Proc. Nall. Acad. Sci. USA
Vol. 84, pp. 7473-7477, November 1987
Isolation and sequence of a eDNA clone for human tyrosinase tha
maps at the mouse c-albino locus
BYOUNG S. KWON*t, ASIFA K. HAQ*, SEYMOUR H. POMERANTZt, AND RUTH HALABAN§
*Molecular Genetics 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
cDNA library with antibodies against hamster tyrosinase
(monophenol, L-dopa:oxygen oxidoreductase, EC 126.96.36.199)
resulted in the isolation of 16 clones. ThecDNA inserts from 13
of the 16 clones cross-hybridized with each other, indicating
that they were from related mRNA species. One of the cDNA
clones, Pmel34, detected one mRNA species with an approxi-
mate length of 2.4 kilobases that was expressed preferentially
in normal and malignant melanocytes but not in other cell
types. The amino acid sequence deduced from the nucleotide
sequence showed that the putative human tyrosinase is com-
posed of 548 amino acids with a molecular weight of 62,610.
The deduced protein contains glycosylation sites and histidine-
rich sites that could be used for copper binding. Southern blot
analysis of DNA derived from newborn mice carrying lethal
albino deletion mutations revealed that Pmel34maps near or at
the c-albino locus, the position of the structural gene for
Screening of a Xgtll human melanocyte
Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase,
EC 188.8.131.52) is a copper-based oxidoreductase that cata-
lyzes the oxidation of tyrosine to dopa and the oxidation of
dopa to dopaquinone (1). It is a key enzyme in melanin
biosynthesis. Oculocutaneous albinism, a group of auto-
somal-recessive diseases in humans (2) and animals,
characterized by reduced or absent melanin in skin, hair, and
eyes. Tyrosinase-negative albino melanocytes have no
tyrosinase activity in vitro.
Genetic control of pigmentation has been extensively
studied in mice. There is evidence that the c-albino locus at
chromosome 7 codes for the structural gene for tyrosinase (3,
4). Mutations at this locus affect both tyrosinase activity and
coat color (5, 6).
A nucleic acid probe for tyrosinase would be an invaluable
tool for studies of the regulation of tyrosinase, of the
molecular basis of human albinism, and of various mouse
mutations affecting coat and eye color. We report here the
isolation and sequence ofacDNA clone forhuman tyrosinase
that maps at or near the mouse c-locus.¶
MATERIALS AND METHODS
Cell Culture. Normal human melanocytes, melanotic mel-
anoma cells (LG), and neuroblastoma cells (SK-N-SH) were
cultured as described (7-9). The murine neuroblastoma cell
line NIE115 was obtained from X. 0. Breakefield (E. K.
Shriver Center, Waltham, MA). Proteins of normal melano-
cytes were radiolabeled with methionine (Amersham)
(100 ,XCi/ml, 1390 Ci/mmol; 1 Ci = 37 GBq) as described
cDNA Libraries and Screening. RNA from normal human
melanocytes was prepared, and poly(A)+ RNA was purified
on an oligo[d(T)I-cellulose column (11, 12). A cDNA li
was prepared employing a Xgtll cloning vector (13-15)
Xgtll library contained 1.7 x 106 independent phages
immunobiological screening and analysis of the fusion
teins produced by Xgtll cDNA clones were carried c
described by Young and Davis (15). The rabbit anti-hal
tyrosinase antibodies and their application in the stun
tyrosinases have been described in detail (10, 16).
RNA Blot Hybridization. Poly(A)+ RNA from n(
human melanocytes, melanoma cells, neuroblastoma
lines, HL-60 (human promyelocytic leukemia cell line)
HepG2 (human hepatocarcinoma cell line) was fractiol
on a 1.2% (wt/vol) formaldehyde denaturing gel (17), t
ferred to a GeneScreenPlus membrane (New England
clear), and hybridized with 32P-labeled cDNA pr(
Pmel14-2, a cDNA clone that was isolated from our hi
melanocyte cDNA library, was used as a control p
because the corresponding RNA was detectable in si5
amounts in all human and murine cells tested.
