Alternative Splicing of the Tyrosinase Gene Transcript in Normal Human Melanocytes and Lymphocytes
2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês
10.1046/j.0022-202x.2001.01549.x
ISSN1523-1747
AutoresJames P. Fryer, William S. Oetting, Marcia J. Brott, Richard A. King,
Tópico(s)RNA and protein synthesis mechanisms
ResumoWe have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific “nested” primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans. We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific “nested” primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans. Tyrosinase (monophenol oxidase, EC1.14.18.1) is a copper-containing enzyme critical to the formation of melanin in the melanosome within the melanocyte (Lerner and Fitzpatrick, 1950Lerner A.B. Fitzpatrick T.B. Biochemistry of melanin formation.Physiol Rev. 1950; 30: 91-126Crossref PubMed Scopus (287) Google Scholar). Its major function is to catalyze the hydroxylation of tyrosine to dopaquinone (Cooksey et al., 1997Cooksey C.J. Garratt P.J. Land E.J. Pavel S. Ramsden C.A. Riley P.A. Smit N.P.M. Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase.J Biol Chem. 1997; 272: 26226-26235Crossref PubMed Scopus (295) Google Scholar). Dopaquinone then forms red–yellow pheomelanin or black–brown eumelanin depending primarily on the availability of sulfhydryl compounds (Prota, 1980Prota G. Recent advances in the chemistry of melanogenesis in mammals.J Invest Dermatol. 1980; 75: 122-127Crossref PubMed Scopus (336) Google Scholar). Tyrosinase functions as part of an enzyme–protein complex within the melanosome and is not active in vivo before vesicular transport to this organelle (Orlow et al., 1994Orlow S.J. Zhou B.-K. Chakraborty A.K. Drucker M. Pifko-Hirst S. Pawelek J.M. High-molecular-weight forms of tyrosinase and the tyrosinase-related proteins: evidence for a melanogenic complex.J Invest Dermatol. 1994; 103: 196-201Crossref PubMed Scopus (126) Google Scholar). Mutations that inactivate this enzyme reduce the amount of melanin synthesized in the melanocyte and produce oculocutaneous albinism (OCA) in mice and humans (Shibahara et al., 1988Shibahara S. Tomita Y. Tagami H. Muller R.M. Cohen T. Molecular basis for the heterogeneity of human tyrosinase.Tohoku J Exp Med. 1988; 156: 403-414Crossref PubMed Scopus (76) Google Scholar,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 (85) Google Scholar;Spritz et al., 1990Spritz R.A. Strunk K.M. Giebel L.B. King R.A. Detection of mutations in the tyrosinase gene in a patient with Type 1A oculocutaneous albinism.N Engl J Med. 1990; 322: 1724-1728Crossref PubMed Scopus (67) Google Scholar;Oetting, 2000Oetting W.S. The tyrosinase gene and oculocutaneous albinism type 1 (OCA1): a model for understanding the molecular biology of melanin formation.Pigment Cell Res. 2000; 13: 320-325https://doi.org/10.1034/j.1600-0749.2000.130503.xCrossref PubMed Scopus (168) Google Scholar). The murine and human tyrosinase genes have a similar structure, with five exons that encode a protein with a putative leader sequence, two copper-binding domains, and a trans-membrane domain (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 tyrosinase gene are generated by alternative splicing.EMBO J. 1988; 7: 2715-2722Crossref PubMed Scopus (136) Google Scholar;Giebel et al., 1991Giebel L.B. Strunk K.M. Spritz R.A. Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment.Genomics. 1991; 9: 435-445Crossref PubMed Scopus (123) Google Scholar). The predominant expression of the murine gene is a full-length mRNA but multiple smaller mRNA are produced (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;Terao et al., 1989Terao M. Tabe L. Garattini E. Sartori D. Studer M. Mintz B. Isolation and characterization of variant cDNAs encoding mouse tyrosinase.Biochem Biophys Res Commun. 