Analysis of the tyrosinase gene of the Japanese pond frog, Rana nigromaculata: cloning and nucleotide sequence of the genomic DNA containing the tyrosinase gene and its flanking regions.

Three genomic DNA fragments containing the tyrosinase-encoding gene (TYR) of the Japanese pond frog, Rana nigromaculata, were cloned. The first, clone I, was isolated from a genomic library of sperm DNA using the mouse TYR cDNA as the probe and contained a DNA segment similar to exon 4 of the mouse TYR gene. Subsequently, the TYR cDNA was isolated by screening a frog embryo cDNA library using clone I as the probe. Two clones that contain genomic DNA of the TYR gene were isolated also from a blood cell DNA library using the frog TYR cDNA as the probe. Comparison of the nucleotide (nt) sequences of the genomic clone II DNA and the cDNA revealed that clone II contained a 3,140-bp DNA fragment consisting of the 5'-flanking region, the first exon, and a part of the first intron. The region upstream of the coding region contained the characteristic sequences for regulatory elements, including TATA- and CAAT-motifs, and also a pigment cell-specific promoter element, which is shared by the promoter regions of the vertebrate TYR genes. A 764-bp segment containing an upstream 748-bp non-coding region and 16-bp coding region was functional for expression of the promoter-less cat gene on a plasmid in the transiently transformed albino frog melanophore. The genomic clone III contained the 3'-untranslated region of the mRNA and its 3'-flanking region. Thus, the cDNA plus genomic DNA fragments isolated here cover the entire TYR gene and its flanking regions.