Cells of the melanocyte lineage are distinguished by their capacity to synthesize the pigment melanin. Production of melanin is primarily regulated by the enzyme tyrosinase (monophenol, 3,4-dihydroxyphenylalanine : oxygen oxidoreductase, EC 1.14 .18 .1), and it is presumed that melanin synthesis can be regulated at a number of levels that control both the amount ofmelanin synthesized and the type of melanin produced. Melanin synthesis occurs principally in specialized organelles, the melanosomes . Thus, the synthesis of melanin is usually restricted to melanocytic cells that contain melanosomes.In this report, we describe isolation of a full-length cDNA clone encoding human tyrosinase by using a probe homologous to the Pmel 34 cDNA sequence described by Kwon et al . (1) . We have transfected and expressed this new human tyrosinase cDNA clone in mouse fibroblasts, and have induced pigmentation in a cell type that does not normally synthesize melanin. Levels of tyrosinase activity in transfected fibroblasts were equivalent to tyrosinase levels in highly pigmented human melanoma cell lines. These tyrosinase-positive fibroblast cell lines demonstrate that melanin synthesis can take place in cells that do not have melanosomes and, therefore, provide a tool for studying the regulation, transport, and processing of tyrosinase.Volume 169 June 1989169 June 2029169 June -2042 Materials and Methods Cell Culture and Cell Lines.Melanoma cell lines were established as previously described (2) . TK-L929 cells (mouse fibroblasts) (3) were used for transfection experiments . Cell lines were maintained in Eagle's MEM supplemented with 2 mM glutamine, 0.1 mM nonessential amino acids (aa)', 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% FCS (complete medium). Cells were passaged with trypsin (1 mg/ml) and EDTA (0.2 mg/ml) . All cultures were checked regularly for the presence ofmycoplasma and contaminated cultures were discarded .
EM.Cell pellets were fixed in Karnofsky's fixative overnight, rinsed in PBS for 1 h, and then post-fixed for 1 h in 1% osmium tetroxide-PBS solution . Cell pellets were dehydrated in graded ethyl alcohol followed by propylene oxide, and embedded in Maraglas-D.E.R. 732 epoxy resin (Dow Corning Corp., Midland, MI) . For orientation, 1-lAm thick sections were
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