1989; 159: 848-853Crossref PubMed Scopus (21) Google Scholar;Porter and Mintz, 1991Porter S. Mintz B. Multiple alternatively spliced transcripts of the mouse tyrosinase-encoding gene.Gene. 1991; 97: 277-282Crossref PubMed Scopus (34) Google Scholar). The smaller mRNA arise from alternative splicing, and do not produce active enzyme (Porter and Mintz, 1991Porter S. Mintz B. Multiple alternatively spliced transcripts of the mouse tyrosinase-encoding gene.Gene. 1991; 97: 277-282Crossref PubMed Scopus (34) Google Scholar;Terao et al., 1989Terao M. Tabe L. Garattini E. Sartori D. Studer M. Mintz B. Isolation and characterization of variant cDNAs encoding mouse tyrosinase.Biochem Biophys Res Commun. 1989; 159: 848-853Crossref PubMed Scopus (21) Google Scholar). Recent studies have also demonstrated alternative splicing in the gene for tyrosinase-related-protein 2/dopachrome tautomerase, another member of the tyrosinase gene family (Pawelek et al., 1980Pawelek J. Korner A. Bergstrom A. Bologna J. New regulators of melanin biosynthesis and the autodestruction of melanoma cells.Nature. 1980; 286: 617-619Crossref PubMed Scopus (191) Google Scholar;Pisarra et al., 2000Pisarra P. Lupetti R. Palumbo A. et al.Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: molecular and functional characterization.J Invest Dermatol. 2000; 115: 48-56Crossref PubMed Scopus (15) Google Scholar). Most of the studies of alternative splicing of the murine tyrosinase or the tyrosinase-related-protein 2 gene were performed with melanoma tissue. We now report our analysis of alternative splicing of the normal human tyrosinase gene. These studies were undertaken to provide information on the biology of this gene and to further our understanding of human OCA resulting from mutations of this gene (OCA1). To overcome the difficulty in obtaining large numbers of melanocytes from normal and affected individuals, a method for analyzing ectopic transcription of the tyrosinase gene in cultured lymphoblastoid cell lines derived from peripheral blood lymphocytes was developed. Lymphocytes were isolated from heparinized whole blood from normally pigmented individuals using a Ficoll gradient (Histopaque, Sigma, St Louis, MO). The isolated lymphocytes were transformed using Epstein–Barr Virus (EBV) and cyclosporine to establish lymphoblastoid cell lines (Pelloquin et al., 1986Pelloquin F. Lamelin J.P. Lenoir G.M. Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A.In Vitro Cell Dev Biol. 1986; 22: 689-694Crossref PubMed Scopus (65) Google Scholar). Normal human primary melanocytes in culture were kindly provided by Raymond Boissy, PhD, Department of Dermatology, University of Cincinnati, OH. A human melanoma cell line, MNT-1, was kindly provided by Vincent Hearing, PhD, NIH and was maintained in Dulbecco's minimal Eagle's medium culture media (Sigma) under standard culture conditions. Total RNA from lymphoblastoid cell lines was isolated using a protocol modified from Glisen and Ullrich (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Extraction purification, and analysis of messenger RNA from eukaryotic cells.in: Molecular Cloning, a Laboratory Manual. Cold Spring Harbor. 87. Cold Spring Harbor Laboratories Press, 1989: 7.1-7Google Scholar). Reverse transcription was performed using 10–15 µg total RNA, 20 pmol tyrosinase specific primers, 20 units of RNasin (Promega, Madison, WI), 200 units of reverse transcriptase with 1 × reverse transcription buffer (Gibco, Rockville, MD), 0.5 mM of each deoxyribonucleoside triphosphate and 0.01 M dithiothreitol (the final reaction volume was 20 μl). The reaction was incubated at 42°C for 2 h and terminated by heating at 70°C for 15 min. Complementary RNA strands were then removed by the addition of 2 units of RNase