Functional Conservation of the Promoter Regions of Vertebrate Tyrosinase Genes
2001; Elsevier BV; Volume: 6; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2001.00008.x
ISSN1529-1774
AutoresShigeru Sato, Mika Tanaka, Hirohito Miura, Takuji Takeuchi, Hiroaki Yamamoto, Kazuho Ikeo, Takashi Gojobori,
Tópico(s)RNA regulation and disease
ResumoTyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5′ flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5′ flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5′ flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed. Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5′ flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5′ flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5′ flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed. basic-helix-loop-helix dopachrome tautomerase L-3,4-dihydroxyphenylalanine microphthalmia-associated transcription factor open reading frame tyrosinase distal element tyrosinase element-1 transfer RNA tyrosinase-related protein unweighted pair-group method with arithmetic mean Skin color has been molded by evolutionary forces to play a variety of adaptive roles in vertebrates. Melanin pigment is the major source of skin color variation, particularly in birds and mammals. Tyrosinase (E.C.1.14.18.1) is the key enzyme for melanin biosynthesis as it initiates the process by catalyzing the oxidation of tyrosine to form DOPA (L-3,4-dihydroxyphenylalanine) and further, oxidizes DOPA to DOPA-quinone (Hearing and Tsukamoto, 1991Hearing V.J. Tsukamoto K. Enzymatic control of pigmentation in mammals.FASEB J. 1991; 5: 2902-2909Crossref PubMed Scopus (646) Google Scholar). Given the importance of melanin, and ultimately of tyrosinase, in the adaptation of vertebrates, it is clear that the genetic network-regulating enzyme function must also be, at least indirectly, scrutinized by natural selection for timely expression during the ontogeny of complex pigmentation patterns and for the effective production of melanin throughout life. Expression of the tyrosinase gene is restricted to specialized cells designated as melanocytes (or melanophores). It has been demonstrated that the 5′ flanking sequences of mammalian tyrosinase genes determine their melanocyte-specific expression (Beermann et al., 1990Beermann F. Ruppert S. Hummler E. Bosch F.X. Muller G. Ruther U. Schutz G. Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice.EMBO J. 1990; 9: 2819-2826Crossref PubMed Scopus (148) Google Scholar;Tanaka et al., 1990Tanaka S. Yamamoto H. Takeuchi S. Takeuchi T. Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene.Development. 1990; 108: 223-227PubMed Google Scholar;Kluppel et al., 1991Kluppel M. Beermann F. Ruppert S. Schmid E. Hummler E. Schutz G. The mouse tyrosinase promoter is sufficient for expression in melanocytes and in the pigmented epithelium of the retina.Proc Natl Acad Sci USA. 1991; 88: 3777-3781Crossref PubMed Scopus (103) Google Scholar;Shibata et al., 1992Shibata K. Muraosa Y. Tomita Y. Tagami H. Shibahara S. Identification of a cis-acting element that enhances the pigment cell- specific expression of the human tyrosinase gene.J Biol Chem. 1992; 267: 20584-20588Abstract Full Text PDF PubMed Google Scholar;Bentley et al., 1994Bentley N.J. Eisen T. Goding C.R. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.Mol Cell Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (400) Google Scholar;Ganss et al., 1994bGanss R. Montoliu L. Monaghan A.P. Schütz G. A cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice.EMBO J. 1994; 13: 3083-3093Crossref PubMed Scopus (114) Google Scholar;Porter and Meyer, 1994Porter S.D. Meyer C.J. A distal tyrosinase upstream element stimulates gene expression in neural-crest-derived melanocytes of transgenic mice: position-independent and mosaic expression.Development. 1994; 120: 2103-2111Crossref PubMed Google Scholar;Yasumoto et al., 1994Yasumoto K. Yokoyama K. Shibata K. Tomita Y. Shibahara S. Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.Mol Cell Biol. 1994; 14: 8058-8070Crossref PubMed Scopus (347) Google Scholar,Yasumoto et al., 1997Yasumoto K. Yokoyama K. Takahashi K. Tomita Y. Shibahara S. Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes.J Biol Chem. 1997; 272: 503-509Crossref PubMed Scopus (309) Google Scholar). We hypothesized that the 5′ regulatory sequences, as well as the coding sequences, of tyrosinase genes have been tightly conserved by selective forces that operate during vertebrate phylogenesis. Our preliminary study showed that coding sequences, except for regions encoding putative signal peptides, in the first exon of vertebrate tyrosinase genes (Japanese quail, snapping turtle, mouse, and human) are highly conserved (Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar). In contrast, only a few short sequence motifs are shared in the 5′ flanking regions of mammalian and nonmammalian tyrosinase genes (Tanaka et al., 1992Tanaka M. Tanaka S. Miura H. Yamamoto H. Kikuchi H. Takeuchi T. Conserved regulatory mechanisms of tyrosinase genes in mice and humans.Pigment Cell Res. 1992; 5: 304-311Crossref PubMed Scopus (8) Google Scholar;Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar). The first such motif was an 11 bp element now termed the M-box (Lowings et al., 1992Lowings P. Yavuzer U. Goding C.R. Positive and negative elements regulate a melanocyte-specific promoter.Mol Cell Biol. 1992; 12: 3653-3662Crossref PubMed Scopus (128) Google Scholar), previously designated as either the upstream element (Shibahara et al., 1991Shibahara S. Taguchi H. Muller R.M. Shibata K. Cohen T. Tomita Y. Tagami H. Structural organization of the pigment cell-specific gene located at the brown locus in mouse. Its promoter activity and alternatively spliced transcript.J Biol Chem. 1991; 266: 15895-15901Abstract Full Text PDF PubMed Google Scholar), an 11-mer motif (Jackson et al., 1991Jackson I.J. Chambers D.M. Budd P.S. Johnson R. The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase.Nucl Acids Res. 1991; 19: 3799-3804Crossref PubMed Scopus (70) Google Scholar), or p-MSE (Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar). This regulatory motif is also found in the proximal promoters of genes encoding the related melanogenic enzymes, tyrosinase-related protein-1 (Tyrp1) and tyrosinase-related protein-2 or dopachrome tautomerase (Dct) (Jackson et al., 1991Jackson I.J. Chambers D.M. Budd P.S. Johnson R. The tyrosinase-related protein-1 gene has a structure and promoter sequence very different from tyrosinase.Nucl Acids Res. 1991; 19: 3799-3804Crossref PubMed Scopus (70) Google Scholar;Shibahara et al., 1991Shibahara S. Taguchi H. Muller R.M. Shibata K. Cohen T. Tomita Y. Tagami H. Structural organization of the pigment cell-specific gene located at the brown locus in mouse. Its promoter activity and alternatively spliced transcript.J Biol Chem. 1991; 266: 15895-15901Abstract Full Text PDF PubMed Google Scholar;Lowings et al., 1992Lowings P. Yavuzer U. Goding C.R. Positive and negative elements regulate a melanocyte-specific promoter.Mol Cell Biol. 1992; 12: 3653-3662Crossref PubMed Scopus (128) Google Scholar;Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar;Yokoyama et al., 1994Yokoyama K. Yasumoto K. Suzuki H. Shibahara S. Cloning of the human DOPAchrome tautomerase/tyrosinase-related protein 2 gene and identification of two regulatory regions required for its pigment cell-specific expression.J Biol Chem. 1994; 269: 27080-27087Abstract Full Text PDF PubMed Google Scholar;Budd and Jackson, 1995Budd P.S. Jackson I.J. Structure of the mouse tyrosinase-related protein-2/dopachrome tautomerase (Tyrp2/Dct) gene and sequence of two novel slaty alleles.Genomics. 1995; 29: 35-43Crossref PubMed Scopus (86) Google Scholar;Miura et al., 1995Miura I. Okumoto H. Makino K. Nakata A. Nishioka M. Analysis of the tyrosinase gene of the Japanese pond frog, Rana nigromaculata: cloning and nucleotide sequence of the genomic DNA containing the tyrosinase gene and its flanking regions.Jpn J Genet. 1995; 70: 79-92Crossref PubMed Scopus (11) Google Scholar;Ferguson and Kidson, 1996Ferguson C.A. Kidson S.H. Characteristic sequences in the promoter region of the chicken tyrosinase-encoding gene.Gene. 1996; 169: 191-195Crossref PubMed Scopus (11) Google Scholar). Functional analyses of mammalian tyrosinase proximal promoters identified the M-box as a positive regulatory element for melanocyte-specific expression (Bentley et al., 1994Bentley N.J. Eisen T. Goding C.R. