0 1992 MUNKSGAARD
Pigment Cell Research 5:162-167 (1992)
Isolation and Characterization of a Chicken Tyrosinase cDNA MAKOTO MOCHI1,l AKIO 110,' HIROAKI YAMAMOTO,' TAKUJI TAKEUCHI,' ~ N GORO D EGUCHI' 'Division of Morphogenesis, Department of Developmental Biolop! National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki 444,Japan, and Biologcal Institute, Faculty of Science, Tohoku University, Aoba-yama, Sendai 980, Japan '
Complementary DNA clones coding for chicken tyrosinase were isolated from retinal pigmented epithelium of chicken embryo. Sequence analysis shows that one of the cDNA clones consisting of 1,997 nucleotides has an open reading frame coding for 529 amino acids. The deduced protein has nine N-glycosylation sites and a transmembrane region. A sequence comparison of the deduced chicken tyrosinase with the mouse and human homologues revealed that amino acid sequences are conserved for the entire polypeptides. Seventy-twopercent and 73% of amino acids in the chicken sequence are identical to that of the mouse and human tyrosinases, respectively. Histidines neighboring the postulated copper-bindingsites and the cysteines are well conserved. RNA blotting analysis showed that a major transcript of 2.5 kb is detected in retinal pigmented epithelium of a 9-day-oldchicken embryo. Key words: Chicken tyrosinase, cDNA cloning, Sequencing, Pigmented epithelium, RNA blotting
INTRODUCTION Tyrosinase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis, and is found in pigmented cells including melanocytes derived from the neural crest and pigmented epithelial cells from the neural plate. Cloning the gene encoding tyrosinase allows us to study the molecular basis of melanin synthesis and the regulatory mechanisms of gene expression during pigment cell differentiation. Tyrosinase cDNA clones from mouse (Yamamoto e t al., 1987, 1989; Miiller et al., 1988)and human (Kwon et al., 1987; Shibahm et al., 1988; Takeda et al., 1989) have been described, and the gene is mapped to the mouse albine locus (Kwon et al., 1987). A cDNA clone encoding tyrosinase-related protein has been isolated (Shibahara et al., 1986), and its gene is mapped to the mouse brown locus (Jackson, 1988). Genetical analyses of these DNAs reveal genetic changes affecting the coat-color differences in mice (Kwon e t al., 1989; Shibahara et al., 1990; Tanaka et al., 1990). There are many species and strains expressing different plumage colors in avians also. Molecular analyses of tyrosinase and other proteins involved in melanosome formation in avians will allow us to study the mechanisms of feather-color differences. Retinal pigmented epithelium of chick embryo is a good material for molecular studies in pigment cell differentiation and pigment formation. We can isolate and cultivate pure populations of pigmented epithelial cells from the chicken embryos. The cells dedifferentiate losing melanosomes and proliferate vigorously in a culture condition
defined by Itoh and Eguchi (1986). These dedifferentiated cells then start to differentiate into pigmented epithelial cells synthesizing much melanin, when cultured under another condition (Itoh and Eguchi, 1986; Eguchi, 1988). Thus, we can obtain a sufficient quantity of cells to study biochemically in any step of pigment cell differentiation. previously we identified and characterized a melanosomal protein, 115-kD melanosomal matrix protein (MMPl15), in the pigmented epithelial cells of chicken (Mochii et al., 1988a).A study with a specific monoclonal antibody showed that the MMP115 is localized in the melanosomal matrix of the pigmented cells and suggested that it has a function in the matrix formation. A complementary DNA clone encoding the MMP115 has been isolated, and the primary structure was determined (Mochii et al., 1988b; 1991). Northern blotting showed that the MMP115 gene was activated in the differentiation process of the pigmented epithelial cell (Mochii et al., 198813). Molecular cloning of a chicken tyrosinase cDNA makes it possible to study the interaction of the MMP115 and tyrosinase in the melanosome formation, and comparison of their primary structures might reveal the functional domains in these proteins.
Address reprint requests to Goro Eguchi, Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki 444, Japan. Received December 28,1991; accepted March 10, 1992.
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Chicken Tyrosinase cDNA In this report, we show the molecular cloning of a chicken tyrosinase cDNA from the pigmented epithelium of chicken embryo. The complete primary structure of the cDNA and the deduced protein, and expression of the gene are described.
MATERIALS AND METHODS Cloning of Chicken 'Qrosinase cDNA A A g t l O cDNA library was constructed with poly(A) RNA from cultured pigmented epithelial cells of White Leghorn embryos as previously described (Mochii e t al., 1991). The library was screened with a DNA fragment of 700 bp encoding the first exon of the quail tyrosinase gene isolated by Yamamoto et al. (in preparation). The probes were made by the PCR technique (Saiki et al., 1988) with Taq DNA polymerase (Perkin-Elmer Cetus) using oligonucleotide primers, 5 '-TTACCATGGGCTTACTGCTG-3' and 5'-GGGATGGTGAAGTTCTCATC-3'. DNA labeling and hybridization were performed as previously described (Mochii et al., 1988b). +
DNA Sequencing EcoRl fragments were subcloned into the Bluescript SK' plasmid (STRATAGENE) and sequenced in both directions by the dideoxy terminator method (Eibor et al., 1987) using Sequenase (United States Biochemical Corporation). Oligonucleotides for sequencing primers were synthesized by an automatic DNA synthesizer (Applied Biosystems). RNA Blot Hybridization Total RNAs were isolated from pigmented epithelium, heart, liver, muscle, and skin of 9-day-old White Leghorn embryos by the guanidiudcesium chloride method (Maniatis et al., 1982). Electrophoresis, blotting, and hybridization were carried out as previously described (Mochii et al., 1988b). RESULTS Isolation and Characterization of the Chicken Qrosinase eDNAs The A g t l O cDNA library constructed from poly(A)+RNA of chicken pigmented epithelial cells was screened with a DNA fragment encoding the first exon of quail tyrosinase gene (Yamamoto e t al., in preparation). Fifteen independent clones were identified from screening of 100,000recombinant phage plaques. Four clones (Ctyl8, Ctyl9, Cty24, and Cty25) containing inserts longer than those contained in the rest were characterized. The inserts were subcloned into plasmids and mapped using endonucleases (Fig. 1). Ctyl8 and Ctyl9 contained 2.0 kb inserts, and Cty 24 and Cty25 contained 1.5 kb inserts. Their restirction maps showed that they were closely related to each other. The nucleotide sequences of both ends of the four inserts were determined and compared with the sequences of human and mouse tyrosinase cDNAs (Shibahara et al., 1988; Yamamot0 e t al., 1988; Takeda et al., 1989). Every insert had the poly(A) tail a t one end which was identified as a 3' terminus. Ctyl8 and Ctyl9 started at the same nucleotide
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Fig. 1. Partial restriction maps of cDNA clones encoding chicken tyrosinase. The protein-coding region is indicated by an open box.
from their 5' termini, and contained ATG codons. I t was assumed that Ctyl8 and Ctyl9 contained enough sequence to encode the complete chicken proteins, and that the entire sequences of Cty24 and Cty25 were contained within the boundaries of the Ctyl8 and Ctyl9 sequences. The complete sequence of the Ctyl8 insert was determined.