H (Gibco) and incubating at 37°C for 20 min. In all cases DEPC treated sterile water was added in place of RNA as a negative control. The cDNA is amplified twice using a series of nested primer sets that allowed amplification of specific regions of the tyrosinase gene (NCBI accession number 0000372). The locations of the primers are shown in Figure 1 and the primer sequences are given in Table I.Table IAmplification primer sequences5′ forward primers F1 5′-ACT CCA ATT AGC CAG TTC C-3′, F2 5′-TTG TGA GGA CTA GAG GAA GA-3′ F3 5′-ATG GAT GCA CTG CTT GGG GGA-3′ F4 5′-ATT GTC TGT AGC CGA TTG GA—3′ F5 5′-TGG AAC GCC CGA GGG ACC TT-3′3′ reverse primers R1 5′-CTT CCA GTG TAT TTC TAA AGC-3′ R2 5′-TCT AAA GCT GAA ATT GGC AGC-3′ R3 5′-CTG TCA ACA AAT GCA TGG TG-3′ R4 5′-GAA GGA AGA TAG GAT CGT T-3′ R5 5′-CTG AAT CTT GTA GAT AGC TA-3′ R6 5′-AGT CAT AGC CCA GAT CTT TG-3′ R7 5′-GGT GCA TTG GCT TCT GGA TA-3′ Open table in a new tab For amplification, the entire reverse transcription reaction was mixed with 1.5 mM MgCl2, 1 × PCR buffer (Cetus, Norwalk, CT), 0.2 mM of each deoxyribonucleoside triphosphate, 5 units of Taq Polymerase (Cetus) and 20 pmol of each forward and reverse amplification primer for the region to be amplified. The amplification reactions were performed with 40 cycles using standard protocols. The reaction was then filtered through a 30,000 NMWL spin filter (Millipore, Bedford, MA) to separate the excess amplification primers prior to the second amplification. The second amplification reaction conditions were the same with the exception that a nested set of primers were utilized, and 10% of the first round reaction was used as template. The negative reverse transcription control was used in the negative control reaction. Amplified products were cloned into a TA vector (Invitrogen, Carlsbad, CA) for the isolation of individual products (Clark, 1988Clark J.M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.Nucleic Acids Res. 1988; 16: 9677-9686Crossref PubMed Scopus (713) Google Scholar;Mead et al., 1991Mead D. Pey N.K. Herrnstadt C. Marcil R.A. Smith L.M. A universal method for the direct cloning of PCR amplified nucleic acid.Biotechnology. 1991; 9: 657-663Crossref PubMed Scopus (196) Google Scholar). The products of the second round of amplification were separated and visualized by electrophoresis of 10 µl sample in 1.2% agarose containing ethidium bromide. The amplification products were sequenced using either fluorescent sequencing primers or primers end-labeled with 32P-adenosine triphosphate. Reaction conditions for both methods were the same. Prior to sequencing, the amplification reactions were filtered through a 30,000 NMWL filter to remove excess PCR primers. Samples were cycle-sequenced as previously described (Oetting et al., 1994Oetting W.S. Fryer J.P. Oofuji Y. Middendorf L.R. Brumbaugh J.A. Summers C.G. King R.A. Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons.Electrophoresis. 1994; 15: 159-164Crossref PubMed Scopus (16) Google Scholar). The isotopically labeled sequence reactions were electrophoresed on a 60 cm acrylamide gel (8%) containing 8 M urea and 1 × trio-boric acid EDTA buffer. The sequencing ladder was visualized using autoradiography. The fluorescent labeled sequence reactions were separated on a 33 cm acrylamide gel (8%) containing 8 M urea and 1 × TBE buffer on a LI-COR Systems S-4000 autosequencer (LI-COR Inc, Lincoln, NE) (Oetting et al., 1994Oetting W.S. Fryer J.P. Oofuji Y. Middendorf L.R. Brumbaugh J.A. Summers C.G. King R.A. Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons.Electrophoresis. 