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.Mol Cell Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (400) Google Scholar;Ganss et al., 1994cGanss R. Schmidt A. Schutz G. Beermann F. Analysis of the mouse tyrosinase promoter in vitro and in vivo.Pigment Cell Res. 1994; 7: 275-278Crossref PubMed Scopus (20) Google Scholar;Yasumoto et al., 1997Yasumoto K. Yokoyama K. Takahashi K. Tomita Y. Shibahara S. Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes.J Biol Chem. 1997; 272: 503-509Crossref PubMed Scopus (309) Google Scholar). We also found sequences homologous to the d-MSE, a 13 bp motif conserved in mouse tyrosinase and Tyrp1 promoters (Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar), and in the turtle flanking region.Bentley et al., 1994Bentley N.J. Eisen T. Goding C.R. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.Mol Cell Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (400) Google Scholar later found two other conserved sequences, a 9 bp motif (termed CR1) adjacent to the M-box and a 15 bp motif (termed CR2) about 90 bp downstream from the M-box. The CR2 contains a basic-helix-loop-helix (bHLH) factor-binding E-box motif, CATGTG, which is also the core sequence of the M-box, and a degenerate octamer element, GTGATAAT, in human, quail, chicken, and turtle genes. The authors further identified a nonconsensus SP1 motif positioned between CR1 and CR2 (Bentley et al., 1994Bentley N.J. Eisen T. Goding C.R. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.Mol Cell Biol. 1994; 14: 7996-8006Crossref PubMed Scopus (400) Google Scholar). In this study, we compared the 5′ flanking regions of vertebrate tyrosinase genes. Despite the overall sequence divergence of the 5′ flanking sequences of the quail and turtle tyrosinase genes, they are capable of activating transcription of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. As a consequence, amelanotic albino melanocytes were induced to synthesize melanin. To verify those results obtained by transfection, we further demonstrate that the 5′ flanking sequences of the quail or turtle tyrosinase genes are able to direct expression of a fused mouse cDNA in transgenic mice, as does the flanking sequence of the human tyrosinase gene (Tanaka et al., 1992Tanaka M. Tanaka S. Miura H. Yamamoto H. Kikuchi H. Takeuchi T. Conserved regulatory mechanisms of tyrosinase genes in mice and humans.Pigment Cell Res. 1992; 5: 304-311Crossref PubMed Scopus (8) Google Scholar). Finally, we identify several conserved sequence motifs in the 5′ flanking regions of other vertebrate tyrosinase genes, including dog (Tang et al, 1995), Japanese pond frog (Miura et al., 1995Miura I. Okumoto H. Makino K. Nakata A. Nishioka M. Analysis of the tyrosinase gene of the Japanese pond frog, Rana nigromaculata: cloning and nucleotide sequence of the genomic DNA containing the tyrosinase gene and its flanking regions.Jpn J Genet. 1995; 70: 79-92Crossref PubMed Scopus (11) Google Scholar), chicken (Ferguson and Kidson, 1996Ferguson C.A. Kidson S.H. Characteristic sequences in the promoter region of the chicken tyrosinase-encoding gene.Gene. 1996; 169: 191-195Crossref PubMed Scopus (11) Google Scholar), and medaka fish (Inagaki et al., 1998Inagaki H. Koga A. Bessho Y. Hori H. The tyrosinase gene from medakafish: transgenic expression rescues albino mutation.Pigment Cell Res. 1998; 11: 283-290Crossref PubMed Scopus (32) Google Scholar). Total RNA fractions were prepared from pigmented epithelium and/or whole eyes of 10-d-old Japanese quail embryos. Single-stranded cDNA were synthesized using an oligo (dT) primer flanked with BamHI, EcoRI, and SmaI restriction sites, T-BES: 5′-GGATCCGAATTCCCCGGGTTTTTTTTTTTTTTTTT-3′. Subsequently, polymerase chain reaction (PCR) amplification was carried out using T-BES and a sense primer specific to quail tyrosinase cDNA. The sequence of the specific primer was 5′-GGAATTCGGATCCCCTGCTGCTCTGTGAGG-3′, which corresponds to bases -20 to -1 of the genomic clone Q63SH1 (Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar) and is also flanked with EcoRI-BamHI sites. In this report, position numbering is relative to the A (+1) for the