STRUCTURAL ORGANIZATION OF THE HUMAN TYROSINASE GENE AND SEQUENCE-ANALYSIS AND CHARACTERIZATION OF ITS PROMOTER REGION

Tyrosinase is the principal enzyme in the biosynthesis of melanin. The expression of tyrosinase is tissue-specific and appears to be regulat ed by various hormonal and environmental factors. Elucidation of the g enomic structure and molecular basis of control of tyrosinase gene exp ression will greatly enhance our understanding of the regulation of hu man pigmentation. To this end, we have isolated and performed restrict ion mapping of recombinant cosmid and lambda phage clones containing t he human tyrosinase gene, sequenced a 2.2-kilobase (kb) region of its promoter, and determined the potential regions regulating the tyrosina se gene expression in transient-expression system. The human tyrosinas e gene is comprised of five exons and four introns. Based on our restr iction mapping studies, the gene spans a distance of over 65-kb on chr omosome 11 (q14 --> q21). We constructed a series of plasmids (pHTY-CA T) that contain 5' sequential deletions of the human tyrosinase 5' fla nking sequence fused to the reporter gene, chloramphenicol acetyltrans ferase (CAT). The plasmids were used to locate promoter regions that a re potential regulators of tyrosinase gene expression in a transient e xpression system using melanoma cell lines. In human melanoma cells, t he plasmid construct with a -2020 base pair (bp) promoter yielded the highest CAT activity. When the deletions reached -1739 bp, the CAT act ivity was dramatically reduced, indicating that important enhancer ele ments for transcription control are present between -1739 and -2020 bp . Further deletions up to -550 bp also resulted in dramatic decreases of CAT activity. However, when the deletion included -550 bp of the 5' flanking sequence, there was 26 percent of the CAT activity compared to that of the -2020 bp promoter. Deletions beyond -550 bp also showed markedly decreased CAT activity. Based on our data, we suggest that h uman tyrosinase gene expression is governed by both tissue-specific an d multiple regulatory elements.