Genomic DNA Blot Analysis. Newborn mice of the g
types C3H/C3H and cch/cH were provided by Sal
Gluecksohn-Waelsch (Albert Einstein College of Medic
Newborn mice provided by M. Lynn Lamoreux (Texas A
University, College Station, TX) were littermates of
were descended from three mice of the genotype C6H/C
that were given to M. L. Lamoreux by S. Gluecks
Waelsch. These stocks were crossed twice onto JU/Ct/
+c/+c mice, then once onto C57BL/6J mice, then for e
generations onto a C57BL/6J-cch/cch stock descended I
mice provided by D. Townsend (University of Minnes
Minneapolis), and then sibling mated for six generation
High molecular weight DNAs of newborn mice of var
genotypes were prepared as described (18). Restric
endonuclease digests ofDNA were electrophoresed in a0
agarose gel at 40C, transferred (19) to GeneScreenPlus,
hybridized with the 32P-labeled cDNA probes. Pmell7-
cDNA clone that was isolated from our human melanoi
cDNA library, was utilized as a control probe for the purr
of estimating the amounts of DNA in each lane bect
Pmell7-1 detected a single EcoRI fragment in all mouse D
tested. The intensity of the hybridizing bands was meast
by a densitometer at 500 nm (Beckman DU-8BUV/
DNA Sequencing. DNA restriction fragments subclone
M13 vectors (20) were sequenced by the dideoxy ch
termination technique (21), with modifications made to
commodate 2'-deoxyadenosine 5'-[a-[35S]thio]triphospt
(22). A forward primer (New England Biolabs) compleir
tary to the lacZ sequence adjacent to the 5'-side ofthe Ec
cCh/c14CWs, and cch/cch. These
tTo whom reprint requests should be addressed.
9This sequence is being deposited in the EMBL/GenBank data t
(Bolt, Beranek, and Newman Laboratories, Cambridge, MA,
Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03581).
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.
Proc. Natl. Acad. Sci. USA 84 (1987)
site in Xgtll was used for the direct sequencing of cDNA
insert end points in Xgtll (23).
Initial screening of 500,000 recombinant phage plaques with
the rabbit anti-tyrosinase antibodies identified 16 indepen-
dent clones. The cDNA inserts varied in size from 0.2 to 1.6
kilobase pairs. The longest cDNA insert (-1.6 kilobase pairs)
from Xme134 hybridized to 12 other cDNA inserts and shared
an overlapping restriction enzyme pattern (Fig. la). The
other three cDNA inserts (not included in the figure) were not
related to the 13 cDNA clones. A cDNA insert contained in
one of these, Xmell7-1, was utilized as a control probe. The
cDNA inserts ofXmell6, Xmel34, Xmel4O, and Xmell7-1 were
subcloned into pBR322 to yield Pmell6, Pmel34, Pmel4O, and
Pmell7-1, respectively, and used for further characterization
800 1000 1200 1400 1600 180
2000 2200 2400
A me 33
A me 16
A mM 52
A me 20
2 3 4
ofthe cDNA clones. A lysogen ofXmell6 was prepared, and
the bacterial lysate was analyzed by 6% NaDodSO4/poly-
acrylamide gel electrophoresis and by competitive immuno-
precipitation assay. Xmell6 was used for this experiment
because the Xmell6 plaques produced the strongest signal in
plaque screening with anti-tyrosinase antibodies even though
Xmel6 contained an incomplete cDNA of -0.7 kilobase
pairs. A fusion protein was produced in Y1089/Xmell6 that
had a relative size of 140 kDa (Fig. lb). The fusion protein
was 25 kDa larger than Escherichia coli 8-galactosidase,
indicating that the incomplete cDNA in Xmell6 was fused to
.3-galactosidasegene in frame. Synthesis of both /3-galacto-
sidase and the fusion protein was dependent on induction
with isopropyl 13-D-thiogalactopyranoside. The production of
immunologically reactive tyrosinase protein in the lysate of
Xmell6 lysogen was tested by competition assay using
metabolically labeled melanocyte cell extracts as a source for
tyrosinase. Bacterial lysates of the Xmell6 lysogen (2 x 107
bacterial cells in 100 pil) competed with roughly 70% of the
anti-tyrosinase antibodies asjudged from the intensity of the
tyrosinase bands and the radioactivity in the relevant gel
slices (Fig. lc).
