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Regulatory expression of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle

参与苹果酸-天冬氨酸穿梭的线粒体和胞质同工酶基因的调节表达

基本信息

批准号：
01480149
负责人：
SHIMADA Kazunori
金额：
$ 4.16万
依托单位：
Osaka University (1990-1991)Kumamoto University (1989)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

The malate-aspartate shuttle, consisting of mitochondrial and cytosolic aspartate aminotransferase (mAsPAT & cAsPAT) and mitochondrial and cytosolic malate dehydrogenase (mMdh & cMDH), is a major pathway for the transport of reducing eqivalents. from cytosol to mitochondria in mammals. To elucidate molecular mechanisms regulating metabolic coordination between the nitochondria and the cytosol, we analyzed the genomic structures of the complete set of mouse isoenzyme genes playing pivotal role in the shuttle.1. The genomic DNA structures showed remarkable conservation of intron positions not only between mAsPAT and cAspAT genes, but also between MMDH and CMDH genes, findings which can be interpreted to mean that the introns were in place before the divergence of cytosolic and mitochondrial isoenzyme genes. We also compared their 5'-flanking regions and found that several highly homologous regions are present between the mouse mAsPAT and cAspAT, mAspAT and MMDH, and cAspAT and CMDH genes … More .2. Deletion analysis and an in vivo transfection assay, using NIH3T3 cells, revealed that all the promoter regions are located within the 300-base pair regions upstream from the initiation codon DNase I footprinting analyses using NlH3T3 cell nuclear extracts led to identification of several protein binding sites within these regions.3. A hynthetic oligomer containing the consensus binding site sequence for CTF/NFI, a transcription factor for RNA polymerase II, competed for the binding of proteins to the promoter regions of cAspAT, MMDH and cmDH genes, but not for that of the mAspAT gene. A synthetic oligoner containing the consensus binding site sequence for Spl, which activates transcription from promoters containing properly positioned GC boxes, competed for protein (s) binding to the promoter region of the mAspAT gene.4. Structural organization of the human cAsPAT gene was also determined by analyzing the phage clones obtained from two kinds of genomic DNA libraries, using mouse cAspAT CDNA as a probe. The gene is more t)ian 32 kb long and is split into 9 exons by 8 introns of various sizes. The 5' and 3'-flanking regions and the exact sizes and boundaries of tlie exon blocks were determined. The S' end of the gene lacks tlie TATA and CAAT boxes, but contains G+C i-ich sequences and one potential binding site for the transcription factor, Spl. Comparison of the nucleotide sequence of 250 bp upstream from the translation-initiation site revealed that the sequences of binding sites for the nuclear proteins, identified in the mouse, are highly conserved between human and nouse cAspAT genes. Less

苹果酸-天冬氨酸穿梭由线粒体和细胞质内的天冬氨酸转氨酶（mAsPAT & cAsPAT）以及线粒体和细胞质内的苹果酸脱氢酶（mMdh & cMDH）组成，是还原等价物运输的主要途径。从哺乳动物的细胞质到线粒体。为了阐明调节线粒体和细胞质间代谢协调的分子机制，我们分析了在线粒体和细胞质间代谢协调中起关键作用的整套小鼠同工酶基因的基因组结构。基因组DNA结构不仅在mAsPAT和cAspAT基因之间，而且在MMDH和CMDH基因之间都显示出显著的内含子位置守恒，这一发现可以解释为内含子在细胞质和线粒体同工酶基因分化之前就存在了。我们还比较了它们的5'-侧翼区域，发现在小鼠mAsPAT和cAspAT、mAsPAT和MMDH以及cAspAT和CMDH基因之间存在几个高度同源的区域。使用NIH3T3细胞进行的缺失分析和体内转染实验显示，所有启动子区域都位于起始密码子DNase I上游的300个碱基对区域内。使用NlH3T3细胞核提取物进行的足迹分析鉴定了这些区域内的几个蛋白质结合位点。含有CTF/NFI （RNA聚合酶II的转录因子）的一致结合位点序列的人工低聚物竞争cAspAT、MMDH和cmDH基因启动子区域的蛋白质结合，但不竞争mAspAT基因的启动子区域。含有Spl的一致结合位点序列的合成寡聚物，可以激活含有正确定位的GC盒的启动子的转录，竞争与mAspAT基因启动子区域结合的蛋白质。以小鼠cAsPAT CDNA为探针，通过分析从两种基因组DNA文库中获得的噬菌体克隆，确定了人类cAsPAT基因的结构组织。该基因长度超过32 kb，由8个不同大小的内含子分成9个外显子。确定了5‘和3’侧翼区域以及外显子块的确切大小和边界。该基因的S端缺乏TATA和CAAT盒子，但含有富含G+C i的序列和转录因子Spl的一个潜在结合位点。比较翻译起始位点上游250bp的核苷酸序列发现，在小鼠中鉴定的核蛋白结合位点序列在人和小鼠cAspAT基因之间高度保守。少