Nucleotide and Deduced Protein Sequences of the Ctyl8 cDNA The complete nucleotide sequence of the Ctyl8 cDNA and the deduced amino acid sequence are shown in Figure 2. The insert consists of 1,997 nucleotides. The sequence of the coding strand has an open reading frame of 1,587 nucleotides, which begins with an ATG codon at 108 nucleotides from the extreme 5' terminus and terminates by a stop codon TAA. The open reading frame is followed by 240 nucleotides of the 3' untranslated sequence and 52 nucleotides of the poly(A) tail. A potential poly-adenylation signal (ATTAAA) is identified. The open reading frame codes a protein of 529 amino acids. The deduced protein contains nine potential N-glycosylation sites (Asn-X-Thr or Asn-X-Ser). A hydrophobic signal peptide is identified a t the N-terminus, and a potential membrane-spanning region is near the C-terminus. Sequence Comparison of Chicken and Mammalian 'Qrosinases The cDNA clone Ctyl8 shows about 60% and 68% DNA similarities to the cDNAs of the mouse and human tyrosinases, respectively. The deduced amino acid sequence of the chicken tyrosinase was compared with that of mouse (Yamamoto e t al., 1988) and human (Shibahara e t al., 1988; 'Ihkeda e t al., 1989). An alignment of these sequences is shown in Figure 3. Seventy-two percent and 73% of amino acids in the chicken tyrosinase are identical to those of mouse and human, respectively. About 30 amino acids a t the N-terminus and 80 amino acids at the C-terminus show 40-50% homologies with the mouse and human sequences. All six potential N-glycosylation sites identified in the mouse and human sequences are conserved in the chicken protein which contains three additional sites. The chicken tyrosinase is four amino acids shorter than the mouse homol o p e but has the same number of amino acids as the human one. The chicken sequence lacks four amino acids in a region connecting the transmembrane sequence and the following region in the mouse sequence. This deletion was con-
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481 CAGCTCACCA1'CAGTGAGAAGGACAAGTTCCTTCCTTGCCTACCTTAACCTAGCA~GRAC~TC Q L T I S E K D K F L A Y L N L A K N I l 4
CCCATGTTCAG~TATCAACGTGTACGATC?'CTTCGTCTGGAI'(;CATTATTATGCCTCT P k l F R N I N V Y D L F V W M H Y Y A S 184
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Fig. 2. Nucleotide sequence of a cDNA, Ctyl8 encoding chicken tyrosinase, and the deduced amino acid sequence. The predicted amino acid sequence (in one-letter code) is shown below the nucleotide se-
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1021 GACAAATCAAGGACCCCAAGACTCCCATCTTCATCAGAAGTCGA'4TTTTGTCTTACACTC U K S R T P R L P S S S E V E F C L T L 3 2
961 CAACAGGCTTTATGCAATGCCACTAGTGAAGGGCCTATAC'TTCGAAATCCTGGAAACAAT
901 CCAGCATCATTTTTCTCCTCATGGCAGG1'AA'TCTGCACTCAATCTGAAGAGTACAACAGC P A S F F S S W Q V I C T Q S E E Y N S 2 8
841 G T G A T C T G C A C T G A T G A A T A C A T G G G T G G C C h A C A C C C C A C C i \ A C C C T ~ ~ n ' A ~ ~ C A G C v I c T D E Y M G G Q II P 'r N P N L L s 2 6 4
781 ATAACAGGTGATGAGAATTTCACCATCCATCCCCTACTGGGACTGGCGAGATGCAGAGG~CTGT I T G D E N I: T 1 I ' Y I B D W R D A E D C 244
7 2 1 CCTGCTTTTCTGCCTTGGCATCGTGCTTTTCTGCTGCTGTGGGA~CGTG~G,~T,~C~G~~~~G P G F L P IY f f R A I: L L L \V E H E I Q K 2 2 4
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661 CCAGACACACTCTTAGGTGGCTCCAATCTGTGG~~GAGACATTGATTTTGCCC~TGA~GCC
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541 C C C A G C A A G G A C T A T G T M T T G C T A C T G G C A C A T A T G C T C A G A T G A A C ~ C G G C T C C A ~ ~ C P S K D Y V I A T G T Y A 6) M N N G S N 164
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421 TTTGGCTTCTCAGGACAAAACTGCACTGAAAGGCGACTGAGAACGAGAAGAAACATCTTC F G F S G Q N C T E R R L R T R R N I F l 2
361 TACAACCGGACATGCAGATGCAGAGGCAATTTCATGGGGTTCAACTGTGGGGAGTGCMG Y N I~ T C R C R G N F M G F N C G E C K 104
301 CTGGGACCACAGTTCCCTTTCTCAGGAGTGGATGACAGAGAGGATTGGCCTrCTGrCTTT L G P Q F P F S G V D D R E D W P S V F 8 4
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241 CCTTGCGGGGAGCGTTCCAACAGAGGAACCTGCCAGCGCATCCTTCTCTCTCAGGCTCCT
181 GCAAACACGCAGAGCTTGCTGAGGAAGGAGTGCTGTCCGCCCTGGGATGGAGATGGGACC A N T Q S L L R K E C C P P W D G D G T 4
121 GCCATGGGCTTACTGCTGGTCATCCTTCAGCCGTCCACTGGGCACTTCCCCAGAGTCTGT A M G L L L V I L Q I ' S T G Q F P R V C 2 4
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6 1 TCCATGCACTGAAGCATAACACTGATGCTCTCCTGCTGCTCTGTGAGGATGT~CTGTTT
1 CCCAGTTGGGAGGAAAAGTTTCTTTCACACTCAGGTCATTGGAGCGGTCAGAGAGAACTC
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quence and numbered on the right side. A putative signal peptide, membiane spanning region, potential N-glycosylation sites, and a potential poly-adenylation signal are underlined. Asterisk indicates a stop codon.