To examine whether mRNA homologous to Pmel34 is ex-
pressed preferentially in melanocytes, RNA gel blot analysis of
poly(A)+ mRNAfrom cells ofmelanocytic and nonmelanocytic
lineage was performed. Pmel34 hybridized to 21S (=2.4
kilobase) mRNA species from normal human melanocytes and
human melanotic melanoma cells (LG) but not from HepG2,
HL-60, human or murine neuroblastoma (Fig. 2), human and
mouse fibroblasts, or lymphocytes (data not shown).
The skin of mice carrying the radiation-induced albino
alleles, such asc3Hlc3H,had no tyrosinase activity (4). The
tyrosinase activity levels in the skin of mice heterozygous
between the lethal albino deletion and chinchilla were shown
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.
Gluecksohn-Waelsch, reviewed in ref. 4). If the c-albino
locus codes for the structural gene for tyrosinase, tyrosinase
cDNA should not detect any hybridizing band in DNA
extracted from c3H/c3H or c14Cos/c14Cos mice but should detect
hybridizing bands of normal and half intensity in DNA from
homozygote (cch/cch) and heterozygotes (cch/c3H and cch/
ing. (a) Alignment of Xmel34-related cDNA inserts. Inserts from 13
cDNA clones whose gene products bound to anti-tyrosinase anti-
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) ofisopropyl
f3D-thiogalactopyranoside. Samples were electrophoresed on a 6%
NaDodSO4/polyacrylamide gel and stained with Coomassie brilliant
blue. The isopropyl /3-D-thiogalactopyranoside-dependent produc-
tion of 3-galactosidase (lane 2) and a
(lane 3, arrow) was noted. Protein sizes in kDa (Kd) are at the left.
(c) Competition immunoprecipitation assay. The lysate ofXmell6 or
Xgtll lysogen was used to compete with metabolically labeled human
cell extract for anti-tyrosinase antibodies. Equal
amounts of lysates of Xgtll lysogen and Xmell6 lysogen (-2 x 107
bacterial cells in 100-1,l volume) were incubated with 5 ,ul of a 1:100
dilution of anti-tyrosinase antibodies. The respective supernatants
were used to immunoprecipitate [35S]methionine-labeled human
melanocyte extract (6 x 106 cpm in protein per tube). The eluted
immune complexes were separated on 8.5% NaDodSO4/polyacryl-
amide gel. In lanes
preabsorbed with lysogens Xgtll and Xmell6, respectively.
Isolation ofhuman tyrosinase cDNA by antibody screen-
-140-kDa Xmell6 fusion protein
1 and 2, the antibody preparation was
melanocytic and nonmelanocytic lineage. Four micrograms of
poly(A)+ RNA of normal human melanocyte (lane 1), LG (lane 2),
HepG2 (lane 3), and HL-60 (lane 4) cells, and 10 ,ug ofpoly(A)+ RNA
of normal human melanocytes (lane 5), LG (lane 6), human neuro-
blastoma (lane 7), and murine neuroblastoma cells (lane 8) were
fractionated on a 1.2% (wt/vol) formaldehyde denaturing agarose
gel, blotted, and hybridized with 32P-labeled Pmel34. The same filter
was used to hybridize to 32P-labeled Pmel14-2 to show that each lane
contained RNA as indicated above and that the RNA was relatively
Blot analysis of poly(A)+ RNA derived from cultures of
Biochemistry:Kwon et al.
