The tyrosinase gene from medakafish: transgenic expression rescues albino mutation.
The tyrosinase gene from medakafish: transgenic expression rescues albino mutation.
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DOI：
10.1111/j.1600-0749.1998.tb00737.x
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发表时间：
1998-10
期刊：
影响因子：
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通讯作者：
H. Inagaki;A. Koga;Y. Bessho;H. Hori
中科院分区：
文献类型：
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作者：
H. Inagaki;A. Koga;Y. Bessho;H. Hori
测定了水稻酪氨酸酶基因及其上游的9.8kb的核苷酸序列，结果表明该基因的编码区由5个外显子和4个内含子组成，全长4.7kb。虽然外显子的数量和大小与哺乳动物的酪氨酸酶基因相似，但编码区的总长度(4.7kb)不到老鼠(约70kb)和人类(约70kb)基因的十分之一。引物延伸分析表明，转录起始点从Aug起始密码子的一个长的未翻译的前导序列(340个核苷酸长)开始。在起始点上游的第131个碱基上存在一个特征的CATGTG序列，称为黑素细胞特异性基因的假定调控基序，而其他典型的调控元件如陆生脊椎动物常见的TATA-box或M-box是缺失的。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：一种含有酪氨酸酶基因，上游10kb5；另一种含有酪氨酸酶基因，上游3kb5。结果表明，在114枚和118枚转基因卵中，分别有53枚和45枚在孵化后发育正常，15枚和10枚呈现马赛克花纹。尽管在这两种情况下都没有像TATA-box这样的典型调控元件，但获得了正确的黑色素沉着，没有异位表达。因此，从白化-I(L)突变中挽救的转基因表达，以及青冈基因组的I基因座，可以推断为编码酪氨酸酶基因。
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.