1981 P
1921 TGCTTGATTAAACAACACTCCTGTGAAAAAAAMAAAAAAAAAAAAAAAAAAAAAAAAAA
1861 C T C C M A A T C T G T C A T A T T C T A A G T T A C C A T G C C A C C C A A G
1801 TGTCCCACTCCTTGTGCCTCTTGTAACATCCACTGTTGTAGTACCTTATCAGCTCCAGTC
1741 GCATGCAGTGTTTATGTTTACAATGACC?'TGTTGAACTGTGCTGCATAAGCTGGCTGGGC
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1681 TATCMTCCCATTTCThhRGACCTCCAGTATCTCAATCATGTTTCTAAGGTGCTAGTCAA
1621 GGAACTTC'TCCAGAAATACAACCCTTACTCACAGAAAGTGAAGATTACAACAATGTATCT G T S P E I Q P L L T E S E D Y N N V S 5 2
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1561 G G A G G C A T A A T T A C T G C T G T G C T C I ' C T G G G C T C A T C C T G G C C T G C A G G A A G ~ A G A ~ A G G I I T A V L S G L I L A C R K K R K 5 0 4
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1501 ATCCCCTACCTCAAGCAAGCCCATCAGATCTGGCCCTGGCTGGTTGGCGCAGCTGTGATC
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1441 CTGGGGTATGACTATGAGTATCTGCAAGAACCAGCACTTGGTTCTTTCCAGGACTTTTA
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1381 TACATGGTTCCCTTTATCCCTCTTTACAGATGGAGAATTTTTTATATCATC~GAGAG
1321 A G A C C C A T G C T A G A A G T T T A C C C A G C A G C C A A C G C A C C C A T T G G G C A C ~ T C G A G ~ T R P M L E V Y P A A N A P I G H N R E N 4 2 4
1261 ATCTTCATCCTACACCATGCATTTGTTGACAGCATTTTTGAGCGGTGGTTAAGAAGACAC I F I L II H A F V D S I F E R W L R R H 404
1201 GCCCTTCACATCTACATGAATGGCTCAATGTCCCAAGTACAA~GCTCTGCGAATGATCCT A L II I Y M N G S M S Q V Q G S A N D P 384
1141 GAAGGCTTTGCTGATCCACACACTGCRATATC~CATATCTCAAAGTGGTTTGCATAAT E G F A D P H T A I S N I S Q S G L H N 3 6
I081 ACTCAGTATGMTCTGGATCCATGGATAAAATGGCCAATGGCCRATTACAGC~rCCG~CACTTTG T Q Y E S G S M D K M A N Y S F R N T L 3 4
Chicken Qrosinase cDNA
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FIFLFAMGLL1.VILQPSTGQFPRVCANTQSL.LRKECCPPIVDGDGTPCGEll~ILI. 60 . . .AVLYC . . IVSF.1.D.I~ . .A .. . SSKN . . A . . . . . . . M . . . S . . . Q L . G . . S . . D . . . .L.AVLYC. . WSF.T.A.H . . .A.VSSKN.ME . . . . . . . S..RS...QL.G..S..N... + + + + + SQAPLGPQFPFSGVDDREDWPSVFYNRTCRCRGNFMGFNCGECKFGFSGQNCTERKLRTR 1 2 0 . S . . S . . . . . . K . . . . . . S . . . . . . . . . . Q . S . . . . . . . . . N . . . . . G . P . . . . KI. .VL . N . . . . . . . . . T......S..........Q.S.........N.....W.P.......LV .
RNIFQLTISEKDKFLAYLNLAKNIPSKDYVIATGTYAQMNNGSNP~lFRNI~~DLFV~~ltl 180 . . . . D.SV . . . N . . FS . . T...HTI.SV...P....G......T...ND..I........ .D.SAP . . . . .F...T...HTI.S....PI...G..K...T...ND.. I WASRDTLLGGSNV\VRDIDFAHEAPCFLPIVIIRAFLL.I~WEREIQKITGDENFTII’~VD\~R~ 240 . . . . . . .L . . . . . . Q . . R E L . . . . . . . V . . . . . . . . . V . . . . . . . . . EI . . . . . . A . . . . . .L...R..Q....L............... . . V.M.A . . . . .EI . . . . . . I
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GLHNALHIYMNGSMSQVQGSANDPIFILHHAFVDSIFEK\~I..RRHRP~ll~E~P~~Al’lGII 420 SF1 . . . . . . F ...T.............L...........Q.......L.....E....... . . . .Q . . . . . . . L Q . . . . E....... SM U NRENYMVPFIPLYRNGEFFISSREL YEYLQEPALGSFQDFLIPYLKQAHQI\VI’\VLVG 180 . . DS . . . . . . . . . . . . D...T.KD. . S . . . . SI)P.FYRNYIE. . . E . . SR. . . . .I.. . . .S . . . . . . . . . . . .D.....KD.....S...DSDPD....YIKS..E..SR..S..L.
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-1LAC---RKKHKGTSPEIQPLLTESEDYNNVSYQSHF 5 2 8 SSRL.LQKK . . K.QPQE.R . . . . MDKD . . HSLL . . . . L 5 3 3 .MV.AVL..L.A..VS.L.---.H...QLPE.K. . . M .K. . . HS-L. . . . L 529
Fig. 3. Alignment of the deduced amino acid sequence of the chicken tyrosinase with that of mouse and human. The sequence is shown in one-letter code. The top line indicates the amino acid sequence deduced from a chicken cDNA, Ctyl8, which is aligned with the sequences of the mouse tyrosinase (mictdle Iine) and the human tyro-
sinase (bottom line). Dots indicate the conserved amino acids. Gaps (-) are introduced for optimal alignment. Cysteine ( + ) and histidine (*) residues aligned in the three sequences are indicated. I and I1 show the highly homologous regions identified by Muller et al. (1988).
firmed by sequencing four independent clones (Ctyl8, Ctyl9, Cty24, and Cty25). The human sequence also lacks three amino acids a t the same position as the chicken sequence, and lacks a single amino acid near the C-terminus (Fig. 3). The chicken tyrosinase has 16 cysteine residues which are completely conserved in mouse and human sequences, but a cysteine in t.he N-terminal signal sequence of mammalian proteins is exchanged for a leucine in the chicken sequence. There are 13 histidine residues in the chicken sequence, and 17 histidines in the mouse and human sequences. Eleven of the histidines are conserved in these sequences. Muller et al. (1988) identified two homologous regions in tyrosinases and hemocyanins, and some histidines in these regions were suggested to serve as copperbinding sites (Lerch, 1988). Four histidines neighboring the homologous region I1 are completely conserved in the chicken sequence (Fig. 3). This finding supports the idea that these histidines play a key role in the enzymatic activities.
prepared from several tissues of embryos. A strong signal of a single transcript of 2.5 kb was detected in RNA of the pigmented epithelium, but no signals were observed in RNAs from heart, liver, muscle, or skin of White Leghorn embryos (Fig. 4). This result shows the tissue-specific expression of the tyrosinase gene.