Proc. Natl. Acad. Sci. USA 84 (1987)
albino deletion mutants. Genomic DNA from newborn mice of
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-
ferred to GeneScreenPlus, and hybridized to Pmel34 (a). The same
filter was probed with Pmell7-1 after removal of the Pmel34 probe
(b). Spectrophotometric analysis of the intensity of Pmell7-1 bands
revealed that there was -20% more cch/cch DNA compared with that
in the other four lanes. Kb, kilobases.
Southern blot analysis ofgenomic DNA of murine lethal
As shown in Fig. 3a, the Pmel34 probe detected three
hybridizing cch/cch genomic DNA fragments whose sizes
were 4.5, 12.0, and 14.5 kb. The different extent of hybrid-
ization of the three bands appears to be due to the hetero-
geneity between the human probe and the mouse genes. The
same three bands were detected at half intensity in cch/c3H
and cch/c14Cos DNA when normalized with respect to the
Pmell7-1 band (Fig. 3b). No hybridizing fragments were
detected in c3H/C3H or c14CoS/c14C0s DNA even after pro-
longed exposure. Therefore, the murine genes whose se-
quences are homologous to cDNA contained in Pmel34 are
located at or near the albino locus.
The nucleotide sequence of three overlapping cDNA clones
(Pmel34, Pmell6, and Pmel40) was determined according to the
strategy shown in Fig. 4. Most ofthe other 10 clones were also
sequenced. The nucleotide sequence of tyrosinase cDNA re-
vealed a single long open reading frame, beginning with the first
nucleotide after theEcoRI 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
found that other clones were also fused in frame with the IacZ
gene. This property was helpful in assigning the open reading
frame even though the cDNAs did not start with the first ATG
codon. The open reading frame coded for a polypeptide of566
amino acids with a molecular weight of 63,549 (Fig. 5). The
codon specifying carboxyl-terminal leucine wasfollowed by the
translation termination codon TAA (nucleotide residues
1645-1647). No nucleotide differences were observed among
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
The deduced sequence of the first 12 amino acid residues
of the tyrosinase has characteristics of the signal peptide of
secretory and membrane-associated proteins (26), which
mainly contains hydrophobic amino acids (10 out of 12
residues) and terminates with serine (27) (Fig. 5). We puta-
tively assign the first 12 amino acids as a part of a signal
peptide. A possible site cleavage of the signal peptide of the
tyrosinase precursor is after the serine residue at phenylal-
anine-1 (Fig. 5). Thus, the protein backbone of processed
tyrosinase is composed of 548 amino acids with a molecular
weight of 62,160.
As tyrosinase is a glycoprotein, we have looked for
possible N-glycosylation sites and found five potential as-
paragine-linked glycosylation signals (28, 29) at positions 73,
98, 148, 217, and 324 as underlined in Fig. 5.
The protein contains two cysteine-rich domains (Fig. 6a,
Pmel34P). There are 17 cysteine residues; 10 residues are
clustered within the first 100 amino acids, and 5 residues are
clustered between amino acids 231 and 308.
The tyrosinase molecule contains two copper atoms (30,
31), and histidine residues serve as copper binding sites (31,
32). There are 15 histidine residues in the deduced amino acid
sequence of human tyrosinase. Four of these appeared
between the two cysteine-rich domains (histidine-130, -167,
-189, and -198), and 6 residues appeared after the second
cysteine-rich domain (histidine-350, -354, -360, -377, -390,
and -406). Histidine residues at positions 350, 354, 360, and
377 had an arrangement similar to that of bovine superoxide
dismutase, another copper-containing enzyme (32). There is
a stretch of 27 amino acids that contains only hydrophobic
map and sequencing strategy for
human tyrosinase cDNA. The
tyrosinase-coding region is indi-
cated by an open box. Horizon-
tal arrows underthe three inserts
show the direction and extent of
sequencing used to generate the
sequence presented in Fig. 5.
Restriction sites used for se-
quencing are indicated. The
scale at the top indicates the
X mel 34
X mel 16
X mel 40
Biochemistry:Kwon et al.
Biochemistry: Kwon et al.