first methionine unless otherwise stated. The double-stranded cDNA were digested with EcoRI, electrophoretically purified, and cloned into the EcoRI site of pUC119. Sequence data from multiple independent clones were accumulated and compared with each other, and then a consensus sequence was released because the original data were obtained by PCR. Total RNA was prepared from whole eyes of 20-d-old-snapping turtle embryos. Single-stranded cDNA were prepared as described above and then the second-strand was synthesized with a specific sense primer, T-FP: 5′-GGAATTCTAGAAAATTGCCTGCTGTTGT-3′ (bases -25 to -7) flanked with EcoRI and XbaI sites. The cDNA were digested with EcoRI and cloned into λgt 11 phage vector because PCR-amplification was somewhat unsatisfactory for cloning into plasmid vectors under the conditions employed. Recombinant phages were screened by PCR using T-FP and λgt 11 primers, and subcloned into the pUC119 vector. A consensus sequence was determined as described above. Primer extension was performed using primers end-labeled with 32P (about 5 × 104 cpm by Cherenkof counting). The sequences of the primers are Q-RP: 5′-CATGGTAAACAGAAGCATCCTCACAGAGCAGCA-3′ (complementary to bases +18 to -15 of the quail tyrosinase gene) and T-RP: 5′-TCGAGGGAACTGCCCGGATGCAGGCTGCAG-3′ (complementary to bases +66 to +37 of the turtle tyrosinase gene). Total RNA prepared from pigmented epithelium or from whole eyes were annealed with primers and extension reactions were performed as previously described (McKnight and Kingsbury, 1982McKnight S.L. Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene.Science. 1982; 217: 316-324Crossref PubMed Scopus (725) Google Scholar). In the control extension reaction, the same amount of E. coli tRNA was used. The products were separated by electrophoresis on 6% sequencing gels and visualized by autoradiography. Hybrid minigenes were constructed as follows: The quail tyrosinase genomic clone pQ12Xb8 (EMBL/GenBank/DDBJ Accession number: AB024279) was digested with Fnu4HI, and the resulting fragment (bases -13 to -1781) was blunted and ligated with the mouse tyrosinase cDNA Tyrs-J (Yamamoto et al., 1989Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.Jpn J Genet. 1989; 66: 121-135Crossref Scopus (90) Google Scholar), which had been digested with EcoRI and blunted. This quail-mouse hybrid minigene, which contained 1.8 kb of the quail tyrosinase promoter, was designated as qg-Tyrs-J. The snapping turtle genomic clone SB1-1 (EMBL/GenBank/DDBJ Accession number: AB024281) was digested with XhoI. As this clone contained an additional XhoI site in the far upstream region, two XhoI-digested fragments were simultaneously cloned into the XhoI site of Tyrs-J. This turtle-mouse hybrid minigene, which contained about 4.3 kb of the turtle tyrosinase promoter, was designated as tg-Tyrs-J. As a positive control, mg-Tyrs-J, which is a minigene composed of 2.6 kb of the mouse tyrosinase promoter fused to Tyrs-J, was used. This minigene (mg-Tyrs-J) has been shown to be capable of expressing tyrosinase activity in cultured albino melanocytes (Yamamoto et al., 1989Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.Jpn J Genet. 1989; 66: 121-135Crossref Scopus (90) Google Scholar) and in transgenic mice (Tanaka et al., 1990Tanaka S. Yamamoto H. Takeuchi S. Takeuchi T. Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene.Development. 1990; 108: 223-227PubMed Google Scholar). The plasmid vector pUC118 and a construct carrying only Tyrs-J were used as negative controls. Cultured albino melanocytes were prepared from C57BL/6 J-Tyrc-2J/Tyrc-2J newborn mice according to the method ofSato et al., 1985Sato C. Ito S. Takeuchi T. Establishment of a mouse melanocyte clone which synthesizes both eumelanin and pheomelanin.Cell Struct Funct. 1985; 10: 421-425Crossref PubMed Scopus (25) Google Scholar and transfections were carried out as previously described (Yamamoto et al., 1989Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.Jpn J Genet. 1989; 66: 121-135Crossref Scopus (90) Google Scholar). In order to detect tyrosinase enzyme activity, transfected melanocytes were fixed with periodate-lysine-paraformaldehyde (McLean and Nakane, 1974McLean I.W. Nakane P.K. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy.J Histochem Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3151) Google Scholar) for 30–60 min at 4°C, washed with 0.1 M sodium phosphate buffer (pH 7.4) and then incubated with 1 mg per ml DOPA in 0.1 M phosphate buffer (pH 7.4) at 37°C for 3–5 h. After completion of DOPA-reaction, cells were washed with phosphate buffer and surveyed by light microscopy for the accumulation of dark DOPA-melanin in order to localize tyrosinase activity. Microinjection of minigenes into fertilized eggs of BALB/C albino mice was performed as described byTanaka et al., 1990Tanaka S. Yamamoto H. Takeuchi S. Takeuchi T. Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene.Development. 1990; 108: 223-227PubMed Google Scholar. Multiple alignments of amino acid and nucleotide sequences were generated either by the CLUSTAL W program version 1.7 (Thompson et al., 1994Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucl Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (53470) Google Scholar) or by the MALIGN program of the ODEN package (Ina, 1990Ina Y. Molecular Evolutionary Analysis of Hepatitis B Viruses and its Application to the Design of the Anti-Virus Drugs. Master thesis, Shizuoka University, Shizuoka, Japan1990Google Scholar). Amino acid substitutions were inferred using the aligned sequences and used to construct phylogenetic trees by the Neighbor-joining method (Saitou and Nei, 1987Saitou N. Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees.Mol Biol Evol. 1987; 4: 406-425PubMed Google Scholar). Bootstrapping resampling was performed to create multiple (n = 1000) randomly sampled data sets from the original alignment. To date, vertebrate tyrosinase cDNA have been cloned from mouse (Yamamoto et al., 1987Yamamoto H. Takeuchi S. Kudo T. Makino K. Nakata A. Shinoda T. Takeuchi T. Cloning and sequencing of mouse tyrosinase cDNA.Jpn J Genet. 1987; 62: 271-274Crossref Scopus (99) Google Scholar,Yamamoto et al., 1989Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.Jpn J Genet. 1989; 66: 121-135Crossref Scopus (90) Google Scholar;Kwon et al., 1988Kwon B.S. Wakulchik M. Haq A.K. Halaban R. Kestler D. Sequence analysis of mouse tyrosinase cDNA and the effect of melanotropin on its gene expression.Biochem Biophys Res Commun. 1988; 153: 1301-1309Crossref PubMed Scopus (123) Google Scholar;Müller et al., 1988Müller G. Ruppert S. Schmid E. Schütz G. Functional analysis of alternatively spliced tyrosinase gene transcripts.EMBO J. 1988; 7: 2723-2730Crossref PubMed Scopus (223) Google Scholar;Ruppert et al., 1988Ruppert S. Müller G. Kwon B. Schütz G. Multiple transcripts of the mouse tyrosinasegene are generated by alternative splicing.EMBO J. 1988; 7: 2715-2722Crossref PubMed Scopus (136) Google Scholar), human (Kwon et al., 1987Kwon B.S. Haq A.K. Pomerantz S.H. Halaban R. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus.Proc Natl Acad Sci USA. 1987; 84: 7473-7477Crossref PubMed Scopus (381) Google Scholar;Shibahara et al., 1988Shibahara S. Tomita Y. Tagami H. Muller R. Cohen T. Molecular basis for the heterogeneity of human tyrosinase.Tohoku J Exp Med. 1988; 156: 403-414Crossref PubMed Scopus (76) Google Scholar), chicken (Mochii et al., 1992Mochii M. Iio A. Yamamoto H. Takeuchi T. Eguchi G. Isolation and characterization of a chicken tyrosinase cDNA.Pigment Cell Res. 1992; 5: 162-167Crossref PubMed Scopus (40) Google Scholar), Japanese pond frog (Takase et al., 1992Takase M. Miura I. Nakata A. Takeuchi T. Nishioka M. Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.Gene. 1992; 121: 359-363Crossref PubMed Scopus (31) Google Scholar), and medaka fish (Inagaki et al., 1994Inagaki H. Bessho Y. Koga A. Hori H. Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes.Gene. 1994; 150: 319-324Crossref PubMed Scopus (53) Google Scholar). Partial cDNA sequences are also available for dog (Tang et al., 1996Tang Q. Williams R.W. Hogan D. Valentine V. Goldowitz D. Canis familiaris tyrosinase (tyr) gene, exon 1, partial cds.DDBJ/EMBL/GenBank nucleotide sequence databases (accession number U42219). 