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Expression of tyrosinase gene introduced into the albino mouse embryo.

引入白化小鼠胚胎的酪氨酸酶基因的表达。

基本信息

批准号：
60480514
负责人：
TAKEUCHI Takuji
金额：
$ 0.64万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480514/
关键词：
mouse tyrosinase cDNA gene expression gene cloning melanin アルビノ

项目摘要

Tyrosinase is a copper containing monooxygenase and is known as the key enzyme for melanin biosynthesis in pigment cells. Deficiency in the enzme activity results in albinism.An attempt was made in the present sutdy to express mouse tyrosinase cDNAs in albino melanocytes. A cDNA library was constructed from poly(A)^+mRNA from mouse melanocytes and screened using anti-tyrosinase antibody and oligonucleotide probes whith corresponded to amino acid sequence of tyrosinase. The first cloned cDNA for tyrosinase was 1838 bp long (designated tyrs-33) and was found to encode a protein of 51 kDa. The second cdNA clone (Tyrs-J) was 1978 kbp and contained an open reading frame which encoded a protein of 60 kDa.On the other hand, 5' non-coding region was screened from a genomic DNA library derived from mouse embryo. A clone, G3L, was about 4.8 kbp long and was shown to contain the first exon (819 bp) and 5' non-coding sequence. In the putative regulatory sequence of the 5' non-coding region, two sets of promoter-like structure consisting of TATA box and CCAAT box. In order to examine the expressibility of these cDNAs and the function of the regulatory region, we fused the cDNAs with the 5' non-coding region and transfected into cultured melanocytes from albino mouse. One of the fused genes, 5' sequence-Tyrs-J, was found to express in the cells and to direct melanin production.

酪氨酸酶是一种含铜的单加氧酶，是色素细胞中黑色素生物合成的关键酶。本研究试图在白化病黑素细胞中表达小鼠酪氨酸酶cDNA。以小鼠黑素细胞poly（A）^+mRNA为模板构建cDNA文库，用抗酪氨酸酶抗体和与酪氨酸酶氨基酸序列相对应的寡核苷酸探针进行筛选。第一个克隆的酪氨酸酶cDNA长1838 bp（命名为tyrs-33），并被发现编码51 kDa的蛋白质。第二个cdNA克隆（Tyrs-J）为1978 kbp，含有一个开放阅读框，编码60 kDa的蛋白质。克隆G3 L长约4.8kbp，含有第一外显子（819 bp）和5'端非编码序列。在推测的5'端非编码区调控序列中，有两套启动子样结构，分别由TATA box和CCAAT box组成。为了检测这些cDNA的表达能力和调控区的功能，我们将它们与5'非编码区融合，并转染到培养的白化病小鼠黑素细胞中。其中一个融合基因，5'序列-Tyrs-J，被发现在细胞中表达并指导黑色素的产生。