GAA TTC CTG CTC CTG GCT GTT TTG TAC TGC CTG CTG TGG AGT
------- Leu Leu Leu AlaVal Leu Tyr Cys Leu Lett TrpSr
TTCCAGACC TCC GCTGGCCAT TTC CCT AGA GCC TGT GTC TCC TCT AAG AAC CTC ATG GAG
Phe GlnThrSerAlaGly His Phe Pro Arg Ala Cys Val Ser Ser Lvs Asn Leu Met Gltt
AAG GM TGC TGTCCA CCGTGG AGC GGG ACAGGA GTC TGT(GC CAG CTT TCA GGC AGA GGT
Lys Glu Cys CysPro Pro Trp Ser Gly ThrGly Val Cys Gly Gln Leu Ser Gly Are
TCCTGT CAG AATATC CTTCTG TCC MT GCA CCA CTT GCG CCT CM TTT CCCTTC ACA GGG
SerCys Gln Asn Ile Leu Leot Ser Asn Ala Pro Leu GlyPro Gln Phe Pro Phe Ther Glv
GTG GAT GAC CGG GAG TCG TGG CCT TCC GTC TTT TAT AAT AGG ACC TGC CAG TGC TCT GGC
Val Asp Asp ArgGlu Ser Trp Pro Ser Val Phe Tyr Asn ArgThr Cys Gln Cys Ser Glv
MC TTCATG GGATTC MC TGT GGA MC TGC MCG
Asn Phe Met GlyPheAsn Cys Gly Asn Cys Lys Phe Gly Phe Trp Gly Pro Asn Cys Thr
GAG AGA CGACTC TTG GTG AGA AGA MC ATC TTC GAT TTG AGT GCC CCA GAG MG GAC AA
Glu Arg Arg Leu Leu Val Arg Arg Asn Ile Phe Asp Leu Ser Ala Pro Glu Lys Asp Lys
mTT TTT GCCTAC CTC ACTTTA GCA AAG CAT ACC ATC AGC TCA GAC TAT GTC ATC CCC ATA
Phe PheAlaTyr Leu Thr Leu AlaLys His Thr Ile Ser Ser Asp Tyr Val Ile Pro Ile
GGG ACC TAT GGC CAA ATG MA AAT GGA TCA ACA CCC ATG mr MC GAC ATC MT ATT TAT
Gly Thr Tyr Gly Gln Met L-s Asn Gly Ser Thr Pro Met Phe Asn Asp Ile Asn Ile Tyr
GAC CTC TmC GTC TGG ATGCAT TAT TATGT,
Asp Leu PheVal Trp Met His Tyr Tyr Val SerMet Asp Ala Leu Leu Gly Gly Tyr Glu
ATCTGG AGA GAC ATTGAT TTT GCC CATGAA GCA CCA GCT TTT CTG CCT TGG CAT AGA CTC
Ile Trp Arg Asp Ile Asp PheAla His GluAla Pro Ala Phe Leu ProTrp His Arg Lett
TTCTTG TTG CGG TGG GAA CM CAAATC CAG MG (TGACA GGA GAT GM MC TTC ACT ATT
Phe Lett Leu Arg Trp Glu Gln Glt Ile Gln Lys Leu Thr Gly Asp Gli
mTT GGC TmT TGG GGA CCA MC TGC ACA
TCAATG CAT GCA CTG CTT GGG GGA TAT CAA
Asn Phe Thr Ile
CCA TATTGC GAC TGG CGG GAT GCA GM MtC TGT GAC ATTTGC ACA GAT
Pro Tyr Trp Asp Trp Arg AspAlaGlu Lys Cos Asp Ile Cys Thr Asp Glu Tyr Met Gly
GGT CAGCAC CCC ACA MT CCT MC TTA CTC AGC CCA GCA TCA TTC TTC TCC TCT TGG CAG
Gly Gln HisProThr AsnPro Asn Leu Leu Ser Pro Ala Ser Phe Phe Ser Ser Trp Gln
ATTGTC TGT AGC CGA TTG GAG GAGTAC MC AGC CATCAGTCT TTA TGC MT GIA ACG CCC
Ile Val Cys Ser Arg Leu Glu Glu Tyr Asn Ser His Gln Ser Leu Cys Asn Gly Thr Pro
GAG GGACCT TTACGG CGT AATCCT GGA MC CAT GAC MA
