (Pmel34, Pmell6, and Pmel40) was determined according to the a stretch of 27 amino acids that contains only hydrophobic

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 I~ .: .;a2.-I -C > 2 - .2* 2 : A.< < -i co <: A 5, F

FIG. 4. Partial restriction X mel 34 map and sequencing strategy for No di_ _ la _ IN__10 ON 4 - human tyrosinase cDNA. The tyrosinase- is indi- so t-o * l cated by an open box. Horizon- X mel 16 tal arrows under the three inserts show the direction and extent of sequencing used to generate the sequence presented in Fig. 5. X mel 40 Restriction sites used for se- quencing are indicated. The scale at the top indicates the nucleotide number. Downloaded by guest on October 5, 2021 7476 Biochemistry: Kwon et al. Proc. Natl. Acad. Sci. USA 84 (1987)

-42 GAA TTC CTG CTC CTG GCT GTT TTG TAC TGC CTG CTG TGG AGT -1 -12 ------Leu Leu Leu Ala Val Leu Tyr Cys Leu Lett TrpSr - near the c-albino locus that is considered to be the structural 1 TTC CAG ACC TCC GCT GGC TTC CCT AGA GCC TGT GTC TCC TCT AAG AAC CTC ATG GAG 60 gene for tyrosinase. Southern blot analysis of DNAs from 1 Phe Gln Thr Ser Ala Gly His Phe Pro Arg Ala Cys Val Ser Ser Lvs Asn Leu Met Gltt 20 mouse-Chinese hamster somatic cell hybrids has confirmed 61 AAG GM TGC TGT CCA CCG TGG AGC GGG ACA GGA GTC TGT (GC CAG CTT TCA GGC AGA GGT 12n 21 Lys Glu Cys Cys Pro Pro Trp Ser Gly Thr Gly Val Cys Gly Gln Leu Ser Gly Are Glv 4n the localization of the sequence homologous to Pmel34 on 121 TCC TGT CAG AAT ATC CTT CTG TCC MT GCA CCA CTT GCG CCT CM TTT CCC TTC ACA GGG 100 mouse chromosome 7 (B.S.K., D. Barton, U. Francke, R.H., 41 Ser Cys Gln Asn Ile Leu Leot Ser Asn Ala Pro Leu Gly Pro Gln Phe Pro Phe Ther Glv 60 L. Lamoreux, B. Whitney, and A.K.M., unpublished obser- 181 GTG GAT GAC CGG GAG TCG TGG CCT TCC GTC TTT TAT AAT AGG ACC TGC CAG TGC TCT GGC 240 61 Val Asp Asp Arg Glu Ser Trp Pro Ser Val Phe Tyr Asn Arg Thr Cys Gln Cys Ser Glv 80 vations). Since the 5' end of Pmel34 mRNA has not been 241 MC TTC ATG GGA TTC MC TGT GGA MC TGC MCG mTT GGC TmT TGG GGA CCA MC TGC ACA 300 cloned the assignment of signal peptide is putative. 81 Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr 100 Shibahara et al. (25) reported a cDNA sequence for murine 301 GAG AGA CGA CTC TTG GTG AGA AGA MC ATC TTC GAT TTG AGT GCC CCA GAG MG GAC AA 360 101 Glu Arg Arg Leu Leu Val Arg Ser Ala Pro tyrosinase. A comparison of our amino acid sequence and Arg Asn Ile Phe Asp Leu Glu Lys Asp Lys 12C that 361 mTT TTT GCC TAC CTC ACT TTA GCA AAG CAT ACC ATC AGC TCA GAC TAT GTC ATC CCC ATA 420 deduced by Shibahara et al. (25) shows extensive 121 Phe Phe Ala Tyr Leu Thr Leu Ala Lys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile 141 homology. Fig. 6 shows the regions ofhomology between the 421 GGG ACC TAT GGC CAA ATG MA AAT GGA TCA ACA CCC ATG mr MC GAC ATC MT ATT TAT 080 two and the of the 141 Gly Thr Tyr Gly Gln Met L-s Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr 160 tyrosinase sequences optimal alignment peptide sequences in the of There are four 481 GAC CTC TmC GTC TGG ATG CAT TAT TAT GT, TCA ATG CAT GCA CTG CTT GGG GGA TAT CAA 541 regions homology. 