1996Google Scholar) and cat (van der Linde-Sipman et al., 1997van der Linde-Sipman J.S. de Wit M.M. van Garderen E. Molenbeek R.F. van der Velde-Zimmermann D. de Weger R.A. Cutaneous malignant melanomas in 57 cats. identification of (amelanotic) signet-ring and balloon cell types and verification of their origin by immunohistochemistry, electron microscopy, and in situ hybridization.Vet Pathol. 1997; 34: 31-38Crossref PubMed Scopus (43) Google Scholar) tyrosinases (reviewed inShibahara et al., 1998Shibahara S. Yasumoto K. Takahashi K. Genetic regulation of the pigment cell.in: Norlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortone J-P. The Pigmentary System. Physiology and Pathophysiology. Oxford University Press, New York1998: 251-273Google Scholar). We have isolated cDNA clones encoding tyrosinase from Japanese quail and snapping turtle. As expected from our previous analyses of the first exons of quail and turtle tyrosinase genes (Yamamoto et al., 1992Yamamoto H. Kudo T. Masuko N. et al.Phylogeny of regulatory regions of vertebrate tyrosinase genes.Pigment Cell Res. 1992; 5: 284-294Crossref PubMed Scopus (32) Google Scholar), the entire nucleotide and deduced amino acid sequences were highly homologous to those of other vertebrate tyrosinases. As shown in Figure 1, quail tyrosinase cDNA (1894 bp) has an open reading frame (ORF) encoding 529 amino acids (Mr 60 333) and the turtle cDNA (1909 bp) also contains an ORF encoding 529 amino acids (Mr 60 167); these ORF are similar in length to those of tyrosinase cDNA isolated from other vertebrates. In addition, the sizes of the major transcripts of quail and turtle tyrosinases were determined to be approximately 2.1 kb by northern hybridization (data not shown). In each of the seven vertebrate tyrosinases aligned in Figure 1, a signal peptide (von Heijne, 1986von Heijne G. A new method for predicting signal sequence cleavage sites.Nucl Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3665) Google Scholar) and a transmembrane domain (Klein et al., 1985Klein P. Kanehisa M. DeLisi C. The detection and classification of membrane-spanning proteins.Biochim Biophys Acta. 1985; 815: 468-476Crossref PubMed Scopus (624) Google Scholar) were identified at the N-terminus and near the C-terminus, respectively. These two regions, together with a cytoplasmic tail at the C-terminal end, show relatively low similarity among aligned sequences compared with the overall high homology of other regions. As expected, two putative copper-binding sites (CuA and CuB) are highly conserved in all vertebrate tyrosinases. In these regions, which are also found in plant and fungal tyrosinases, six conserved histidine residues that are symmetrically arranged are believed to form a binuclear copper center and are crucial for specific copper-binding and/or enzymatic activity (Hearing and Tsukamoto, 1991Hearing V.J. Tsukamoto K. Enzymatic control of pigmentation in mammals.FASEB J. 1991; 5: 2902-2909Crossref PubMed Scopus (646) Google Scholar;Oetting and King, 1994aOetting W.S. King R.A. Molecular basis of oculocutaneous albinism.J Invest Dermatol. 1994; 103: 131S-136SCrossref PubMed Scopus (47) Google Scholar;van Gelder et al., 1997van Gelder C.W. Flurkey W.H. Wichers H.J. Sequence and structural features of plant and fungal tyrosinases.Phytochemistry. 1997; 45: 1309-1323Crossref PubMed Scopus (392) Google Scholar). There are 14 cysteine residues at conserved positions distributed unevenly in the sequences (Figure 1). The first 10 cysteine residues cluster near the N-termini and the other five (four in medaka fish tyrosinase) residues reside between two copper-binding sites. Amino acid substitutions within the N-terminal cysteine rich region are found in BALB/C albino (Tyrc/Tyrc) mouse (C103S) (Shibahara et al., 1990Shibahara S. Okinaga S. Tomita Y. Takeda A. Yamamoto H. Sato M. Takeuchi T. A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine to serine substitution at position 85.Eur J Biochem. 1990; 189: 455-461Crossref PubMed Scopus (86) Google Scholar;Yokoyama et al., 1990Yokoyama T. Silversides D.W. Waymire K.G. Kwon B.S. Takeuchi T. Overbeek P.A. Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice.Nucl Acids Res. 1990; 18: 7293-7298Crossref PubMed Scopus (167) Google Scholar) and among patients diagnosed with O
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