Glu Gly Pro Leu Arg Arg Asn Pro Gly Asn His Asp Lys Ser Thr Thr Pro Arg Leu Pro
TCT TCAGCT GAT GTA GM('C
TGC CTG AGT TTG ACC CM TAT GAATCT GGT TCC ATG GAT
Ser SerAlaAsp Val GluPheCys Leu Ser Leu Thr Oln Tyr Glu Ser Gly Ser Met Asp
GCT GCC MT TTCAGCTTTAGA MT ACA CTG GM GGA TTT GCT AGT CCA CTT ACT GGG
LysAla AlaAsnPheSer Phe Arg Asn Thr Leu Glu Gly PheAla Ser Pro Leu Thr Gly
ATAGCG GAT GCCTCTCAA AGC AGC ATGCAC AAT (CC TTGCAC ATC TAT ATG MT GGA CAT
IleAlaAspAla Ser Gln Ser SerMet His AsnAla Leu His Ile Tyr Met Asn Gly Hit
GTCCCAGGTACAGGATCT GCC MC GAT CGT ATC TTC CTT CTC ACC ATGCAT TTG TTG ACA
Val Pro Gly Thr Gly SerAlaAsn Asp Arg Ile Phe Leu Leu ThrMet Hit Leu Leu Thr
GTATTTTTG AGG CAG TGG CTC CM AGGCAC CGT CCTCTT CM GM GTT TAT CCA GM (CC
Val Phe Leu Arg Gln Trp Leu Gln Arg His Arg Pro Leu Gln Glu Val Tyr Pro GluAla
MT GCACCC ATTGGACAT AAC CGG GMTCC TACATG GTT CCT TTT ATA CCA CTGTAC AGA
AsnAlaPro Ile Gly His Asn Arg Glu Ser Tyr tet Val Pro Phe Ile Pro Leu Tyr Arg
MT GGT GATTTC
Asn Gly Asp Phe Phe Ile Ser Ser Lys Asp Leu Gly Tyr Asp Tyr Ser Tyr Leu Gln Asp
TCAGACCCA GAC TCT TTT CM GAC TAC ATT MGTCC TAT TTG GAA CAA GCG AGT CGGATC
Ser Asp Pro Asp SerPheGln Asp Tyr Ile Lys Ser Tyr Leu Glu GlnAla Ser Arg Ile
TGGTCATGGCTC CTTGGG GCG GCG ATG GTA GGG GCC GTC CTC ACT GCC CTG CTG GCA GGGI
Trp Ser Trp Leu Leu GlyAla Ala Met Val
4AG TAC ATG GGA
TCC ACA ACC CCA AGG CTC CCC
mTIATT TCA TCC MA CATCTG GGC TAT GAC TAT ACC TAT CTA CM GAT
Gly Ala Val Leu ThrAla Leu LeuAla Gly
CCTGTG AGC TTG CTG TGT CGTCAC MG AGA AAG CAGCTT CCT GM GM MG CAG CCA CTC
Pro Val Ser Leu Leu CvsArt His Lys Arg Lys Gln Leu Pro Glu Glu Lys Gln Pro Leu
CTCATG GAG AM GM GGATTA CCACAGCTT GTA TCA GAG CCA TTT ATA AAA GGC TTA
LeuMet Glu Lys Glu Gly Leu Pro Gln Leu Val Ser Glu Pro Phe Ile Lys Gly Leu Gly
AAT AGA GTA GGGCCA MA ACCCCT GAC CTC ACT CTA ACTCAA AGT MT GTC CAG GTT CCA
Asn Arg Val Gly Pro Lys Ser Pro Asp Leu Thr Leu Thr Gln Ser Asn Val Gln Val Pro
GAG AATATCTGC TGGTAT TTTCTG TAA AGACCA TTT GCA AM TTG TM CCT MAT ACA MG 160n
Glu Asn Ile Cys Trp Try Phe Leu ---
TGT AGCCTT CTT CCA ACTCAG GTA GMCAC ACC TGT CTTTGT CTT GCT GTT TTC ACT CAG
CCC TTTTAACAT TTT CCC CTA AGC CCA TAT GTCTM CIAAAG GAT GCT ATT TGGTM TGA
GGAACTGTTATTTGT ATG TGAATTMA AGT GCT CTT AGG MT TC
tyrosinaseand the deduced amino acidsequence.The nucleotide
sequenceofmessagestrand is numbered in the 5' to 3' direction.