161 Asp Leu Phe Val Trp Met His Tyr Tyr Val Ser Met Asp Ala Leu Leu Gly Gly Tyr Glu 180 regions of homology in the two proteins. Homologous do- 541 ATC TGG AGA GAC ATT GAT TTT GCC CAT GAA GCA CCA GCT TTT CTG CCT TGG CAT AGA CTC 605o mains I and III the first and second 181 Ile Trp Arg Asp Ile Ala Ala Pro Ala Pro span cysteine-rich Asp Phe His Glu Phe Leu Trp His Arg Lett 20o regions, respectively. The space between is iden- 601 TTC TTG TTG CGG TGG GAA CM CAA ATC CAG MG (TG ACA GGA GAT GM MC TTC ACT ATT 460 201 Phe Lett Leu Arg Trp Glu Gln Glt Ile Gln Lys Leu Thr Gly Asp Gli Asn Phe Thr Ile 220 tical or similar, and the residues around cysteine are espe- 661 CCA TAT TGC GAC TGG CGG GAT GCA GM MtC TGT GAC ATT TGC ACA GAT 4AG TAC ATG GGA 72n cially similar in the two proteins. Homologous domains II and 221 Pro Tyr Trp Asp Trp Arg Asp Ala Glu Lys Cos Asp Ile Cys Thr Asp Glu Tyr Met Gly 240 IV include residues 149-213 and 326-439, respectively. In 721 GGT CAG CAC CCC ACA MT CCT MC TTA CTC AGC CCA GCA TCA TTC TTC TCC TCT TGG CAG 780 241 Gly Gln His Pro Thr Asn Pro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln 260 homologous domain II, three histidine residues are aligned, 781 ATT GTC TGT AGC CGA TTG GAG GAG TAC MC AGC CAT CAG TCT TTA TGC MT GIA ACG CCC 840 and residues surrounding each histidine are similar. In 261 Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr Pro 280 domain IV there are six in the human sequence and 841 GAG GGA CCT TTA CGG CGT AAT CCT GGA MC CAT GAC MA TCC ACA ACC CCA AGG CTC CCC 900 281 Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Thr Thr Pro Arg Leu Pro 300 five histidines in the murine sequence; four histidines are 901 TCT TCA GCT GAT GTA GM ('C TGC CTG AGT TTG ACC CM TAT GAA TCT GGT TCC ATG GAT 9f0 aligned with similar interspaces. Histidine-406 is surrounded 301 Ser Ser Ala Asp Val Glu Phe Cys Leu Ser Leu Thr Oln Tyr Glu Ser Gly Ser Met Asp 120 by seven residues that are identical in the two proteins. The 961 MA GCT GCC MT TTC AGC TTT AGA MT ACA CTG GM GGA TTT GCT AGT CCA CTT ACT GGG 1020 321 Lys Ala Ala Asn Phe Ser Phe Arg Asn Thr Leu Glu Gly Phe Ala Ser Pro Leu Thr Gly 340 amino acid identity between the homologous domains of the 1021 ATA GCG GAT GCC TCT CAA AGC AGC ATG CAC AAT (CC TTG CAC ATC TAT ATG MT GGA CAT 1080 two proteins is 48.8% in domain I, 54.6% in domain II, 50.0% 341 Ile Ala Asp Ala Ser Gln Ser Ser Met His Asn Ala Leu His Ile Tyr Met Asn Gly Hit 341 in domain III, and 48.0% in domain IV. If we take