RNA Blot Hybridization In order to identify the transcripts for the chicken tyrosinase, RNA blotting analysis was performed on RNAs
DISCUSSION Seventy-two percent and 73% of amino acids in the chicken protein sequence deduced from the nucleotide sequence of the Ctyl8 cDNA clone are identical to that of the mouse (Yamamoto et al., 1988) and human (Shibahara et al., 1988; lhkeda et al., 1989) tyrosinases, respectively. However, there is a 42% similarity between the chicken sequence and that of mouse brown-locus protein (Shibahara et al., 1986). These show that the Ctyl8 insert codes a chicken tyrosinase, and that the amino acid sequence of tyrosinase is highly conserved between avians and mammals. In particular, the cysteine residues in the chicken sequence are completely conserved in the mouse and human sequences (see Fig. 3). These cysteines are also conserved in the mouse brown-locus protein (Shibahara et al., 1986). This suggests that the cysteines possess an important func-
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in any of the proteins, including the N-terminal signal peptide and C-terminus. The transcripts of the tyrosinase gene were not seen in the RNA prepared from the skin of 9-day-old White Leghorn embryo. It seemed that our RNA blotting using total RNA could not detect a small amount of transcripts in the embryonic skin. RNA blotting using RNA from Black Silkie chick shows a weak signal of the tyrosinase gene’s transcripts in the skin (data not shown). It seems that much of the difference between the amounts of the transcripts in the pigmented epithelium and in the skin is ascribed to the population of pigment cells in these tissues. The pigmented epithelium is composed of a single cell type. We thus assume that the pigmented epithelial cells from chicken embryos are good material by which to study the molecular basis of pigment formation.
ACKNOWLEDGMENTS This study was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Cooperative Research (A) to G.E. from the Ministry of Education, Science and Culture, and also in part by the Research Fund of the Naito Foundation to G.E. REFERENCES Eguchi, G. (1988) Cellular and molecular background of Wolffian lens regeneration. Cell Differ., 25:147-158. Itoh, Y,, and G. Eguchi (1986) In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: A unique model system for studying cellular and molecular mechanisms of “transdifferentiation.” Dev. Biol., 115:352-362. Jackson, I.J. (1988)A cDNA encoding tyrosinase-related protein maps to t h e brown locus in mouse. Proc. Natl. Acad. Sci. USA, 85: Fig. 4. RNA blot analysis of chicken tyrosinase. Total RNA (20 4392-4396. pg per lane) from pigmented epithelium (lane l), heart (lane 21, liver Kwon, B.S., A.K. Haq, S.H. Pomerantz, and R. Halaban (1987) Iso(lane 3), muscle (lane 4), and skin (lane 5) of 9-day-old White Leghorn lation and sequence of a cDNA clone for human tyrosinase that maps embryo were electrophoresed, blotted, and probed with 3ZP-labeled a t t h e mouse c-albino locus. Proc. Natl. Acad. Sci. USA, 84: Ctyl8 cDNA. 7473-7477. Kwon, B.S., R. Halaban, and C. Chintamaneni (1989)Molecular basis of mouse himalayan mutation. Biochem. Biophys. Res. Commun., tion in the activities of the tyrosinases and related pro161:252-260. teins. I t was reported that one of cysteines in tyrosinase Lerch, K. (1988) Protein and aetive-site structure of tyrosinase. In is essential for the enzymatic activity (Shibahara et al., J.T. Bagnara (ed): Advances in Pigment Cell Research. Alan R. Liss, New York, pp. 85-98. 1990). Muller et al. (1988)identified two homologousregions in tyrosinases and hernocyanins, and some histidines in Maniatis, T., E.F. Fritsch, and J. Sambrook, eds. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory these regions are thought to serve as the copper-binding Press, Cold Spring Harbor, N.Y. sites (Lerch, 1988).This postulation is consistent with our Mochii, M., T. ’Pakeuchi, R. Xodama, K. Agata, and G. Eguchi (1988a) observation that the histidines near these regions are comThe expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells. Cell Differ., pletely conserved (see Fig. 3). 231133-142. We previously isolated and characterized a cDNA encoding a 115kD melanosomal matrix protein, termed MMP115, Mochii, M., K. Agata, H. Kobayashi, T.S. Yamamoto, and G. Eguchi (1988b) Expression of gene coding for a melanosomal matrix proin chicken (Mochiiet al., 1988a,b). The MMP115 is thought tein transcriptionally regulated in the transdifferentiation of chick to be one of the structural proteins of the matrix of the embryo pigmented epithelial cells. Cell Differ., 24:67-74. melanosome. I t is conceivable that the MMP115 interacts Mochii, M., K. Agata, and G . Eguchi (1991) Complete sequence and with tyrosinase in the process of melanosome formation. expression of a cDNA encoding a chicken 115-kDa melanosomal matrix protein. Pigment Cell Res., 441-47. We expect that there is a common mechanism which localizes newly synthesized tyrosinase and MMP115 to melanosomes. Muller, G., S. Ruppert, E. Schmid, and G. Schiitz (1988) Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO We have suggested the presence of a specific sorting sigJ., 72723-2730. nal for melanosome localization in the MMP115 polypep- Saiki, R., D. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G. Horn, tide (Mochii e t al., 19911, and searched for common K.. Mullis, and H. Erlich (1988) Primer-directed enzymatic amplisequences in the two polypeptides whch might serve as a fication of DNA with a thermostable DNA polymerase. Science, 239~487-491. specific signal, but we identified no sequence similarities
Chicken Tyrosinase cDNA Shibahara, S., Y. Tomita, T. Sakakura, C. Nager, B. Chaudhuri, and R. Muller (1986) Cloning and expression of cDNA encoding mouse tyrosinase. Nucleic Acids Res., 142413-2427. Shibahara, S., Y. Tomta, H. Tagarni,R.M. Muller, andT. Cohen(1988) Molecular basis for the heterogeneity of human tyrosinase. Tohoku J . Exp. Med., 156:403-414. Shibahara. S., S. Okinaga, Y. Tomita, A. Takeda, H. Yamamoto, M. Sato, and T. Takeuchi (1990) A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine serine substitution at position 85. J. Biochem., 189:455-461. Tabor, S., and C.C. Richardson (1987) DNA sequence analysis with a modified T7 polymerase. Proc. Natl. Acad. Sci., USA, 82:4767-4771.