1994; 15: 159-164Crossref PubMed Scopus (16) Google Scholar). Initial studies showed that adequate amounts of tyrosinase mRNA were available for PCR amplification from lymphoblastoid cell lines, and that tyrosinase cDNA could be readily visualized with ethidium bromide and sequenced. To establish that the cDNA represented products of both tyrosinase alleles of an individual, the reverse transcription–PCR product of mRNA from a normally pigmented individual who was heterozygous at the codon 192 polymorphism (Y192S; TCT/TAT) in exon 1 of the tyrosinase gene was analyzed (Giebel et al., 1991Giebel L.B. Strunk K.M. Spritz R.A. Organization and nucleotide sequences of the human tyrosinase gene and a truncated tyrosinase-related segment.Genomics. 1991; 9: 435-445Crossref PubMed Scopus (123) Google Scholar;Oetting et al., 1993Oetting W.S. Witkop C.J. Brown S.A. Colomer R. Fryer J.P. Bloom K.E. King R.A. A frequent mutation in the tyrosinase gene associated with type I-A (tyrosinase-negative) oculocutaneous albinism in Puerto Rico.Am J Hum Genet. 1993; 52: 17-23PubMed Google Scholar). The Y192S mutation represents a common polymorphism with equal frequencies of the two alleles in the Caucasian population. As shown in Figure 2, the cDNA sequence clearly showed both tyrosinase alleles for codon 192, indicating that the transcript from both alleles was ectopically expressed in a lymphoblastoid cell line. Lymphoblastoid cell line RNA from six normally pigmented individuals was used to identify transcripts of the normal tyrosinase gene. Three regions of the tyrosinase cDNA were amplified using the primers in Table I. Amplification of these regions with cDNA from lymphoblastoid cell line mRNA produced products of the expected size (N1-3), as shown in Figure 3(a) for one individual. Five smaller products were also identified, and each was cloned into a TA vector, sequenced and compared with the known tyrosinase cDNA sequence (Mead et al., 1991Mead D. Pey N.K. Herrnstadt C. Marcil R.A. Smith L.M. A universal method for the direct cloning of PCR amplified nucleic acid.Biotechnology. 1991; 9: 657-663Crossref PubMed Scopus (196) Google Scholar). Repeat analysis of reverse transcription–PCR products did not consistently demonstrate all smaller products from each of the six individuals, but each product could be identified with transcript-specific primers Table II in cDNA from all six individuals (data not shown). The five smaller transcripts resulted from alternative splicing, using three alternative splice sites in exon 1 and the normal 3′ and 5′ splice sites of exon 3.Table IISequences for transcript-specific primers (TSP)TSP-A5′-AAG AAT GAT GCA AGA GTC GT-3′TSP-B5′-TCA GGC AGA GAT TGT CTG TA-3′TSP-C5′-ACG ACT CTT ATT GTC TGT AG-3′TSP-D5′-ACA CTG GAA GTA TTT TTG AG-3′TSP-E5′-TCA GGC AGA GTA TTT TTG AG-3′ Open table in a new tab The alternatively spliced transcripts were more readily identified by further amplification using transcript-specific primers (Figure 3b and Figure 4). The design of these primers was based on the sequence of the alternative splice junctions of transcripts A–E, such that the primers would span the splice site junctions. The 5′ and 3′ ends of the primers were on opposite sides of the splice junction, and they would anneal only to the cDNA of the specifically spliced transcripts and not to the normal cDNA sequences. A transcript-specific primer was used with either primer F2 (transcript A), R2 (transcript C), or R6 (transcripts B, D, and E) to amplify the alternative spliced cDNA, illustrated in Figure 4. In transcript A, an alternative donor splice site in codon 118 and an alternative acceptor splice site at codon 266 generated a transcript of 384 bp. In transcript B, an alternative donor splice site in codon 53 and the normal exon 2 acceptor splice site generated a transcript of 534 bp. In transcript C, the codon 118 alternative donor splice site and the normal exon 2 acceptor splice site were used to generate a transcript of 211 bp. In