into 1081 GTC CCA GGT ACA GGA TCT GCC MC GAT CGT ATC TTC CTT CTC ACC ATG CAT TTG TTG ACA 1140 361 Val Pro Gly Thr Gly Ser Ala Asn Asp Arg Ile Phe Leu Leu Thr Met Hit Leu Leu Thr 380 account discrepancies among chemically similar amino acids, 1141 GTA TTT TTG AGG CAG TGG CTC CM AGG CAC CGT CCT CTT CM GM GTT TAT CCA GM (CC 1200 the homology is 60-70% in these regions. When we analyzed 381 Val Phe Leu Arg Gln Trp Leu Gln Arg His Arg Pro Leu Gln Glu Val Tyr Pro Glu Ala 400 nucleotide homology, however, between our human cDNA 1201 MT GCA CCC ATT GGA CAT AAC CGG GM TCC TAC ATG GTT CCT TTT ATA CCA CTG TAC AGA 126(0 401 Asn Ala Pro Ile Gly His Asn Arg Glu Ser Tyr tet Val Pro Phe Ile Pro Leu Tyr Arg 420 and the murine cDNA of Shibahara (PMT4) no significant 1261 MT GGT GAT TTC mTI ATT TCA TCC MA CAT CTG GGC TAT GAC TAT ACC TAT CTA CM GAT 1320 homology was present (<25%). To prove that the poor DNA 421 Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp 440 homology is not due to the species difference, we isolated a 1321 TCA GAC CCA GAC TCT TTT CM GAC TAC ATT MG TCC TAT TTG GAA CAA GCG AGT CGG ATC 1381 mouse 441 Ser Asp Pro Asp Ser Phe Gln Asp Tyr Ile Lys Ser Tyr Leu Glu Gln Ala Ser 460 genomic fragment (PTY-1) corresponding to the Arg Ile 4.5-kilobase EcoRI band (Fig. 3) that hybridized to 1381 TGG TCA TGG CTC CTT GGG GCG GCG ATG GTA GGG GCC GTC CTC ACT GCC CTG CTG GCA GGGI 1440 Pmel34 461 Trp Ser Trp Leu Leu Gly Ala Ala Met Val Gly Ala Val Leu Thr Ala Leu Leu Ala Gly 480 under high-stringency conditions. PTY-1 mapped at or near 1441 CCT GTG AGC TTG CTG TGT CGT CAC MG AGA AAG CAG CTT CCT GM GM MG CAG CCA CTC 1500 481 the mouse c-albino locus (B.S.K., D. Barton, U. Francke, Pro Val Ser Leu Leu Cvs Art His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln Pro Leu 550 R.H., L. Lamoreux, B. Whitney, and A.K.H., 1501 CTC ATG GAG AM GM GGA TTA CCA CAG CTT GTA TCA GAG CCA TTT ATA AAA GGC TTA GGC 1560 unpublished 501 Leu Met Glu Lys Glu Gly Leu Pro Gln Leu Val Ser Glu Pro Phe Ile Lys Gly Leu Gly 520 data). Shibahara reported (33) that PMT4 did not map at the 1561 AAT AGA GTA GGG CCA MA ACC CCT GAC CTC ACT CTA ACT CAA AGT MT GTC CAG GTT CCA 1620 521 mouse c-albino locus. It is of interest that the two proteins Asn Arg Val Gly Pro Lys Ser Pro Asp Leu Thr Leu Thr Gln Ser Asn Val Gln Val Pro 540 that are encoded by what appears to be separate loci have 1621 GAG AAT ATC TGC TGG TAT TTT CTG TAA AGA CCA TTT GCA TTG TM CCT ACA MG 160n 541 AM MAT Glu Asn Ile Cys Trp Try Phe Leu --- similar primary and probably secondary . The in- 1681 TGT AGC CTT CTT CCA ACT CAG GTA GM CAC ACC TGT CTT TGT CTT GCT GTT TTC ACT CAG 17'.0 formation gained from amino acid sug- 1741 CCC TTT TAA CAT TTT CCC CTA AGC CCA TAT GTC TM CIA AAG GAT GCT ATT TGG TM TGA 1800 gests that the two proteins may share functional and