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Takeda, A., Y. Tomita, S. Okinaga, S. Tagami, and S. Shibahara (1989) Functional analysis of t h e cDNA encoding human tyrosinase. Biochem. Biophys. Res. Commun., 162:984-990. Tanaka, S., H. Yamamoto, S. Takeuchi, and T. Takeuchi (1990) Melanization in albino mice transformed by introducing cloned mouse tyrosinase. Development, 108:223-227. Yamamoto, H., S. Takeuchi, T. Kudo, K. Makino, A. Nakata, T. Shinoda, and T. Takeuchi (1987) Cloning and sequencing of mouse tyrosinase cDNA. Jpn. J. Genet., 62271-274. Yamamoto, H., S. Takeuchi, T. Kudo, C. Sato, and T. Takeuchi (1989) Melanin production in cultured albino melanocytes tiansfected with mouse tyrosinase cDNA. Jpn. J. Genet., 64:121-135.
Pigment Cell Research 5:162-167 (1992)
Isolation and Characterization of a Chicken Tyrosinase cDNA MAKOTO MOCHI1,l AKIO 110,' HIROAKI YAMAMOTO,' TAKUJI TAKEUCHI,' ~ N GORO D EGUCHI' 'Division of Morphogenesis, Department of Developmental Biolop! National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki 444,Japan, and Biologcal Institute, Faculty of Science, Tohoku University, Aoba-yama, Sendai 980, Japan '
Complementary DNA clones coding for chicken tyrosinase were isolated from retinal pigmented epithelium of chicken embryo. Sequence analysis shows that one of the cDNA clones consisting of 1,997 nucleotides has an open reading frame coding for 529 amino acids. The deduced protein has nine N-glycosylation sites and a transmembrane region. A sequence comparison of the deduced chicken tyrosinase with the mouse and human homologues revealed that amino acid sequences are conserved for the entire polypeptides. Seventy-twopercent and 73% of amino acids in the chicken sequence are identical to that of the mouse and human tyrosinases, respectively. Histidines neighboring the postulated copper-bindingsites and the cysteines are well conserved. RNA blotting analysis showed that a major transcript of 2.5 kb is detected in retinal pigmented epithelium of a 9-day-oldchicken embryo. Key words: Chicken tyrosinase, cDNA cloning, Sequencing, Pigmented epithelium, RNA blotting
INTRODUCTION Tyrosinase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis, and is found in pigmented cells including melanocytes derived from the neural crest and pigmented epithelial cells from the neural plate. Cloning the gene encoding tyrosinase allows us to study the molecular basis of melanin synthesis and the regulatory mechanisms of gene expression during pigment cell differentiation. Tyrosinase cDNA clones from mouse (Yamamoto e t al., 1987, 1989; Miiller et al., 1988)and human (Kwon et al., 1987; Shibahm et al., 1988; Takeda et al., 1989) have been described, and the gene is mapped to the mouse albine locus (Kwon et al., 1987). A cDNA clone encoding tyrosinase-related protein has been isolated (Shibahara et al., 1986), and its gene is mapped to the mouse brown locus (Jackson, 1988). Genetical analyses of these DNAs reveal genetic changes affecting the coat-color differences in mice (Kwon e t al., 1989; Shibahara et al., 1990; Tanaka et al., 1990). There are many species and strains expressing different plumage colors in avians also. Molecular analyses of tyrosinase and other proteins involved in melanosome formation in avians will allow us to study the mechanisms of feather-color differences. Retinal pigmented epithelium of chick embryo is a good material for molecular studies in pigment cell differentiation and pigment formation. We can isolate and cultivate pure populations of pigmented epithelial cells from the chicken embryos. The cells dedifferentiate losing melanosomes and proliferate vigorously in a culture condition
defined by Itoh and Eguchi (1986). These dedifferentiated cells then start to differentiate into pigmented epithelial cells synthesizing much melanin, when cultured under another condition (Itoh and Eguchi, 1986; Eguchi, 1988). Thus, we can obtain a sufficient quantity of cells to study biochemically in any step of pigment cell differentiation. previously we identified and characterized a melanosomal protein, 115-kD melanosomal matrix protein (MMPl15), in the pigmented epithelial cells of chicken (Mochii et al., 1988a).A study with a specific monoclonal antibody showed that the MMP115 is localized in the melanosomal matrix of the pigmented cells and suggested that it has a function in the matrix formation. A complementary DNA clone encoding the MMP115 has been isolated, and the primary structure was determined (Mochii et al., 1988b; 1991). Northern blotting showed that the MMP115 gene was activated in the differentiation process of the pigmented epithelial cell (Mochii et al., 198813). Molecular cloning of a chicken tyrosinase cDNA makes it possible to study the interaction of the MMP115 and tyrosinase in the melanosome formation, and comparison of their primary structures might reveal the functional domains in these proteins.
Address reprint requests to Goro Eguchi, Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji-cho, Okazaki 444, Japan. Received December 28,1991; accepted March 10, 1992.
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Chicken Tyrosinase cDNA In this report, we show the molecular cloning of a chicken tyrosinase cDNA from the pigmented epithelium of chicken embryo. The complete primary structure of the cDNA and the deduced protein, and expression of the gene are described.
MATERIALS AND METHODS Cloning of Chicken 'Qrosinase cDNA A A g t l O cDNA library was constructed with poly(A) RNA from cultured pigmented epithelial cells of White Leghorn embryos as previously described (Mochii e t al., 1991). The library was screened with a DNA fragment of 700 bp encoding the first exon of the quail tyrosinase gene isolated by Yamamoto et al. (in preparation). The probes were made by the PCR technique (Saiki et al., 1988) with Taq DNA polymerase (Perkin-Elmer Cetus) using oligonucleotide primers, 5 '-TTACCATGGGCTTACTGCTG-3' and 5'-GGGATGGTGAAGTTCTCATC-3'. DNA labeling and hybridization were performed as previously described (Mochii et al., 1988b). +
DNA Sequencing EcoRl fragments were subcloned into the Bluescript SK' plasmid (STRATAGENE) and sequenced in both directions by the dideoxy terminator method (Eibor et al., 1987) using Sequenase (United States Biochemical Corporation). Oligonucleotides for sequencing primers were synthesized by an automatic DNA synthesizer (Applied Biosystems). RNA Blot Hybridization Total RNAs were isolated from pigmented epithelium, heart, liver, muscle, and skin of 9-day-old White Leghorn embryos by the guanidiudcesium chloride method (Maniatis et al., 1982). Electrophoresis, blotting, and hybridization were carried out as previously described (Mochii et al., 1988b). RESULTS Isolation and Characterization of the Chicken Qrosinase eDNAs The A g t l O cDNA library constructed from poly(A)+RNA of chicken pigmented epithelial cells was screened with a DNA fragment encoding the first exon of quail tyrosinase gene (Yamamoto e t al., in preparation). Fifteen independent clones were identified from screening of 100,000recombinant phage plaques. Four clones (Ctyl8, Ctyl9, Cty24, and Cty25) containing inserts longer than those contained in the rest were characterized. The inserts were subcloned into plasmids and mapped using endonucleases (Fig. 1). Ctyl8 and Ctyl9 contained 2.0 kb inserts, and Cty 24 and Cty25 contained 1.5 kb inserts. Their restirction maps showed that they were closely related to each other. The nucleotide sequences of both ends of the four inserts were determined and compared with the sequences of human and mouse tyrosinase cDNAs (Shibahara et al., 1988; Yamamot0 e t al., 1988; Takeda et al., 1989). Every insert had the poly(A) tail a t one end which was identified as a 3' terminus. Ctyl8 and Ctyl9 started at the same nucleotide
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Fig. 1. Partial restriction maps of cDNA clones encoding chicken tyrosinase. The protein-coding region is indicated by an open box.