transcript D, exon 3 was spliced out using the normal donor and acceptor splice sites, and this generated a transcript of 171 bp. In transcript E, the codon 53 alternative donor splice site and the normal exon 4 acceptor splice site were used to generate a transcript of 167 bp. The alternative splicing at codons 53, 118, and 266 appears to result from the presence of cryptic splice sites at these locations, as shown in Figure 5 and Figure 6. Consensus donor splice site sequences (AG|gtragt) are found at codons 53 and 118 (Reed and Maniatis, 1985Reed R. Maniatis T. The role of the mammalian branchpoint sequence in pre-mRNA splicing.Cell. 1985; 41: 95-105Abstract Full Text PDF PubMed Scopus (246) Google Scholar). A consensus acceptor splice site and the upstream branch point (YNYTRAY……. (Y)nNYAG) are found with codon 266. Thus, the alternative splicing that we have identified involved cryptic splice sites that exist in the tyrosinase gene sequence.Figure 6Diagram showing normal and alternatively spliced tyrosinase transcripts identified from human lymphoblastoid RNA. Sequence N represents the normal sequence showing the 5 exons of the tyrosinase gene. (A–E) Represent alternatively spliced transcripts. The mRNA regions removed by alternative splicing are indicated by the stippled regions. Cryptic splice sites are located at codons 53, 118, and 266.View Large Image Figure ViewerDownload (PPT) RNA from normal human primary melanocyte cultures was used to confirm that the splicing observed in lymphoblastoid cell line mRNA was representative of the pigment cell and not an artifact of ectopic expression. Normal melanocyte RNA was reverse transcribed using tyrosinase-specific primers (R2 and R5) and amplified using transcript-specific primers Table II. This analysis showed a single amplified product of the expected size for each alternatively spliced transcript, as shown in Figure 7. All alternatively spliced transcripts found in lymphoblastoid cell lines were readily identified in normal melanocyte RNA, showing that this processing is not the result of ectopic expression or aberrant processing in the transformed lymphocytes. Melanin forms in the melanocyte, a specialized cell found in the skin, hair follicle, eye, and ear. Tyrosinase is the key enzyme in the formation of melanin by catalyzing the initial step in melanin synthesis, which is necessary for the subsequent formation of both black–brown eumelanin and red–yellow pheomelanin (Lerner and Fitzpatrick, 1950Lerner A.B. Fitzpatrick T.B. Biochemistry of melanin formation.Physiol Rev. 1950; 30: 91-126Crossref PubMed Scopus (287) Google Scholar;Prota, 1980Prota G. Recent advances in the chemistry of melanogenesis in mammals.J Invest Dermatol. 1980; 75: 122-127Crossref PubMed Scopus (336) Google Scholar). Mutations of the tyrosinase gene are associated with partially or totally inactive enzyme, and reduced melanin in the melanocyte. The loss of melanin produces the clinical phenotype known as OCA. Human OCA is complex, with at least seven different gene loci being involved (King et al., 1995King R.A. Hearing V.J. Creel D.J. Oetting W.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Metabolic and Molecular Bases of Inherited Disease.in: 8. McGraw-Hill, New York1995: 4353-4392Google Scholar;Dell'Angelica et al., 1999Dell'Angelica E.C. Shotelersuk V. Aguilar R.C. Gahl W.A. Bonifacino J.S. Altered trafficking of lysosomal proteins in Hermansky–Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor.Mol Cell. 1999; 3: 11-21Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar;Shotelersuk et al., 2000Shotelersuk V. Dell'Angelica E.C. Hartnell L. Bonifacino J.S. Gahl W.A. A new variant of Hermansky–Pudlak syndrome due to mutations in a gene responsible for vesicle formation.Am J Med. 2000; 108: 423-427https://doi.org/10.1016/s0002-9343(99)00436-2Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Mutations of the tyrosinase gene producing OCA1 are usually identified by sequence analysis of the coding region of the gene with DNA from affected individuals. The majority are missense mutations. One large tyrosinase gene deletion has been identified as well as several nonsense, frameshift, and splice-site mutations (Oetting and King, 1999Oetting W.S. King R.A. Molecular basis of albinism: mutations and polymorphisms of pigment genes associated with albinism.Hum Mutat. 