|Budget Amount *help
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1989: ¥4,200,000 (Direct Cost: ¥4,200,000)
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
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