from their 5' termini, and contained ATG codons. I t was assumed that Ctyl8 and Ctyl9 contained enough sequence to encode the complete chicken proteins, and that the entire sequences of Cty24 and Cty25 were contained within the boundaries of the Ctyl8 and Ctyl9 sequences. The complete sequence of the Ctyl8 insert was determined.
Nucleotide and Deduced Protein Sequences of the Ctyl8 cDNA The complete nucleotide sequence of the Ctyl8 cDNA and the deduced amino acid sequence are shown in Figure 2. The insert consists of 1,997 nucleotides. The sequence of the coding strand has an open reading frame of 1,587 nucleotides, which begins with an ATG codon at 108 nucleotides from the extreme 5' terminus and terminates by a stop codon TAA. The open reading frame is followed by 240 nucleotides of the 3' untranslated sequence and 52 nucleotides of the poly(A) tail. A potential poly-adenylation signal (ATTAAA) is identified. The open reading frame codes a protein of 529 amino acids. The deduced protein contains nine potential N-glycosylation sites (Asn-X-Thr or Asn-X-Ser). A hydrophobic signal peptide is identified a t the N-terminus, and a potential membrane-spanning region is near the C-terminus. Sequence Comparison of Chicken and Mammalian 'Qrosinases The cDNA clone Ctyl8 shows about 60% and 68% DNA similarities to the cDNAs of the mouse and human tyrosinases, respectively. The deduced amino acid sequence of the chicken tyrosinase was compared with that of mouse (Yamamoto e t al., 1988) and human (Shibahara e t al., 1988; 'Ihkeda e t al., 1989). An alignment of these sequences is shown in Figure 3. Seventy-two percent and 73% of amino acids in the chicken tyrosinase are identical to those of mouse and human, respectively. About 30 amino acids a t the N-terminus and 80 amino acids at the C-terminus show 40-50% homologies with the mouse and human sequences. All six potential N-glycosylation sites identified in the mouse and human sequences are conserved in the chicken protein which contains three additional sites. The chicken tyrosinase is four amino acids shorter than the mouse homol o p e but has the same number of amino acids as the human one. The chicken sequence lacks four amino acids in a region connecting the transmembrane sequence and the following region in the mouse sequence. This deletion was con-
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481 CAGCTCACCA1'CAGTGAGAAGGACAAGTTCCTTCCTTGCCTACCTTAACCTAGCA~GRAC~TC Q L T I S E K D K F L A Y L N L A K N I l 4
CCCATGTTCAG~TATCAACGTGTACGATC?'CTTCGTCTGGAI'(;CATTATTATGCCTCT P k l F R N I N V Y D L F V W M H Y Y A S 184
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Fig. 2. Nucleotide sequence of a cDNA, Ctyl8 encoding chicken tyrosinase, and the deduced amino acid sequence. The predicted amino acid sequence (in one-letter code) is shown below the nucleotide se-
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1021 GACAAATCAAGGACCCCAAGACTCCCATCTTCATCAGAAGTCGA'4TTTTGTCTTACACTC U K S R T P R L P S S S E V E F C L T L 3 2
961 CAACAGGCTTTATGCAATGCCACTAGTGAAGGGCCTATAC'TTCGAAATCCTGGAAACAAT
901 CCAGCATCATTTTTCTCCTCATGGCAGG1'AA'TCTGCACTCAATCTGAAGAGTACAACAGC P A S F F S S W Q V I C T Q S E E Y N S 2 8
841 G T G A T C T G C A C T G A T G A A T A C A T G G G T G G C C h A C A C C C C A C C i \ A C C C T ~ ~ n ' A ~ ~ C A G C v I c T D E Y M G G Q II P 'r N P N L L s 2 6 4
781 ATAACAGGTGATGAGAATTTCACCATCCATCCCCTACTGGGACTGGCGAGATGCAGAGG~CTGT I T G D E N I: T 1 I ' Y I B D W R D A E D C 244
7 2 1 CCTGCTTTTCTGCCTTGGCATCGTGCTTTTCTGCTGCTGTGGGA~CGTG~G,~T,~C~G~~~~G P G F L P IY f f R A I: L L L \V E H E I Q K 2 2 4
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661 CCAGACACACTCTTAGGTGGCTCCAATCTGTGG~~GAGACATTGATTTTGCCC~TGA~GCC
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541 C C C A G C A A G G A C T A T G T M T T G C T A C T G G C A C A T A T G C T C A G A T G A A C ~ C G G C T C C A ~ ~ C P S K D Y V I A T G T Y A 6) M N N G S N 164
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421 TTTGGCTTCTCAGGACAAAACTGCACTGAAAGGCGACTGAGAACGAGAAGAAACATCTTC F G F S G Q N C T E R R L R T R R N I F l 2
361 TACAACCGGACATGCAGATGCAGAGGCAATTTCATGGGGTTCAACTGTGGGGAGTGCMG Y N I~ T C R C R G N F M G F N C G E C K 104
301 CTGGGACCACAGTTCCCTTTCTCAGGAGTGGATGACAGAGAGGATTGGCCTrCTGrCTTT L G P Q F P F S G V D D R E D W P S V F 8 4
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241 CCTTGCGGGGAGCGTTCCAACAGAGGAACCTGCCAGCGCATCCTTCTCTCTCAGGCTCCT
181 GCAAACACGCAGAGCTTGCTGAGGAAGGAGTGCTGTCCGCCCTGGGATGGAGATGGGACC A N T Q S L L R K E C C P P W D G D G T 4
121 GCCATGGGCTTACTGCTGGTCATCCTTCAGCCGTCCACTGGGCACTTCCCCAGAGTCTGT A M G L L L V I L Q I ' S T G Q F P R V C 2 4
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6 1 TCCATGCACTGAAGCATAACACTGATGCTCTCCTGCTGCTCTGTGAGGATGT~CTGTTT
1 CCCAGTTGGGAGGAAAAGTTTCTTTCACACTCAGGTCATTGGAGCGGTCAGAGAGAACTC
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quence and numbered on the right side. A putative signal peptide, membiane spanning region, potential N-glycosylation sites, and a potential poly-adenylation signal are underlined. Asterisk indicates a stop codon.
