Nishimura K, Liisanantti M, Muta Y, Kashiwagi K, Shirahata A, Jänne M, Kankare K, Jänne O A, Igarashi K
Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263, Japan.
Biochem J. 1998 Jun 15;332 ( Pt 3)(Pt 3):651-9. doi: 10.1042/bj3320651.
The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library. One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2). There was no sequence identity between the two promoters. The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters. After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter. Similar results were obtained by using stably transfected mouse FM3A cells. In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold. AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells. When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities. The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57. Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small. Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene.
从小鼠基因组文库中分离出两个S-腺苷甲硫氨酸脱羧酶基因（AMD基因）的启动子区域。一个启动子来自真正的小鼠AMD基因（AMD1），而另一个来自无内含子的AMD基因（AMD2）。这两个启动子之间没有序列同一性。AMD1启动子的序列与人AMD1和大鼠Amd1B启动子高度同源。在各种细胞系中进行瞬时转染后，AMD1启动子比AMD2启动子强1至2个数量级。使用稳定转染的小鼠FM3A细胞也得到了类似的结果。在过量产生S-腺苷甲硫氨酸脱羧酶（AdoMetDC）的SAM-1细胞中，AMD1基因扩增了5倍以上。与AMD1基因编码的蛋白质相比，无内含子的AMD2基因编码的AdoMetDC有两个氨基酸替换（第70位密码子的Met替换为Ile，第139位密码子的Ala替换为Val），并且在AdoMetDC缺陷的大肠杆菌细胞中表达时，其催化活性降低（<50%），加工活性也降低。当将AMD2编码的蛋白质的Ile-70转换为Met时，催化活性和加工活性均明显恢复，这表明与酶原加工位点相邻的Met-70对这两种活性都很重要。FM3A细胞中的第三个AMD位点（AMD3）包含一个假基因，其中两个碱基的缺失在第57位产生了一个提前终止密码子。由于AMD2启动子的强度仅为真正的AMD1基因的1-10%，且AMD2蛋白的比活性较低，因此AMD2编码的酶对总AdoMetDC活性的相对贡献较小。因此，小鼠细胞中的AdoMetDC活性被认为主要归因于AMD1基因的产物。
