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Isolation and Sequence of a Edna Clone for Human Tyrosinase Tha Maps at the Mouse C-Albino Locus BYOUNG S

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Isolation and Sequence of a Edna Clone for Human Tyrosinase Tha Maps at the Mouse C-Albino Locus BYOUNG S Proc. Nall. Acad. Sci. USA Vol. 84, pp. 7473-7477, November 1987 Biochemistry Isolation and sequence of a eDNA clone for human tyrosinase tha maps at the mouse c-albino locus BYOUNG S. KWON*t, ASIFA K. HAQ*, SEYMOUR H. POMERANTZt, AND RUTH HALABAN§ *Molecular Genetics Laboratory, Guthrie Research Institute, Sayre, PA 18840; tDepartment of Biological Chemistry, University of Maryland School of Medicine, Baltimore, MD 21201; and §Department of Dermatology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Aaron B. Lerner, July 16, 1987 ABSTRACT Screening of a Xgtll human melanocyte on an oligo[d(T)I-cellulose column (11, 12). A cDNA li cDNA library with antibodies against hamster tyrosinase was prepared employing a Xgtll cloning vector (13-15) (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) Xgtll library contained 1.7 x 106 independent phages resulted in the isolation of 16 clones. The cDNA inserts from 13 immunobiological screening and analysis of the fusion of the 16 clones cross-hybridized with each other, indicating teins produced by Xgtll cDNA clones were carried c that they were from related mRNA species. One of the cDNA described by Young and Davis (15). The rabbit anti-hal clones, Pmel34, detected one mRNA species with an approxi- tyrosinase antibodies and their application in the stun mate length of 2.4 kilobases that was expressed preferentially tyrosinases have been described in detail (10, 16). in normal and malignant melanocytes but not in other cell RNA Blot Hybridization. Poly(A)+ RNA from n( types. The amino acid sequence deduced from the nucleotide human melanocytes, melanoma cells, neuroblastoma sequence showed that the putative human tyrosinase is com- lines, HL-60 (human promyelocytic leukemia cell line) posed of 548 amino acids with a molecular weight of 62,610. HepG2 (human hepatocarcinoma cell line) was fractiol The deduced protein contains glycosylation sites and histidine- on a 1.2% (wt/vol) formaldehyde denaturing gel (17), t rich sites that could be used for copper binding. Southern blot ferred to a GeneScreenPlus membrane (New England analysis of DNA derived from newborn mice carrying lethal clear), and hybridized with 32P-labeled cDNA pr( albino deletion mutations revealed that Pmel34 maps near or at Pmel14-2, a cDNA clone that was isolated from our hi the c-albino locus, the position of the structural gene for melanocyte cDNA library, was used as a control p tyrosinase. because the corresponding RNA was detectable in si5 amounts in all human and murine cells tested. Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, Genomic DNA Blot Analysis. Newborn mice of the g EC 1.14.18.1) is a copper-based oxidoreductase that cata- types C3H/C3H and cch/cH were provided by Sal lyzes the oxidation of tyrosine to dopa and the oxidation of Gluecksohn-Waelsch (Albert Einstein College of Medic dopa to dopaquinone (1). It is a key enzyme in melanin Newborn mice provided by M. Lynn Lamoreux (Texas A biosynthesis. Oculocutaneous albinism, a group of auto- somal-recessive diseases in humans (2) and animals, is University, College Station, TX) were littermates of characterized by reduced or absent melanin in skin, hair, and genotypes C14COs/C14Cos cCh/c14CWs, and cch/cch. These eyes. Tyrosinase-negative albino melanocytes have no were descended from three mice of the genotype C6H/C tyrosinase activity in vitro. that were given to M. L. Lamoreux by S. Gluecks Genetic control of pigmentation has been extensively Waelsch. These stocks were crossed twice onto JU/Ct/ studied in mice. There is evidence that the c-albino locus at +c/+c mice, then once onto C57BL/6J mice, then for e chromosome 7 codes for the structural gene for tyrosinase (3, generations onto a C57BL/6J-cch/cch stock descended I 4). Mutations at this locus affect both tyrosinase activity and mice provided by D. Townsend (University of Minnes coat color (5, 6). Minneapolis), and then sibling mated for six generation A nucleic acid probe for tyrosinase would be an invaluable High molecular weight DNAs of newborn mice of var tool for studies of the regulation of tyrosinase, of the genotypes were prepared as described (18). Restric molecular basis of human albinism, and of various mouse endonuclease digests ofDNA were electrophoresed in a 0 mutations affecting coat and eye color. We report here the agarose gel at 40C, transferred (19) to GeneScreenPlus, isolation and sequence ofa cDNA clone for human tyrosinase hybridized with the 32P-labeled cDNA probes. Pmell7- that maps at or near the mouse c-locus.