1981 P
1921 TGCTTGATTAAACAACACTCCTGTGAAAAAAAMAAAAAAAAAAAAAAAAAAAAAAAAAA
1861 C T C C M A A T C T G T C A T A T T C T A A G T T A C C A T G C C A C C C A A G
1801 TGTCCCACTCCTTGTGCCTCTTGTAACATCCACTGTTGTAGTACCTTATCAGCTCCAGTC
1741 GCATGCAGTGTTTATGTTTACAATGACC?'TGTTGAACTGTGCTGCATAAGCTGGCTGGGC
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1681 TATCMTCCCATTTCThhRGACCTCCAGTATCTCAATCATGTTTCTAAGGTGCTAGTCAA
1621 GGAACTTC'TCCAGAAATACAACCCTTACTCACAGAAAGTGAAGATTACAACAATGTATCT G T S P E I Q P L L T E S E D Y N N V S 5 2
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1561 G G A G G C A T A A T T A C T G C T G T G C T C I ' C T G G G C T C A T C C T G G C C T G C A G G A A G ~ A G A ~ A G G I I T A V L S G L I L A C R K K R K 5 0 4
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1381 TACATGGTTCCCTTTATCCCTCTTTACAGATGGAGAATTTTTTATATCATC~GAGAG
1321 A G A C C C A T G C T A G A A G T T T A C C C A G C A G C C A A C G C A C C C A T T G G G C A C ~ T C G A G ~ T R P M L E V Y P A A N A P I G H N R E N 4 2 4
1261 ATCTTCATCCTACACCATGCATTTGTTGACAGCATTTTTGAGCGGTGGTTAAGAAGACAC I F I L II H A F V D S I F E R W L R R H 404
1201 GCCCTTCACATCTACATGAATGGCTCAATGTCCCAAGTACAA~GCTCTGCGAATGATCCT A L II I Y M N G S M S Q V Q G S A N D P 384
1141 GAAGGCTTTGCTGATCCACACACTGCRATATC~CATATCTCAAAGTGGTTTGCATAAT E G F A D P H T A I S N I S Q S G L H N 3 6
I081 ACTCAGTATGMTCTGGATCCATGGATAAAATGGCCAATGGCCRATTACAGC~rCCG~CACTTTG T Q Y E S G S M D K M A N Y S F R N T L 3 4
Chicken Qrosinase cDNA
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FIFLFAMGLL1.VILQPSTGQFPRVCANTQSL.LRKECCPPIVDGDGTPCGEll~ILI. 60 . . .AVLYC . . IVSF.1.D.I~ . .A .. . SSKN . . A . . . . . . . M . . . S . . . Q L . G . . S . . D . . . .L.AVLYC. . WSF.T.A.H . . .A.VSSKN.ME . . . . . . . S..RS...QL.G..S..N... + + + + + SQAPLGPQFPFSGVDDREDWPSVFYNRTCRCRGNFMGFNCGECKFGFSGQNCTERKLRTR 1 2 0 . S . . S . . . . . . K . . . . . . S . . . . . . . . . . Q . S . . . . . . . . . N . . . . . G . P . . . . KI. .VL . N . . . . . . . . . T......S..........Q.S.........N.....W.P.......LV .
RNIFQLTISEKDKFLAYLNLAKNIPSKDYVIATGTYAQMNNGSNP~lFRNI~~DLFV~~ltl 180 . . . . D.SV . . . N . . FS . . T...HTI.SV...P....G......T...ND..I........ .D.SAP . . . . .F...T...HTI.S....PI...G..K...T...ND.. I WASRDTLLGGSNV\VRDIDFAHEAPCFLPIVIIRAFLL.I~WEREIQKITGDENFTII’~VD\~R~ 240 . . . . . . .L . . . . . . Q . . R E L . . . . . . . V . . . . . . . . . V . . . . . . . . . EI . . . . . . A . . . . . .L...R..Q....L............... . . V.M.A . . . . .EI . . . . . . I
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-1LAC---RKKHKGTSPEIQPLLTESEDYNNVSYQSHF 5 2 8 SSRL.LQKK . . K.QPQE.R . . . . MDKD . . HSLL . . . . L 5 3 3 .MV.AVL..L.A..VS.L.---.H...QLPE.K. . . M .K. . . HS-L. . . . L 529
Fig. 3. Alignment of the deduced amino acid sequence of the chicken tyrosinase with that of mouse and human. The sequence is shown in one-letter code. The top line indicates the amino acid sequence deduced from a chicken cDNA, Ctyl8, which is aligned with the sequences of the mouse tyrosinase (mictdle Iine) and the human tyro-
sinase (bottom line). Dots indicate the conserved amino acids. Gaps (-) are introduced for optimal alignment. Cysteine ( + ) and histidine (*) residues aligned in the three sequences are indicated. I and I1 show the highly homologous regions identified by Muller et al. (1988).
firmed by sequencing four independent clones (Ctyl8, Ctyl9, Cty24, and Cty25). The human sequence also lacks three amino acids a t the same position as the chicken sequence, and lacks a single amino acid near the C-terminus (Fig. 3). The chicken tyrosinase has 16 cysteine residues which are completely conserved in mouse and human sequences, but a cysteine in t.he N-terminal signal sequence of mammalian proteins is exchanged for a leucine in the chicken sequence. There are 13 histidine residues in the chicken sequence, and 17 histidines in the mouse and human sequences. Eleven of the histidines are conserved in these sequences. Muller et al. (1988) identified two homologous regions in tyrosinases and hemocyanins, and some histidines in these regions were suggested to serve as copperbinding sites (Lerch, 1988). Four histidines neighboring the homologous region I1 are completely conserved in the chicken sequence (Fig. 3). This finding supports the idea that these histidines play a key role in the enzymatic activities.
prepared from several tissues of embryos. A strong signal of a single transcript of 2.5 kb was detected in RNA of the pigmented epithelium, but no signals were observed in RNAs from heart, liver, muscle, or skin of White Leghorn embryos (Fig. 4). This result shows the tissue-specific expression of the tyrosinase gene.
