Functional domains of chicken mitochondrial transcription factor a for the maintenance of mitochondrial DNA copy number in lymphoma cell line DT40
Functional domains of chicken mitochondrial transcription factor a for the maintenance of mitochondrial DNA copy number in lymphoma cell line DT40
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DOI：
10.1074/jbc.m303842200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Kitagawa, Y
中科院分区：
文献类型：
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作者：
Matsushima, Y;Matsumura, K;Kitagawa, Y
克隆了鸡线粒体转录因子A(c-TFAM)的核型和线粒体型基因(c-tfam)。位于6q1.1-q1.2的c-tfam具有与小鼠tfam相似的外显子/内含子结构，只是编码核和mt形式特异序列的第一个外显子的位置相反。用c-Myc标记c-Myc基因在鸡淋巴瘤DT40细胞中瞬时表达核型时，产物定位于细胞核内，而DT40细胞中唯一的内源性mt形式仅在线粒体内免疫染色。C-TFAM与非洲爪哇(XL-)TFAM最相似之处在于，除了两个高迁移率基团(HMG)盒外，还有扩展的C-末端区域，它们之间有一个连接区，以及一个C-末端尾巴，这也在人和鼠TFAM中发现。C-和xl-TFAM在连接区和C-末端的相似性高于HMG盒。DT40细胞中两个tfam等位基因的破坏阻止了细胞的增殖。Tfam(+)/tfam(-)细胞mtDNA及其转录本分别减少50%和40%~60%。外源野生型c-tfam基因在tfam(+)/tfam(-)细胞中的表达以剂量依赖的方式使mtDNA增加4倍，而其转录水平仅略有增加。缺少第一个HMG盒的缺失突变体失去了该活性，而在第二个HMG盒的缺失突变体中仅观察到该活性的边缘降低。尽管C末端在线粒体DNA体外转录中起着重要的作用，但c-TFAM在这一区域的缺失仅使维持线粒体DNA水平的活性降低了50%。尾部的一系列缺失突变提示该区域存在维持线粒体DNA水平的刺激和抑制序列。
Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam(+)/tfam(-) cells showed a 50 and 40-60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c- tfam cDNA in the tfam(+)/tfam(-) cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level.