Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9228-9232, October 1991 Genetics A melanocyte-specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chromosome 12 (pigmentation/silver locus) BYOUNG S. KWON*t, CHAYA CHINTAMANENI*, CHRISTINE A. KOZAKt, NEAL G. COPELAND§, DEBRA J. GILBERT§, NANCY JENKINS§, DAVID BARTON II, UTA FRANCKE¶**, YVONNE KOBAYASHI*, AND KACK K. KIM* *Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; tLaboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; §Mammalian Genetics Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702; and IDepartment of Human Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Communicated by Elizabeth S. Russell, July 8, 1991 ABSTRACT Melanocytes preferentially express an mRNA was referred to as Pmel 17-1.tt We demonstrated that the species, Pmel 17, whose protein product cross-reacts with gene for Pmel 17-1 did not map to the c locus on mouse anti-tyrosinase antibodies and whose expression correlates with chromosome 7 (10) and that the transcript of Pmel 17-1 was the melanin content. We have now analyzed the deduced preferentially detected in melanocytes but not in nonpig- protein structure and mapped its chromosomal location in mented cells. mouse and human. The amino acid sequence deduced from the Mouse molecular genetic linkage maps have proved in- nucleotide sequence of the Pmel 17 cDNA showed that the valuable for the structural and functional characterization of protein is composed of645 amino acids with a molecular weight the mouse genome. They have been used to determine of 68,600. The Pmel 17 protein contains a putative leader whether newly identified genes are homologous to known sequence and a potential membrane anchor segment, which genes or classic mutations (11-15). In this study, we have indicates that this may be a membrane-associated protein in analyzed interspecies crosses to map the mouse homologue melanocytes. The deduced protein contains five potential of the Pmel 17 gene to chromosome 10 near si (silver), a N-glycosylation sites and relatively high levels of serine and mouse coat color locus. threonine. Three repeats ofa 26-amino acid motifappear in the middle of the molecule. The human Pmel 17 gene, designated MATERIALS AND METHODS D12S53E, maps to chromosome 12, region 12pter-q21; and the Isolation ofcDNA Clones. Because the initial isolate ofPmel mouse homologue, designated D12S53Eh, maps to the distal 17-1 was only 1.0 kilobase (kb), which was clearly not region of mouse chromosome 10, a region also known to carry full-length, the human melanocyte cDNA library (9) was the coat color locus si (silver). rescreened using a cDNA insert of clone Pmel 17-1 to obtain a longer cDNA. We isolated nine overlapping cDNA clones, A major obstacle in understanding the biology of pigmenta- and the nucleotide sequences of all ofthem were determined. tion and pigment-related diseases is the lack of molecular The full-length cDNA of Pmel 17-1 is referred to as Pmel 17. probes specific for molecules involved in melanin biosynthe- DNA Sequencing. Restriction fragments of DNA subcloned sis. Tyrosinase catalyzes the first two steps of melanin in M13 vectors were sequenced by the dideoxynucleotide biosynthesis (1-3) and, therefore, is a key enzyme. Tyrosi- chain-termination technique (16). A forward primer (New nase-negative oculocutaneous albinism is due to inactivating England Biolabs) complementary to the lacZ sequence adja- mutations of the tyrosinase gene (4-6). Other albino mutants cent to the 5' side ofthe EcoRI site in Agtll was used for direct may, however, be positive for tyrosinase activity (7), which sequencing of the end point of the cDNA insert in Agtll (17). indicates that a block in melanin synthesis is not necessarily Probes. Probes for Pmel 17-1 have been described (10). due to the absence oftyrosinase. Elucidation ofthe influence Probes for the Gli, Ifg, Pah, Igf-J, pg, and SI loci have been of additional factors in the control of melanization could be described (11, 18-20). significant for the understanding of tyrosinase-positive albi- Human-Hamster Somatic Cell Hybrids and Filter Hybrid- nism and variations in skin and hair pigmentation. For ization. The primary chromosome assignment of Pmel 17-1 in example, the silver (si) mutation induces hypopigmentation, the human was carried out with 16 hybrid clones that were which in the mouse involves the premature loss of many derived from eight independent fusion experiments between melanocytes from the hair bulb (8). Chinese hamster cell lines and human diploid fibroblasts or When we screened a Agtll cDNA library of a primary lymphocytes. The origin and characterization ofthese hybrids culture of pigmented human melanocytes with anti- have been summarized (21, 22). For regional mapping ofPmel tyrosinase antibodies, 16 cDNA clones whose gene products 17-1, we used