Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene
Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene
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DOI：
10.1074/jbc.272.3.1842
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发表时间：
1997-01-17
影响因子：
4.8
通讯作者：
Shane, B
中科院分区：
文献类型：
--
作者：
Stover, PJ;Chen, LH;Shane, B
人线粒体丝氨酸羟甲基转移酶（mSHMT）基因的分离，测序，并进行了表征。该基因含有10个内含子和11个外显子，所有的剪接点符合GT/AG规则。5'启动子区含有几种调控蛋白的共有基序，包括PEA-3、Sp-1、AP-2和肝脏中表达的许多基因共有的CCCTCCC基序。不存在共有TATA或CAAT序列基序，引物延伸和cDNA末端的5 '-快速扩增研究表明转录起始发生在多个位点。推导的mRNA的线粒体前导序列区域包含两个潜在的ATG起始位点，其由单独的外显子编码。插入的891个碱基对的内含子含有一致的启动子元件，这表明mSHMT可以从交替启动子转录。5 '-快速扩增cDNA末端分析表明，第一个ATG在人MCF-7细胞中转录。然而，用缺乏外显子1的人mSHMT基因转染缺乏mSHMT活性的中国仓鼠卵巢细胞克服了细胞的甘氨酸营养缺陷型，并将细胞内甘氨酸浓度恢复到野生型细胞中观察到的水平，表明外显子1对mSHMT定位或活性不是必需的，并且从第二个ATG开始的翻译足以使mSHMT输入线粒体。MCF-7细胞中的线粒体SHMT mRNA水平在细胞周期中没有变化，并且不受培养基中缺乏甘氨酸、丝氨酸、叶酸、胸苷酸或嘌呤的影响。
The human mitochondrial serine hydroxymethyltransferase (mSHMT) gene was isolated, sequenced, and characterized. The 4.5-kilobase gene contains 10 introns and 11 exons, with all splice junctions conforming to the GT/AG rule. The 5' promoter region contains consensus motifs for several regulatory proteins including PEA-3, Sp-1, AP-2, and a CCCTCCC motif common to many genes expressed in liver. Consensus TATA or CAAT sequence motifs are not present, and primer extension and 5'-rapid amplification of cDNA ends studies suggest that transcription initiation occurs at multiple sites. The mitochondrial leader sequence region of the deduced mRNA contains two potential ATG start sites, which are encoded by separate exons. The intervening 891-base pair intron contains consensus promoter elements suggesting that mSHMT may be transcribed from alternate promoters. 5'-Rapid amplification of cDNA ends analysis demonstrated that the first ATG is transcribed in human MCF-7 cells. However, transfection of Chinese hamster ovary cells deficient in mSHMT activity with the human mSHMT gene lacking exon 1 overcame the cell's glycine auxotrophy and restored intra cellular glycine concentrations to that observed in wildtype cells, showing that exon 1 is not essential for mSHMT localization or activity and that translation initiation from the second ATG is sufficient for mSHMT import into the mitochondria. Mitochondrial SHMT mRNA levels in MCF-7 cells did not vary during the cell cycle and were not affected by the absence of glycine, serine, folate, thymidylate, or purines from the media.