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

由线粒体和胞质天冬氨酸氨基转移酶（mAsPAT和cAsPAT）以及线粒体和胞质苹果酸脱氢酶（mMdh和cMDH）组成的苹果酸-天冬氨酸穿梭是还原性物质转运的主要途径。从细胞质到线粒体。为了阐明调节线粒体和胞质之间代谢协调的分子机制，我们分析了在穿梭中起关键作用的小鼠同工酶基因的完整基因组结构.基因组DNA结构显示，不仅mAsPAT和cAspAT基因之间，而且MMDH和CMDH基因之间的内含子位置显着的保守性，这可以解释为这意味着内含子的位置之前，细胞溶质和线粒体同工酶基因的分歧。我们还比较了它们的5 ′-侧翼区，发现小鼠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 关于我们 .2.使用NIH 3 T3细胞进行的缺失分析和体内转染测定揭示了所有启动子区域均位于起始密码子DNA酶I上游的300个碱基对区域内，使用NIH 3 T3细胞核提取物进行的足迹分析导致在这些区域内鉴定出几个蛋白质结合位点。含有CTF/NFI（RNA聚合酶II的转录因子）共有结合位点序列的合成寡聚体竞争蛋白质与cAspAT、MMDH和cmDH基因启动子区的结合，但不竞争mAspAT基因的结合。含有Spl的共有结合位点序列的合成寡聚体（其激活从含有适当定位的GC盒的启动子的转录）竞争与mAspAT基因的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，也确定了人cAsPAT基因的结构组织。该基因长约32 kb，由8个大小不等的内含子组成，分为9个外显子。确定了外显子块的5'和3'侧翼区以及确切的大小和边界。该基因的S'端缺少TATA和CAAT盒，但含有G+C i-ich序列和一个转录因子Spl的潜在结合位点。比较的250 bp的核苷酸序列上游的抑制起始位点显示，在小鼠中确定的核蛋白的结合位点的序列，是高度保守的人和鼻cAspAT基因之间。少

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