a Chinese hamster-human hybrid clone with a reacted to anti-tyrosinase antibodies were isolated (9). Thir- spontaneous deletion of the distal long arm of human chro- teen clones that cross-hybridized to each other were mapped mosome 12. The remaining region is 12pter-q21. Genomic to the mouse albino locus (c) and were characterized as the DNA was extracted from cultured hybrid cells by routine cDNA encoding human tyrosinase (9). The three remaining clones also cross-hybridized but did not share homology with tTo whom reprint requests should be addressed. the other group of clones. The representative cDNA clone Present address: Department of Pathology, University of Cam- bridge, Cambridge CB2 1QP, United Kingdom. **Present address: Department of Genetics, Stanford University The publication costs of this article were defrayed in part by page charge Medical Center, Stanford, CA 94305. payment. This article must therefore be hereby marked "advertisement" ttThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M77348). 9228 Downloaded by guest on September 30, 2021 Genetics: Kwon et al. Proc. NatL. Acad. Sci. USA 88 (1991) 9229 procedures. Ten micrograms of hybrid and each parental RESULTS control cell DNA was digested with a 4-fold excess ofHindIII Sequence Analysis of Pmel 17. Fig. LA shows the amino acid under conditions recommended by the manufacturer. sequence deduced from the longest open reading frame of DNA fragments were separated by agarose gel electropho- Pmel 17. The open readingframe encodes 668 amino acids with resis and transferred to Hybond-N membranes (Amersham) an Mr of70,944. The first 23 amino acids show characteristics by standard methods. After hybridization, the filters were of a signal peptide of secretory or membrane-associated pro- washed and autoradiographed as described (23). teins and fit the -1, -3 rule (28) (Fig. 1). Inclusion ofa histidine Mouse Gene Mapping and Filter Hybridization. Southern in a signal peptide is unusual but not unknown (28). Thus, the blot analysis was used to type the DNAs from a panel of protein backbone ofprocessed Pmel 17 would be composed of Chinese hamster and mouse somatic cell hybrids (24) and 645 amino acids with an Mr of 68,600. from the progeny of the intersubspecies backcross [(NFS/N Five potential asparagine-linked glycosylation sites are or C58/J x Mus musculus musculus)Fj x M. m. musculus] located at amino acid positions 81, 106, 111, 321, and 568 as (19), and the interspecies backcross [(C57BL/6J x Mus indicated in Fig. 1 A and C. There is a stretch of 26 amino spretus)Fj x C57BL/6J] (25). DNAs were digested by re- acids that constitutes a hydrophobic domain toward the C striction endonucleases, electrophoresed on 0.4% agarose terminus of the protein (amino acids 598-623) (Fig. 1). This gels, transferred to nylon membranes, hybridized with 32p- hydrophobic region is bordered by charged residues at either labeled probes, and washed as described (24, 26). end (Glu597and Arg624-626), consistent with a transmembrane segment that makes a single helical span. One of the striking Recombination distances were calculated as described by features of the protein is the relatively high percentage of Green (27) using the computer program SPRETUS MADNESS serines (9.3%) and threonines (9.3%) and their peculiar developed by D. Dave (Data Management Services, Freder- arrangements. Although serine/threonine residues appear ick, MD) and A. M. Buchberg (National Cancer Institute- throughout the protein, we observed two sets of His-Ser- Frederick Cancer Research and Development Center, Fred- Ser-Ser (amino acids 202-205 and 639-642), one Gly-Ser-Ser erick, MD). Gene order was determined by minimizing the (amino acids 302-304), four sets of Asp(Asn, Glu)-Thr/Ser- number ofrecombination events required to explain the allele Thr/Ser (amino acids 264-266, 321-323, 370-372, and 425- distribution patterns. 427), six sets of Leu (Ile, Val, Ala)-Thr/Ser-Thr/Ser (amino A 1 MDLVLKRCLL_LAVIGALLAVGATKVPRNQ DWLGVSRQLR TKAWNRQLYP 51 EWTEAQRLDC WRGGQVSLKV SNDGPTLIGA NASFSIALNF PGSQKVLPDG 101 QVIWVNNTII NGSQVWGGQP VYPQETDDAC IFPDGGPCPS GSWSQKRSFV 151 YVWKTWGQYW QVLGGPVSGL SIGTGRAMLG THTMEVTVYH RRGSRSYVPL 201 AHSSSAFTIT DQVPFSVSVS QLRALDGGNK HFLRNQPLTF ALQLHDPSGY 251 LAEADLSYTW DFGDSSGTLI SRAPVVTHTY LEPGPVTAQV VLQAAIPLTS 301 CGSSPVPGTT DGHRITAEAP NTTAGQVPTT EVVGTTPGQA PTAEPSGTTS 351 VQVPTTEVIS TAPVQMPTAE STGMTPEKVP VSEVMGTTLA EMSTPEATGM 401 TPAEVSIVVL SGTTAAQVTT TEWVETTARE LPIPEPEGPD ASSIMSTESI 451 TGSLGPLLDG TATLRLVKRQ VPLDCVLYRY GSFSVTLDIV QGIESAEILQ 501 AVPSGEGDAF ELTVSCQGGL PKEACMEISS PGCQPPAQRL CQPVLPSPAC 551 QLVLHQILKG GSGTYCLNVS LADTNSLAVV STQLIMPVPG ILLTGQEAGL 601 GQVPLIVGIL LVLMAVVLAS LIYRRRLMKQ DFSVPQLPHS SSHWLRLPRI 651 FCSCPIGENS PLLSGQQV B 0 200 400 600 3- A A -I *A. * * A.A- - A . I .k. .Y . -3 d 0 ~~~~~~200400 600 C 1 2 3 456 7891011121314151617 1819 20 21.