Numbers above each line refer to nucleotideposition.Aportionof
aputative signal peptideis indicatedby negativenumbers. Nucleo-
tide residue 1 is the first nucleotide of the cDNA insert ofputative
maturetyrosinase. The predicted amino acid sequenceis shown
below the nucleotidesequence.Positive numbers below the amino
acidsequencerefer to amino acidposition, beginningwith the amino
terminus of the mature tyrosinase. The preceding residues of a
portion of a putative signal peptide are indicated by negative
numbers. Potentialglycosylation signalsandpotential polyadenyl-
ylation signalsare underlined.Stopcodon is indicatedby (---).
Nucleotidesequenceof a cDNAencodingthe human
and neutralamino acid residues toward thecarboxylterminus
of the protein (amino acids at positions 460-486). Our
predictionis that thisregionserves as amembrane-spanning
domain. The transmembrane domain-like sequence is fol-
lowedbythe 62 amino acids at thecarboxylterminus.
We describe here a cDNA clone for human tyrosinase. The
murine gene corresponding to Pmel34 cDNA is mapped at or
Proc. Natl. Acad. Sci. USA 84(1987)
near the c-albino locus that is considered to be the structural
gene for tyrosinase. Southern blot analysis of DNAs from
mouse-Chinese hamster somatic cell hybrids has confirmed
the localization of the sequence homologous to Pmel34 on
mouse chromosome 7 (B.S.K., D. Barton, U. Francke, R.H.,
L. Lamoreux, B. Whitney, and A.K.M., unpublished obser-
vations). Since the 5' end of Pmel34 mRNA has not been
cloned the assignment of signal peptide is putative.
Shibahara et al. (25) reported acDNA sequence for murine
tyrosinase. A comparison of our amino acid sequence and
that deduced by Shibahara et al. (25) shows extensive
homology. Fig. 6 shows the regions ofhomology between the
two tyrosinase sequences and the optimal alignment of the
peptide sequences in the regions ofhomology. There are four
regions of homology in the two proteins. Homologous do-
mains I and III span the first and second cysteine-rich
regions, respectively. The space between cysteines is iden-
tical or similar, and the residues around cysteine areespe-
cially similar in the twoproteins. Homologous domains II and
IV include residues 149-213 and 326-439, respectively. In
homologous domain II, three histidine residues arealigned,
and residues surrounding each histidine are similar. In
domain IV there are six histidines in the human sequence and
five histidines in the murine sequence; four histidines are
aligned with similar interspaces. Histidine-406 is surrounded
by seven residues that are identical in the two proteins. The
amino acid identity between the homologous domains of the
two proteins is 48.8% in domain I, 54.6% in domain II, 50.0%
in domain III, and 48.0% in domain IV. If we take into
account discrepancies amongchemically similar aminoacids,
the homology is 60-70% in these regions. When weanalyzed
nucleotide homology, however, between our human cDNA
and the murine cDNA of Shibahara (PMT4) no significant
homology was present (<25%). To prove that the poor DNA
homology is not due to the species difference, we isolated a
mouse genomic fragment (PTY-1) corresponding to the
4.5-kilobase EcoRI band (Fig. 3) that hybridized to Pmel34
under high-stringency conditions. PTY-1 mapped at or near
the mouse c-albino locus (B.S.K., D. Barton, U. Francke,
R.H., L. Lamoreux, B. Whitney, and A.K.H., unpublished
data). Shibahara reported (33) that PMT4 did not map at the
mouse c-albino locus. It is of interest that the two proteins
that are encoded by what appears to be separate loci have
similar primary and probably secondary structure. The in-
formation gained from amino acid sequence alignment sug-
gests that the two proteins may share functional and immu-
Note Added in Proof. The exon sequence of PTY-1 was identical to
the corresponding region of a mouse tyrosinase cDNA sequence
reported by Yamamoto et al. (34).