immu- 1801 GGA ACT GTT ATT TGT ATG TGA ATT MA AGT GCT CTT AGG MT TC nological properties. Note Added in Proof. The sequence of PTY-1 was identical to FIG. 5. Nucleotide sequence of a cDNA encoding the human the corresponding region of a mouse tyrosinase cDNA sequence tyrosinase and the deduced amino acid sequence. The nucleotide reported by Yamamoto et al. (34). sequence of message strand is numbered in the 5' to 3' direction. Numbers above each line refer to nucleotide position. A portion of The authors thank Dr. S. Glueckson-Waelsch and Dr. M. Lynn a putative signal peptide is indicated by negative numbers. Nucleo- Lamoreux for providing mutant mice; Dr. B. Knowles for HepG2 tide residue 1 is the first nucleotide of the cDNA insert of putative cells; Dr. D. Levitt for HL-60 cells; Dr. G. Kim and Mr. H. Robbins mature tyrosinase. The predicted amino acid sequence is shown III for technical help; and Dr. G. Moellmann, Dr. S. Litwin, and Dr. below the nucleotide sequence. Positive numbers below the amino D. Kestler for critical comments and editing. The authors also thank acid sequence refer to amino acid position, beginning with the amino Helen Kelley and Elaine Wall for typing the manuscript. This work terminus of the mature tyrosinase. The preceding residues of a was supported in part by Grants lR23AI23058-01 (B.S.K.), CA07093 portion of a putative signal peptide are indicated by negative (S.H.P.), and CA04679 (R.H., to Aaron B. Lerner) from the National numbers. Potential glycosylation signals and potential polyadenyl- Institutes of Health. ylation signals are underlined. is indicated by (---). 1. Mason, H. S. (1948) J. Biol. Chem. 172, 83-99. 2. Witkop, C. J., Jr. (1984) in The Clinics in Dermatology (Lippincot, and neutral amino acid residues toward the carboxyl terminus Philadelphia), Vol. 2, pp. 70-134. of the protein (amino acids at positions 460-486). Our 3. Coleman, D. E. (1962) Arch. Biochem. Biophys. 69, 562-568. prediction is that this region serves as a membrane-spanning 4. Gluecksohn-Waelsch, S. (1979) Cell 16, 225-237. domain. The transmembrane domain-like is fol- 5. Green, M. C. (1961) J. Hered. 52, 73-75. sequence 6. Kidson, S. H. & Fabian, B. C. (1981) J. Exp. Zool. 215, 91-97. lowed by the 62 amino acids at the carboxyl terminus. 7. Halaban, R. & Alfano, F. D. (1984) In Vitro 20, 447-450. DISCUSSION 8. Halaban, R., Ghosh, S., Duray, P., Kirkwood, J. M. & Lerner, A. B. (1986) J. Invest. Dermatol. 87, 95-101. We describe here a cDNA clone for human tyrosinase. The 9. Ross, R. A. & Biedler, J. L. (1985) in Advances in Neuroblastoma murine gene corresponding to Pmel34 cDNA is mapped at or Research, eds. Evans, A. E., D'Ansio, G. J. & Seeser, R. C. Downloaded by guest on October 5, 2021 Biochemistry: Kwon et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7477

