Inagaki H, Koga A, Bessho Y, Hori H
Division of Biological Sciences, Graduate School of Science, Nagoya University, Japan.
Pigment Cell Res. 1998 Oct;11(5):283-90. doi: 10.1111/j.1600-0749.1998.tb00737.x.
We have determined the 9.8 kb genomic nucleotide sequence of the tyrosinase gene and its 5 upstream region from a teleost, medakafish (Oryzias latipes), and shown that the coding region is composed of five exons and four introns, spanning 4.7 kb. While the number and sizes of the exons were found to be similar to those of mammalian tyrosinase genes, however, the total size 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 analysis revealed that the transcription initiation site starts with a long untranslated leader sequence (340 nucleotide long) from the AUG start codon. A characteristic CATGTG sequence known as a putative regulatory motif in melanocyte-specific genes was present in the 131st base upstream from the initiation site, while other typical regulatory elements such as the TATA-box or M-box common to terrestrial vertebrates were lacking. Transgenic experiments were carried out by microinjecting two kinds of plasmid clones into fertilized eggs of the albino i(l) mutant: one consisting of the genomic tyrosinase gene with the 10 kb 5 upstream region and the other the tyrosinase cDNA with the 3 kb 5 upstream region. The results showed that 53 and 45 of 114 and 118 transgenic eggs, respectively, developed normally beyond hatching and 15 and 10 exhibited a mosaic pattern of pigmentation. Despite the absence of typical regulatory elements like a TATA-box in both cases correct melanin pigmentation was obtained without ectopic expression. Thus, transgenic expression rescued from the albino-i(l) mutation, and the i locus of the medaka genome can be concluded to encode the tyrosinase gene.
我们已经确定了硬骨鱼青鳉（Oryzias latipes）酪氨酸酶基因及其5'上游区域的9.8 kb基因组核苷酸序列，并表明编码区由5个外显子和4个内含子组成，跨度为4.7 kb。虽然发现外显子的数量和大小与哺乳动物酪氨酸酶基因相似，但是，编码区的总大小（4.7 kb）被证明小于小鼠（约70 kb）和人类（> 70 kb）基因的十分之一。引物延伸分析表明，转录起始位点从AUG起始密码子开始有一个长的非翻译前导序列（340个核苷酸长）。在起始位点上游第131个碱基处存在一个称为黑素细胞特异性基因推定调控基序的特征性CATGTG序列，而缺乏陆地脊椎动物共有的其他典型调控元件，如TATA盒或M盒。通过将两种质粒克隆显微注射到白化i(l)突变体的受精卵中进行转基因实验：一种由具有10 kb 5'上游区域的基因组酪氨酸酶基因组成，另一种由具有3 kb 5'上游区域的酪氨酸酶cDNA组成。结果表明，114个和118个转基因卵分别有53个和45个在孵化后正常发育，15个和10个表现出色素沉着的镶嵌模式。尽管在这两种情况下都没有像TATA盒这样的典型调控元件，但仍获得了正确的黑色素沉着且没有异位表达。因此，从白化i(l)突变体中拯救了转基因表达，可以得出结论，青鳉基因组的i位点编码酪氨酸酶基因。