The authors thank Dr. S. Glueckson-Waelsch and Dr. M. Lynn
Lamoreux for providing mutant mice; Dr. B. Knowles for HepG2
cells; Dr. D. Levitt for HL-60 cells; Dr. G. Kim and Mr. H. Robbins
III for technical help; and Dr. G. Moellmann, Dr. S. Litwin, and Dr.
D. Kestler for critical comments and editing. The authors also thank
Helen Kelley and Elaine Wall for typing the manuscript. This work
was supported in part by Grants lR23AI23058-01 (B.S.K.), CA07093
(S.H.P.), and CA04679 (R.H., to Aaron B. Lerner) from the National
Institutes of Health.
Mason, H. S. (1948) J. Biol. Chem. 172, 83-99.
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Philadelphia), Vol. 2, pp. 70-134.
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Proc. Natl. Acad. Sci. USA 84 (1987) Download full-text
Homologous Region I
Pnei 34 P:
(8-138) F P R A
V S S K N L
-- M E r
PMT 4 P:
(2-137) F P R E
A N I E A L R R G V
P D L L PS
P G T D P
CG Q L S G R G S
SSS G R G R
I L L S N A P L G P Q F P F T G V
P H S R H Y P H D G K
V A V
I A D S R
D D R E S W P S V F Y N R T
D D R E A W P L R F F N R T C Q
QS G N F M G F N
N D N F S G H N
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
- A Y L T L A K H T I S S D Y V I
+* *+ *
S H F V R A- L D M A K R T T H P Q F V
+ +* *
Homologous Region II
Pmel 34 P:(149-213) G S T PM F
- N D
I S V Y N Y F V W T H Y Y
I Y D L F V W M
Y Y V S M D A L
S V K K T F L
PMT 4 P;
(150-215) G N T P Q F E N
GTCGQ ES FCGDV D F SJEGPA FLT W HR YNHL LQL
G Y E I W R D I D F A
E A P A F L P W
'R L F L L R W
E Q E I Q K
+ + *
E R DMQ E ML
Pmel 34 P: (231-309)
T D E Y M G G Q H P T N P N L L S P A S F F S S W Q
D V C T D D L M GS R S N F D S T L
I S P N S V F S Q W R
PMT 4 P:
S R L E E Y N S H Q S L
* ** * ++
N G T P E G
- P L R R N P
- E G G P I R R N P
- L E E Y D T L G T L|N S T
-G N H D K S T T P R L P S S A D V E F
A G N V G R P A V Q R L P E P Q D V T Q
Homologous Region IV
S F R N T L E G F
S F R N T V E G Y S A
- A S P L T G I A D
- A S Q S S M
i N A L
- T G K Y D P A V R S
I Y M N G H V P G- T G
L F L N G T - G G Q T H L S P N D P I F V L L -
S A N D R I F
- L L T M
T --V F L R Q W L Q R H R P L Q E V Y P
T D A V F D
- E A N APIG
- E W L R R Y N A D I S T F P L E - N A P I G i
N R E S Y - M V P F I P L Y R N G D F F I S S K D
* * +
* * * *
N R Q
- L G Y D
* * *
* + +
- Y N M V P F W P P V T N T E M F V T A P D N L G Y A
Y S Y L Q
Y E V
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 ofcysteine residues (*), positions ofpossible 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 ofPmel34P and PMT4P in the homologous regions. (*) Identicalamino
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).
Primary structure of the deduced protein of Pmel34 in the single-letter amino acid code. (a) Diagram showing the primary structure
(Raven, New York), pp. 249-259.
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