RNA Blot Hybridization In order to identify the transcripts for the chicken tyrosinase, RNA blotting analysis was performed on RNAs
DISCUSSION Seventy-two percent and 73% of amino acids in the chicken protein sequence deduced from the nucleotide sequence of the Ctyl8 cDNA clone are identical to that of the mouse (Yamamoto et al., 1988) and human (Shibahara et al., 1988; lhkeda et al., 1989) tyrosinases, respectively. However, there is a 42% similarity between the chicken sequence and that of mouse brown-locus protein (Shibahara et al., 1986). These show that the Ctyl8 insert codes a chicken tyrosinase, and that the amino acid sequence of tyrosinase is highly conserved between avians and mammals. In particular, the cysteine residues in the chicken sequence are completely conserved in the mouse and human sequences (see Fig. 3). These cysteines are also conserved in the mouse brown-locus protein (Shibahara et al., 1986). This suggests that the cysteines possess an important func-
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in any of the proteins, including the N-terminal signal peptide and C-terminus. The transcripts of the tyrosinase gene were not seen in the RNA prepared from the skin of 9-day-old White Leghorn embryo. It seemed that our RNA blotting using total RNA could not detect a small amount of transcripts in the embryonic skin. RNA blotting using RNA from Black Silkie chick shows a weak signal of the tyrosinase gene’s transcripts in the skin (data not shown). It seems that much of the difference between the amounts of the transcripts in the pigmented epithelium and in the skin is ascribed to the population of pigment cells in these tissues. The pigmented epithelium is composed of a single cell type. We thus assume that the pigmented epithelial cells from chicken embryos are good material by which to study the molecular basis of pigment formation.
ACKNOWLEDGMENTS This study was supported by Grants-in-Aid for Scientific Research on Priority Areas and for Cooperative Research (A) to G.E. from the Ministry of Education, Science and Culture, and also in part by the Research Fund of the Naito Foundation to G.E. REFERENCES Eguchi, G. (1988) Cellular and molecular background of Wolffian lens regeneration. Cell Differ., 25:147-158. Itoh, Y,, and G. Eguchi (1986) In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: A unique model system for studying cellular and molecular mechanisms of “transdifferentiation.” Dev. Biol., 115:352-362. Jackson, I.J. (1988)A cDNA encoding tyrosinase-related protein maps to t h e brown locus in mouse. Proc. Natl. Acad. Sci. USA, 85: Fig. 4. RNA blot analysis of chicken tyrosinase. Total RNA (20 4392-4396. pg per lane) from pigmented epithelium (lane l), heart (lane 21, liver Kwon, B.S., A.K. Haq, S.H. Pomerantz, and R. Halaban (1987) Iso(lane 3), muscle (lane 4), and skin (lane 5) of 9-day-old White Leghorn lation and sequence of a cDNA clone for human tyrosinase that maps embryo were electrophoresed, blotted, and probed with 3ZP-labeled a t t h e mouse c-albino locus. Proc. Natl. Acad. Sci. USA, 84: Ctyl8 cDNA. 7473-7477. Kwon, B.S., R. Halaban, and C. Chintamaneni (1989)Molecular basis of mouse himalayan mutation. Biochem. Biophys. Res. Commun., tion in the activities of the tyrosinases and related pro161:252-260. teins. I t was reported that one of cysteines in tyrosinase Lerch, K. (1988) Protein and aetive-site structure of tyrosinase. In is essential for the enzymatic activity (Shibahara et al., J.T. Bagnara (ed): Advances in Pigment Cell Research. Alan R. Liss, New York, pp. 85-98. 1990). Muller et al. (1988)identified two homologousregions in tyrosinases and hernocyanins, and some histidines in Maniatis, T., E.F. Fritsch, and J. Sambrook, eds. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory these regions are thought to serve as the copper-binding Press, Cold Spring Harbor, N.Y. sites (Lerch, 1988).This postulation is consistent with our Mochii, M., T. ’Pakeuchi, R. Xodama, K. Agata, and G. Eguchi (1988a) observation that the histidines near these regions are comThe expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells. Cell Differ., pletely conserved (see Fig. 3). 231133-142. We previously isolated and characterized a cDNA encoding a 115kD melanosomal matrix protein, termed MMP115, Mochii, M., K. Agata, H. Kobayashi, T.S. Yamamoto, and G. Eguchi (1988b) Expression of gene coding for a melanosomal matrix proin chicken (Mochiiet al., 1988a,b). The MMP115 is thought tein transcriptionally regulated in the transdifferentiation of chick to be one of the structural proteins of the matrix of the embryo pigmented epithelial cells. Cell Differ., 24:67-74. melanosome. I t is conceivable that the MMP115 interacts Mochii, M., K. Agata, and G . Eguchi (1991) Complete sequence and with tyrosinase in the process of melanosome formation. expression of a cDNA encoding a chicken 115-kDa melanosomal matrix protein. Pigment Cell Res., 441-47. We expect that there is a common mechanism which localizes newly synthesized tyrosinase and MMP115 to melanosomes. Muller, G., S. Ruppert, E. Schmid, and G. Schiitz (1988) Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO We have suggested the presence of a specific sorting sigJ., 72723-2730. nal for melanosome localization in the MMP115 polypep- Saiki, R., D. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G. Horn, tide (Mochii e t al., 19911, and searched for common K.. Mullis, and H. Erlich (1988) Primer-directed enzymatic amplisequences in the two polypeptides whch might serve as a fication of DNA with a thermostable DNA polymerase. Science, 239~487-491. specific signal, but we identified no sequence similarities
Chicken Tyrosinase cDNA Shibahara, S., Y. Tomita, T. Sakakura, C. Nager, B. Chaudhuri, and R. Muller (1986) Cloning and expression of cDNA encoding mouse tyrosinase. Nucleic Acids Res., 142413-2427. Shibahara, S., Y. Tomta, H. Tagarni,R.M. Muller, andT. Cohen(1988) Molecular basis for the heterogeneity of human tyrosinase. Tohoku J . Exp. Med., 156:403-414. Shibahara. S., S. Okinaga, Y. Tomita, A. Takeda, H. Yamamoto, M. Sato, and T. Takeuchi (1990) A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine serine substitution at position 85. J. Biochem., 189:455-461. Tabor, S., and C.C. Richardson (1987) DNA sequence analysis with a modified T7 polymerase. Proc. Natl. Acad. Sci., USA, 82:4767-4771.
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