The tyrosinase gene from medakafish: Transgenic expression rescues albino mutation

We have determined the 9.8 kb genomic nucleotide sequence of the tyrosinase gene and its 5' upstream region from a teleost, medakafish (Oryzias latipe s), and shown that the coding region is composed of five exons and four int rons, spanning 4.7 kb. While the number and sizes of the exons were found t o be similar to those of mammalian tyrosinase genes, however, the total siz e of the coding region (4.7 kb) was demonstrated to be less than one tenth those of mouse (ca. 70 kb) and human (> 70 kb) genes. Primer extension anal ysis revealed that the transcription initiation site starts with a long unt ranslated leader sequence (340 nucleotide long) from the AUG start codon. A characteristic CATGTG sequence known as a putative regulatory motif in mel anocyte-specific genes was present in the 131st base upstream from the init iation site, while other typical regulatory elements such as the TATA-box o r M-box common to terrestrial vertebrates were lacking. Transgenic experime nts were carried out by microinjecting two kinds of plasmid clones into fer tilized eggs of the albino i(1) mutant: one consisting of the genomic tyros inase gene with the 10 kb 5' upstream region and the other the tyrosinase c DNA with the 3 kb 5' upstream region. The results showed that 58 and 45 of 114 and 118 transgenic eggs, respectively, developed normally beyond hatchi ng and 15 and 10 exhibited a mosaic pattern of pigmentation. Despite the ab sence of typical regulatory elements like a TATA-box in both cases correct melanin pigmentation was obtained without ectopic expression. Thus, transge nic expression rescued from the albino-i(1) mutation, and the i locus of th e medaka genome can be concluded to encode the tyrosinase gene.