Molecular Genetics of Human Albinism

1.以小鼠酪氨酸酶基因为探针，从人正常肝脏文库中克隆了一个含有5‘侧翼区和第一外显子的人酪氨酸酶基因。在转录起始点的5‘上游区域，存在一个可能的TATA盒和CAAT盒以及一些回文序列。应用酶DNA扩增和DNA直接测序技术检测了一例日本酪氨酸酶阴性白化患者的酪氨酸酶基因突变。通过直接测序可以检测到单碱基的变化。这种碱基改变(G到A)被认为是导致患者酪氨酸酶的氨基酸改变(Arg到GLN)。突变的等位基因也可以通过使用MspI的Souome印迹杂交来鉴定，因为这种变化破坏了MspI位点。我们将人-鼠融合微型基因HG-TYRS-J引入白化小鼠受精卵中，其中人基因组5‘非编码侧翼序列与小鼠酪氨酸酶基因融合。我们获得了两只有色小鼠，并建立了一些转基因品系。组织学分析表明，在转基因小鼠中，黑化仅限于毛球和眼睛中的黑素细胞。这些结果表明，该微型基因编码活性的酪氨酸酶蛋白，其5‘侧翼区含有表达的调控序列，并且在酪氨酸酶基因的调控方面，小鼠和人类之间存在共同或非常相似的机制。

1. A human tyrosinase gene containing a 5'-flanking region and the first exon was isolated from a normal human liver library using mouse tyrosinase cDNA as a probe for screening. In the 5'-upstream region of transcription start sites, there is a putative TATA box and CAAT box and some palindromic sequences.2. Enzymatic DNA amplification and direct DNA sequencing were used to detect a mutation in the tyrosinase gene of a Japanese patient with tyrosinase-negative albinism. Single-base change could be detected by direct sequencing. This base change (G to A) is thought to result in an amino acid change (Arg to Gln) in tyrosinase of the patient. The mutant allele can be also identified by Southem blot hybridization using MspI because the change destroys the MspI site.3. We introduced a human-mouse fused minigene, hg-tyrs-J, in which the human genomic 5' non-coding flanking sequence was fused to a mouse tyrosinase cDNA, into fertilized eggs of albino mice. We obtained two pigmented mice and established some transgenic lines. Histological analysis revealed that melanization was restricted to melanocytes in hair bulbs and eyes in transgenic mice. These results suggested that this minigene encodes active throsinase protein and that its 5'-flanking region contains the regulatory sequences for the expression and that there is a common or very similar mechanism between mice and humans in tyrosinase gene regulation.