¶ cDNA clone that was isolated from our human melanoi cDNA library, was utilized as a control probe for the purr MATERIALS AND METHODS of estimating the amounts of DNA in each lane bect Pmell7-1 detected a single EcoRI fragment in all mouse D Cell Culture. Normal human melanocytes, melanotic mel- tested. The intensity of the hybridizing bands was meast anoma cells (LG), and neuroblastoma cells (SK-N-SH) were by a densitometer at 500 nm (Beckman DU-8BUV/ cultured as described (7-9). The murine neuroblastoma cell Spectrophotometer). line NIE115 was obtained from X. 0. Breakefield (E. K. DNA Sequencing. DNA restriction fragments subclone Shriver Center, Waltham, MA). Proteins of normal melano- M13 vectors (20) were sequenced by the dideoxy ch cytes were radiolabeled with [355]methionine (Amersham) termination technique (21), with modifications made to (100 ,XCi/ml, 1390 Ci/mmol; 1 Ci = 37 GBq) as described commodate 2'-deoxyadenosine 5'-[a-[35S]thio]triphospt (10). (22). A forward primer (New England Biolabs) compleir cDNA Libraries and Screening. RNA from normal human tary to the lacZ sequence adjacent to the 5'-side of the Ec melanocytes was prepared, and poly(A)+ RNA was purified tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge 9This sequence is being deposited in the EMBL/GenBank data t payment. This article must therefore be hereby marked "advertisement" (Bolt, Beranek, and Newman Laboratories, Cambridge, MA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03581). Downloaded by guest on October 5, 2021 7474 Biochemistry: Kwon et al. Proc. Natl. Acad. Sci. USA 84 (1987) site in Xgtll was used for the direct sequencing of cDNA of the cDNA clones. A lysogen of Xmell6 was prepared, and insert end points in Xgtll (23). the bacterial lysate was analyzed by 6% NaDodSO4/poly- acrylamide gel electrophoresis and by competitive immuno- RESULTS precipitation assay. Xmell6 was used for this experiment because the Xmell6 plaques produced the strongest signal in Initial screening of 500,000 recombinant phage plaques with plaque screening with anti-tyrosinase antibodies even though the rabbit anti-tyrosinase antibodies identified 16 indepen- Xmel6 contained an incomplete cDNA of -0.7 kilobase dent clones. The cDNA inserts varied in size from 0.2 to 1.6 pairs. A fusion protein was produced in Y1089/Xmell6 that kilobase pairs. The longest cDNA insert (-1.6 kilobase pairs) had a relative size of 140 kDa (Fig. lb). The fusion protein from Xme134 hybridized to 12 other cDNA inserts and shared was 25 kDa larger than Escherichia coli 8-galactosidase, an overlapping restriction enzyme pattern (Fig. la). The indicating that the incomplete cDNA in Xmell6 was fused to other three cDNA inserts (not included in the figure) were not .3-galactosidase gene in frame. Synthesis of both /3-galacto- related to the 13 cDNA clones. A cDNA insert contained in sidase and the fusion protein was dependent on induction one of these, Xmell7-1, was utilized as a control probe. The with isopropyl 13-D-thiogalactopyranoside. The production of cDNA inserts of Xmell6, Xmel34, Xmel4O, and Xmell7-1 were immunologically reactive tyrosinase protein in the lysate of subcloned into pBR322 to yield Pmell6, Pmel34, Pmel4O, and Xmell6 lysogen was tested by competition assay using Pmell7-1, respectively, and used for further characterization metabolically labeled melanocyte cell extracts as a source for tyrosinase. Bacterial lysates of the Xmell6 lysogen (2 x 107 a 0 200 400 600 800 1000 1200 1400 1600 180 2000 2200 2400 bacterial cells in 100 pil) competed with roughly 70% of the anti-tyrosinase antibodies as judged from the intensity of the tyrosinase bands and the radioactivity in the relevant gel slices (Fig. lc). A"34 To examine whether mRNA homologous to Pmel34 is ex- A me 33 Xmel44 pressed preferentially in melanocytes, RNA gel blot analysis of A me 16 poly(A)+ mRNA from cells ofmelanocytic and nonmelanocytic Xmel 4 lineage was performed. Pmel34 hybridized to 21S (=2.4 A mM 52 Xmel 16 kilobase) mRNA species from normal human melanocytes and me 106 human melanotic melanoma cells (LG) but not from HepG2, mel? HL-60, human or murine neuroblastoma (Fig. 2), human and A me 20 mouse fibroblasts, or lymphocytes (data not shown). The skin of mice carrying the radiation-induced albino b C alleles, such as c3Hlc3H, had no tyrosinase activity (4). The Mr tyrosinase activity levels in the skin of mice heterozygous 1 2 3 4 IKdI 1 2 between the lethal albino deletion and chinchilla were shown Mr IKdl to be intermediate between the normal and mutant homo- zygotes, consistent with the murine albino locus encoding the structural gene of tyrosinase (unpublished observation of S. 200 - Gluecksohn-Waelsch, reviewed in ref. 4). If the c-albino 94- locus codes for the structural gene for tyrosinase, tyrosinase 116 - cDNA should not detect any hybridizing band in DNA 67-b4 - 66.2 - extracted from c3H/c3H or c14Cos/c14Cos mice but should detect 40 -- hybridizing bands of normal and half intensity in DNA from 43- homozygote (cch/cch) and heterozygotes (cch/c3H and cch/ c)4Cos) respectively.
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