IIa 100 200 300 400 500 AA a I I I S 8,CHO CH pmelT34P NH2 COOH

pMT4P NH2 I

cys-nch region cys-rich region b I II in w

Homologous Region I Homologous Region III

34 P: (8-138) F P R A V S S K N L - - E r P P W S G -- TG V Pnei M Pmel 34 P: (231-309) D I T D E Y M G G Q H P T N P N L L S P A S FF S S W Q *** * I** * * ** * I*I*+I*I**+ **+ ++ *+** * ** * PMT 4 P: F A N I D L P S S P T D (2-137) P R E E A L R R G V P L G G P PMT 4 P: (234-313) D V C T DD L M GS R S N F D S T L I S P N S V F S Q W R

CG Q L S G R G S Q N - I L L S N A P L G P Q F P FT V G I V S R L E E Y N S H Q S L N G T P E G - P L R R N P ***** 1*1 * * * + +* * I*i* + *I*lI** * ** * ++ +5*1*15*+ * * *5 s+s* * G SSS G R G R V A V I A D S R H S P R H Y P H D G K V V E S - L E E Y D T L G T L|N S T - E G G P I R R N P

D D R E S W P S V F Y NRT Q S G N F M G F N G N K -G N H DK S T T P R L P S S A DV E F L ****+** * ** * + ~*+***J* 1i *l*j * * ** +++ *5* + D D R E ** 11 A W P L R F F N R T C Q N D N F S G H N G T R A G N V G R P A V Q R L P E P Q D V T Q L

F G F W - G P N T E R R L L V R R N I F D L S A P E K D K * * ++ * *+ * *** *** ** +* Homologous Region IV PG-WRGAA NQ+KIL*TV*R*RNLLD*LS;- E-E+K Pmel 34-P:(326-439) S F R N TL E G F - A S P LT G I A D - A S Q S S M i N A L 5** * * * * +1*1* --F F - A Y L T L A K H T I S S D Y V I ** +*.* * PMT 4-P: (330-443) S F R NT V E G Y S A - P - T G KY D P A V R S - L N - L * * * +* *+ * + +* * S H F V R A- L D M A K R T T H P Q F V I

- I Y M NG H V P G- T G - S A N DR I F - L LT M L L 11+++* * + . i * * * * * * 1* A L F L N GT - G G T H L S P N D P I F V L L - T F Homologous Region II Q

Pmel 34 P:(149-213) G S T PM F - N D I N I YD L F V W M Y Y V S M D A LL T --V F L R Q W L R HR P L E V Y P - E A N APIG * ** * * * +*+ *** 1*1** * * Q Q * ** + ** * + + * * * *** *1* PMT 4 P; (150-215) G N T P Q F E N - I S V Y N Y F V W T H Y Y - S V K K T F L T D A V F D - E W L R R Y N A D I S T F P L E - N A P I G i

G G Y E I W R D I D F A E A P A F 'R L F L L R W L P W NR E S Y - M V P F I P L Y RN G D F F I - * * * + *+**+I I*+*s***+* * * ** S S K D L G Y D * * + * * * * * * * + + * . * *** G TCGQ ES FCGDV D F SJEGP A FLT W HR YNHL LQL N R Q - Y N M V P F W P P V T N T E M F V T A P DN L G Y A

E Q E I Q K - L YS Y L Q * + + * *

E R DMQ E ML Y E V - Q FIG. 6. Primary structure of the deduced protein of Pmel34 in the single-letter amino acid . (a) Diagram showing the primary structure of the deduced protein of Pmel34 (Pmel34P) and regions of homology between Pmel34P and the reported putative mouse tyrosinase (PMT4P). The putative signal sequence (S), positions of cysteine residues (*), positions of possible copper ligands (H), possible glycosylation sites (CHO), and a putative transmembrane region (TM) are indicated. NH2, amino-terminus; COOH, carboxyl terminus. The numbers of identical residues (expressed as percentage) in a given segment are indicated between the two proteins. The numbers along with Pmel34P and PMT4P indicate the positions ofamino acids in the putative mature proteins. (b) Alignment of Pmel34P and PMT4P in the homologous regions. (*) Identical amino acids in these proteins. (+) Chemically similar amino acids found in both sequences. Residues in boxes with asterisks are registered residues used for optimum alignment (cysteine or histidine).

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