1999; 13: 99-115Crossref PubMed Scopus (259) Google Scholar;Schnur et al., 1996Schnur R.E. Selling B.T. Holmes S.A. Wick P.A. Tatsumura Y.O. Spritz R.A. Type 1 oculocutaneous albinism associated with a full-length deletion of the tyrosinase gene.J Invest Dermatol. 1996; 106: 1137-1140Crossref PubMed Scopus (21) Google Scholar;Matsunaga et al., 1999Matsunaga J. Dakeishi-Hara M. Tanita M. et al.A splicing mutation of the tyrosinase gene causes yellow oculocutaneous albinism in a Japanese patient with a pigmented phenotype.Dermatology. 1999; 199: 124-129Crossref PubMed Scopus (19) Google Scholar;Oetting, 2000Oetting W.S. The tyrosinase gene and oculocutaneous albinism type 1 (OCA1): a model for understanding the molecular biology of melanin formation.Pigment Cell Res. 2000; 13: 320-325https://doi.org/10.1034/j.1600-0749.2000.130503.xCrossref PubMed Scopus (168) Google Scholar). There are, however, a number of individuals with an OCA1 phenotype (i.e., white hair, white skin, and blue eyes) who do not have two identifiable mutations in the coding region of the gene, and it is hypothesized that some of these individuals have mutations that affect the transcription of the gene or processing of the transcription product (Oetting, 2000Oetting W.S. The tyrosinase gene and oculocutaneous albinism type 1 (OCA1): a model for understanding the molecular biology of melanin formation.Pigment Cell Res. 2000; 13: 320-325https://doi.org/10.1034/j.1600-0749.2000.130503.xCrossref PubMed Scopus (168) Google Scholar;Gilhar et al., 1995Gilhar A. Gershoni-Baruch R. Margolis A. Benderly A. Brandes J.M. DOPA reaction of fetal melanocytes before and after skin transplantation on to nude mice.Br J Dermatol. 1995; 133: 884-889Crossref PubMed Scopus (10) Google Scholar). Studies of melanocytes would provide an approach to analyzing tyrosinase transcription products, but melanocytes are generally not available from these individuals. To overcome these problems, we have developed a method that will allow us to analyze ectopic tyrosinase gene transcription products in lymphoblastoid cell lines from individuals with albinism. A similar approach has been used successfully in other genetic diseases (Bidichandani et al., 1995Bidichandani S.I. Lanyon W.G. Shiach C.R. Lowe G.D. Connor J.M. Detection of mutations in ectopic factor VIII transcripts from nine haemophilia A patients and the correlation with phenotype.Hum Genet. 1995; 95: 531-538Crossref PubMed Scopus (20) Google Scholar;Tavassoli et al., 1997Tavassoli K. Eigel A. Pollmann H. Horst J. Mutational analysis of ectopic factor VIII transcripts from hemophilia A patients: identification of cryptic splice site, exon skipping and novel point mutations.Hum Genet. 1997; 100: 508-511https://doi.org/10.1007/s004390050543Crossref PubMed Scopus (20) Google Scholar;Kure et al., 1998Kure S. Sakata Y. Miyabayashi S. et al.Mutation and polymorphic marker analyses of 65K- and 67K-glutamate decarboxylase genes in two families with pyridoxine-dependent epilepsy.J Hum Genet. 1998; 43: 128-131Crossref PubMed Scopus (26) Google Scholar;Ars et al., 2000Ars E. Serra E. Garcia J. Kruyer H. Gaona A. Lazaro C. Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1.Hum Mol Genet. 