Fernando L P, Andrews G K
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City.
Gene. 1989 Sep 1;81(1):177-83. doi: 10.1016/0378-1119(89)90349-1.
The chicken metallothionein gene (cMT), isolated from a chicken genomic DNA phage lambda library, was found to be approximately 1.5 kb in length and to consist of three exons, separated by two intervening sequences. The number and placement of the introns in the cMT gene is precisely the same as that in the mammalian metallothionein-coding genes. S1 nuclease mapping indicated a prominent transcription start point (tsp) 62 bp 5' to the translation start codon. The promoter region analyzed (623 bp) contained three regions of homology (at -47, -488, and -577 bp relative to the tsp) with the metal regulatory element (MRE) consensus sequence, and three potential Sp1 binding sites. Two of the putative MREs (-47, -577) were 12 to 14-bp palindromes, which suggests that they are binding sites for trans-acting proteins. The intact cMT gene was functional in mammalian cells, and the cMT promoter could confer metal responsiveness on the firefly luciferase cDNA (Luc) in transient expression assays. Deletion mutagenesis established that 107 bp of 5'-flanking sequence, containing the proximal MRE and a putative Sp1-binding site, were sufficient for transient expression and metal induction of the cMT promoter-Luc fusion gene.
从鸡基因组DNA噬菌体λ文库中分离出的鸡金属硫蛋白基因（cMT），长度约为1.5 kb，由三个外显子组成，中间被两个间隔序列隔开。cMT基因中内含子的数量和位置与哺乳动物金属硫蛋白编码基因中的完全相同。S1核酸酶图谱显示，在翻译起始密码子5'端62 bp处有一个突出的转录起始点（tsp）。分析的启动子区域（623 bp）包含三个与金属调节元件（MRE）共有序列同源的区域（相对于tsp分别在-47、-488和-577 bp处），以及三个潜在的Sp1结合位点。两个假定的MRE（-47、-577）是12至14 bp的回文序列，这表明它们是反式作用蛋白的结合位点。完整的cMT基因在哺乳动物细胞中具有功能，并且在瞬时表达测定中，cMT启动子可以赋予萤火虫荧光素酶cDNA（Luc）金属反应性。缺失诱变表明，包含近端MRE和一个假定的Sp1结合位点的107 bp 5'侧翼序列足以实现cMT启动子-Luc融合基因的瞬时表达和金属诱导。
