Screening of a Xgtll human melanocyte cDNA library with antibodies against hamster tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were from related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidinerich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is a copper-based oxidoreductase that catalyzes the oxidation of tyrosine to dopa and the oxidation of dopa to dopaquinone (1). It is a key enzyme in melanin biosynthesis. Oculocutaneous albinism, a group of autosomal-recessive diseases in humans (2) and animals, is characterized by reduced or absent melanin in skin, hair, and eyes. Tyrosinase-negative albino melanocytes have no tyrosinase activity in vitro.Genetic control of pigmentation has been extensively studied in mice. There is evidence that the c-albino locus at chromosome 7 codes for the structural gene for tyrosinase (3,4). Mutations at this locus affect both tyrosinase activity and coat color (5, 6).A nucleic acid probe for tyrosinase would be an invaluable tool for studies of the regulation of tyrosinase, of the molecular basis of human albinism, and of various mouse mutations affecting coat and eye color. We report here the isolation and sequence of a cDNA clone for human tyrosinase that maps at or near the mouse c-locus. ¶ MATERIALS AND METHODS Cell Culture. Normal human melanocytes, melanotic melanoma cells (LG), and neuroblastoma cells (SK-N-SH) were cultured as described (7-9). The murine neuroblastoma cell line NIE115 was obtained from X. 0. Breakefield (E. K. Shriver Center, Waltham, MA). Proteins of normal melanocytes were radiolabeled with [355]methionine (Amersham) (100 ,XCi/ml, 1390 Ci/mmol; 1 Ci = 37 GBq) as described (10).cDNA Libraries and Screening. RNA from normal human melanocytes was prepared, and poly(A)+ RNA was purified on an oligo[d(T)I-cellulose column (11,12). A cDNA li was prepared employing a Xgtll cloning vector (13-15) Xgtll library contained 1.7 x 106 independent phages immunobiological screening and analysis of the fusion teins produced by Xgtll cDNA clones were carried c described by Young and Davis (15). The rabbit anti-hal tyrosinase antibodies and their application in the stun tyrosinases have been described in detail (10, 16).RNA Blot Hybridization. ...

Tyrosinase, the enzyme that controls the synthesis of melanin, is a unique product of melanocytes. Normal and malignant human melanocytes grown in culture were used to study the factors that regulate the expression of tyrosinase. Immunoprecipitation experiments showed that newly synthesized tyrosinase appeared as a protein with an apparent molecular weight of 70,000 that was processed to a protein with an apparent molecular weight of 80,000. Neither tunicamycin nor 2-deoxy-D-glucose inhibited this conversion, suggesting that Oglycosylation is the major biochemical event in the posttranslational modification of tyrosinase. Agents that stimulated the proliferation of normal melanocytes also stimulated tyrosinase activity. Melanocytes with low levels of tyrosinase activity synthesized less tyrosinase, processed the enzyme more slowly, and degraded it more rapidly than melanocytes with high levels of tyrosinase activity. We conclude that tyrosinase activity in cultures of human melanocytes derived from different donors is determined predominantly by its abundance.Epidermal melanin is synthesized by melanocytes via oxidation of tyrosine. The amount of melanin in the epidermis is determined, at least in part, by the activity of tyrosinase. For example, the activity of tyrosinase is higher in foreskins taken from black babies than in foreskins taken from caucasian babies (31); and melanocytes in patients with some forms of albinism have extremely low or no detectable tyrosinase activity (17). Knowledge of factors that regulate tyrosinase is important because melanin protects the skin from harmful solar radiation. In murine melanoma cells grown in vitro the activity of tyrosinase is at least partially regulated by the intracellular levels of cyclic adenosine monophosphate (cAMP). Exposure of these cells to substances that increase the intracellular levels of cAMP also causes an increase in tyrosinase activity and melanin content (1, 6-9, 22, 33, 38).Recently, a method for propagating normal human melanocytes in vitro (5) was developed. This achievement made it possible to study the control of tyrosinase activity in nonmalignant human melanocytes and to compare their responses with those of murine and human melanoma cells. Our experiments show that the activity of tyrosinase in cultured melanocytes is stimulated by agents that stimulate the growth of the cells and is determined by the rate of synthesis, processing, and degradation of the enzyme.

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