2000; 9: 237-247https://doi.org/10.1093/hmg/9.2.237Crossref PubMed Scopus (256) Google Scholar;Tassone et al., 2000Tassone F. Hagerman R.J. Taylor A.K. Gane L.W. Godfrey T.E. Hagerman P.J. Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome.Am J Hum Genet. 2000; 66: 6-15Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar). There were two critical points to address in establishing the utility of this approach. First, the transcript pattern for the tyrosinase gene had to be established. We readily identified transcripts of this gene in lymphoblastoid cell lines. In addition to the normal-sized transcripts, we identified five smaller transcripts resulting from variant splicing that removed different regions or exons of the gene from the transcript through the use of normal or alternative splice sites. These transcripts are presumably degraded rather than processed in these cells as there is no evidence that they are involved in melanin synthesis. Similar transcript variants of the tyrosinase gene have been identified in mouse skin melanocytes and melanoma, and these transcripts do not result in active tyrosinase protein (Porter and Mintz, 1991Porter S. Mintz B. Multiple alternatively spliced transcripts of the mouse tyrosinase-encoding gene.Gene. 1991; 97: 277-282Crossref PubMed Scopus (34) 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 tyrosinase gene are generated by alternative splicing.EMBO J. 1988; 7: 2715-2722Crossref PubMed Scopus (136) Google Scholar;Le Fur et al., 1996Le Fur N. Kelsall S.R. Mintz B. Base substitution at different alternative splice donor sites of the tyrosinase gene in murine albinism.Genomics. 1996; 37: 245-248Crossref PubMed Scopus (43) Google Scholar;Kelsall et al., 1997Kelsall S.R. Le Fur N. Mintz B. Qualitative and quantitative catalog of tyrosinase alternative transcripts in normal murine skin melanocytes as a basis for detecting melanoma-specific changes.Biochem Biophys Res Commun. 1997; 236: 173-177https://doi.org/10.1006/bbrc.1997.6925Crossref PubMed Scopus (11) Google Scholar). In the mouse, exon 1 contains two alternative donor splice sites (at bp 291 and 369) and two alternative receptor splice sites (at bp 522 and 858); the normal splice site at the 3′ end of exon 1 is at bp 881 (Kelsall et al., 1997Kelsall S.R. Le Fur N. Mintz B. Qualitative and quantitative catalog of tyrosinase alternative transcripts in normal murine skin melanocytes as a basis for detecting melanoma-specific changes.Biochem Biophys Res Commun. 1997; 236: 173-177https://doi.org/10.1006/bbrc.1997.6925Crossref PubMed Scopus (11) Google Scholar). The locations of the alternative splice sites in the mouse are similar to but not identical to those that produce alternative transcripts in human lymphoblastoid cell line mRNA. Second, the reliability of the transcript pattern in lymphoblastoid cell lines had to be determined by comparing this with the pattern in human melanocytes. Normal melanocytes in primary culture and a human melanoma cell line (MNT1) were found to produce the same pattern of tyrosinase gene transcripts (Figure 6 and Figure 7) indicating that the lymphoblastoid cell lines could be a surrogate for tyrosinase gene transcript analysis when melanocytes are not available. An additional point is raised with these studies. The detection of tyrosinase mRNA in peripheral blood has been suggested as a method for monitoring the clinical course of patients with melanoma (Smith et al., 1991Smith B. Selby P. Southgate J. Pittman K. Bradley C. Blair G.E. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction.Lancet. 1991; 338: 1227-1229Abstract PubMed Scopus (616) Google Scholar;Alao et al., 1999Alao J.P. Mohammed M.Q. Slade M.J. Retsas S. Detection of tyrosinase mRNA by RT-PCR in the peripheral blood of patients with advanced metastatic melanoma.Melanoma Res. 1999; 9: 395-399Crossref PubMed Scopus (23) Google Scholar;Calogero et al., 2000Calogero A. Timmer-Bosscha H. Schraffordt K.H. Tiebosch A.T. Mulder N.H. Hospers G.A. Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes.Br J Cancer. 2000; 83: 184-187Crossref PubMed Scopus (17) Google Scholar;Johansson et al., 2000aJohansson M. Arstrand K. Hakansson A. Lindholm C. Kagedal B. Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction.Melanoma Res. 2000; 10: 213-222Crossref PubMed Scopus (10) Google Scholar;Kulik et al., 2001Kulik J. Nowecki Z.I. Rutkowski P. Ruka W. Rochowska M. Shurzak H. Siedlecki J.A. Detection of circulating melanoma cells in peripheral blood by a two-marker RT-PCR assay.Melanoma Res. 2001; 11: 65-73Crossref PubMed Scopus (27) Google Scholar). Studies have described the extraction of mRNA from peripheral blood mononuclear or buffy coat cells, suggesting that circulating melanoma cells may be present. Our results indicate that the presence of ectopic tyrosinase gene transcripts may arise from lymphocytes in the blood cells obtained for the analysis, rather than from circulating melanoma cells. This may explain why this diagnostic approach for melanoma has proven to be problematic (Calogero et al., 2000Calogero A. Timmer-Bosscha H. Schraffordt K.H. Tiebosch A.T. Mulder N.H. Hospers G.A. Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes.Br J Cancer. 2000; 83: 184-187Crossref PubMed Scopus (17) Google Scholar;Aubin et al., 2000Aubin F. Chtourou M. Teyssier J.R. Laubriet A. Mougin C.H. Blanc D. Humbert P. The detection of tyrosinase mRNA in the peripheral blood of stage I melanoma patients is not of clinical relevance in predicting metastasis risk and survival.Melanoma Res. 2000; 10: 113-118Crossref PubMed Scopus (32) Google Scholar;Tsao et al., 2001Tsao H. Nadiminti U. Sober A.J. Bigby M. A meta-analysis of reverse transcriptase-polymerase chain reaction for tyrosinase mRNA as a marker for circulating tumor cells in cutaneous melanoma.Arch Dermatol. 2001; 137: 325-330Crossref PubMed Scopus (1) Google Scholar). These studies provide the first description of the use of ectopic transcription analysis with the human tyrosinase gene. We have also identified transcripts of the P gene in lymphoblastoid cell lines (data not shown); the product of this gene is involved in melanin biosynthesis and mutations of the gene are associated with OCA2 (Gardner et al., 1992Gardner J.M. Nakatsu Y. Gondo Y. Lee S. Lyon M.F. King R.A. Brilliant M.H. The mouse pink-eyed dilution gene: association with human Prader-Willi and Angelman syndromes.Science. 1992; 257: 1121-1124Crossref PubMed Scopus (181) Google Scholar;Lee et al., 1995Lee S.-T. Nicholls R.D. Jong M.T.C. Spritz R.A. Organization and sequence of the human P gene and identification of a new family of transport pump proteins.Genomics. 1995; 26: 354-363Crossref PubMed Scopus (147) Google Scholar;Oetting et al., 1998Oetting W.S. Gardner J.M. Fryer J.P. Ching A. Durham-Pierre D. King R.A. Brilliant M.H. Mutations in brief: mutations of the human P gene associated with type II oculocutaneous albinism (OCA2).Hum Mutat. 1998; 12: 4333-4434Crossref Google Scholar). This approach to transcript analysis may be useful when the primary transcript of a gene is not available because of the lack of access to the critical tissue. Future studies of human disorders of pigmentation resulting from mutations affecting splicing or transcription of the tyrosinase gene should be possible using this approach. The authors thank Vincent Hearing, PhD, for the MNT-1 cells, and Raymond Boissy, PhD, for the normal human melanocyte cultures. This work was supported in part by funds provided by Bernard and Mary Ellen Black and by NIH grant AR 44649.
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