Inagaki H, Bessho Y, Koga A, Hori H
Department of Biology, Faculty of Science, Nagoya University, Japan.
Gene. 1994 Dec 15;150(2):319-24. doi: 10.1016/0378-1119(94)90445-6.
In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.
在青鳉鱼（Oryzias latipes）中，已分离出许多体色突变体。其中，一种无色黑素细胞突变体b，纯合携带b等位基因，有黑色的眼睛，但皮肤呈橙色，黑素细胞无黑色素，这表明存在一种组织特异性的黑色素形成机制。为了阐明该机制的分子基础，我们从野生型（wt）鱼中克隆了酪氨酸酶（Tyr）的cDNA，酪氨酸酶是黑色素生物合成中的关键酶。DNA序列分析表明，所有克隆编码一个由540个氨基酸组成的蛋白质，具有五个潜在的糖基化位点和两个铜结合位点，这是酪氨酸酶的特征。基因组DNA印迹分析表明，Tyr基因在鱼类基因组中以单拷贝形式存在。以cDNA克隆为探针进行RNA印迹分析。在野生型中，2.2-kb的Tyr mRNA在眼睛和皮肤中表达，但在肝脏中不表达，这与组织特异性黑色素形成相对应。在b突变体中，与预期相反，不仅在眼睛中检测到了mRNA，在无黑色素的皮肤中也检测到了。因此，由b控制的皮肤色素沉着与Tyr基因的表达没有直接关系。
