Human poly(ADP-ribose) glycohydrolase (PARG) gene and the common promoter sequence it shares with inner mitochondrial membrane translocase 23 (TIM23)
Human poly(ADP-ribose) glycohydrolase (PARG) gene and the common promoter sequence it shares with inner mitochondrial membrane translocase 23 (TIM23)
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DOI：
10.1016/s0378-1119(03)00738-8
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发表时间：
2003-09-18
期刊：
影响因子：
3.5
通讯作者：
Jacobson, MK
中科院分区：
文献类型：
--
作者：
Meyer, RG;Meyer-Ficca, ML;Jacobson, MK
聚（ADP-核糖基）化是由聚（ADP-核糖）聚合酶（PARP）家族成员介导的翻译后蛋白质修饰。 ADP-核糖聚合物由多种 PARP 酶通过 NAD(+) 裂解合成，参与多种细胞功能的调节。目前，只有一种酶，聚（ADP-核糖）糖水解酶（PARG），已被确定可以催化细胞中的 ADP-核糖聚合物水解，导致生物聚合物快速周转，最终可能导致细胞 NAD（除以）库的致命消耗。在这项研究中，我们描述了通过逆转录 CF3 人成纤维细胞 RNA 构建第一个人 PARG cDNA 克隆。使用 NCBI“Genome BLAST”程序，将人类 PARG 基因定位到 10 号染色体 (10q11.23)，与早期通过原位杂交获得的结果一致。 cDNA 的体外偶联转录和翻译产生了 111-85 kDa 范围内的几个特异性条带，表明可能使用替代翻译起始位点。通过进一步详细的计算分析来表征基因结构。开放阅读框由 18 个外显子和 17 个内含子组成，其中外显子 9 至 14 形成酶的催化中心，外显子 1 至 3 编码假定的调节域。我们发现人类 PARG 基因与线粒体内膜转位酶 23 (TIM23) 共享一个 470 bp 的共同启动子区域。克隆了人双向启动子区域，并使用 EGFP-荧光素酶报告融合基因 (GFL) 在瞬时转染的 HEK293 细胞中进行表达研究，以量化两个方向的转录激活。发现 TIM23 的启动子活性比 PARG 高 3.7 倍，表明这两个基因以不同的水平表达，尽管这两个基因的共调节仍然是一个有趣的可能性。 (C) 2003 Elsevier B.V. 保留所有权利。
Poly(ADP-ribosyl)ation is a posttranslational protein modification mediated by members of the poly(ADP-ribose) polymerase (PARP) family. The ADP-ribose polymers, synthesized by the diverse PARP enzymes by cleavage of NAD(+), are involved in the regulation of multiple cellular functions. At present, only a single enzyme, poly (ADP-ribose) glycohydrolase (PARG), has been identified to catalyze ADP-ribose polymer hydrolysis in the cell causing a rapid turnover of the biopolymer which may ultimately result in lethal depletion of cellular NAD(divided by) pools. In this study, we describe the construction of the first human PARG cDNA clone by reverse transcription of CF3 human fibroblast RNA. Using the NCBI "Genome BLAST" program, the human PARG gene was mapped to chromosome 10 (10q11.23) in agreement to earlier results obtained by in situ hybridization. In vitro coupled transcription and translation of the cDNA yielded several specific bands in the range of 111-85 kDa, indicating possible usage of alternative translation initiation sites. The gene structure was characterized by further detailed computational analyses. The open reading frame consists of 18 exons and 17 introns with exons 9 to 14 forming the catalytic center of the enzyme and exons 1 to 3 encoding the putative regulatory domain. We show that the human PARG gene shares a 470-bp common promoter region with the inner mitochondrial membrane translocase 23 (TIM23). The human bidirectional promoter region was cloned and expression studies in transiently transfected HEK293 cells was performed using an EGFP-luciferase reporter fusion gene (GFL) to quantify transcription activation in both directions. The activity of the promoter was found to be 3.7 fold higher for TIM23 than for PARG, indicating that the two genes are expressed at different levels, although coregulation of the two genes remains an interesting possibility. (C) 2003 Elsevier B.V. All rights reserved.