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1991 Fiscal Year Final Research Report Summary

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

Research Project

Project/Area Number 01480149
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionOsaka University (1990-1991)
Kumamoto University (1989)

Principal Investigator

SHIMADA Kazunori  Osaka Univ. Res. Inst. for Microb. Dis. Professor, 微生物病研究所, 教授 (40037354)

Co-Investigator(Kenkyū-buntansha) NOMIYAMA Hisayuki  Kumamoto Univ. Dept. of Biochemistry Assist. Prof., 医学部, 講師 (00156225)
TAKIHARA Yoshihiro  Osaka Univ. Res. Inst. Microb. Dis. Instructor, 微生物病研究所, 助手 (60226967)
SETOYAMA Chiaki  Kumamoto Univ. Dept. of Biochemistry Assist. Prof., 医学部, 講師 (60040250)
Project Period (FY) 1989 – 1991
KeywordsMalate-aspartate Shuttle / Isoenzyme / cAspAT / mAspAT / cMDH / mMDH / Gene Expression
Research Abstract

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

  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] Nagashima,F.: "cDNA cloning and expression of pig cytosolic aminotransferase in E.coli:Amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function" Biochemistry. 28. 1153-1160 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nishi,T.: "Import and processing of mitochondrial aspartate aminotransferase:Structure-function relationship of the presequence" J.Biol.Chem.264. 6044-6061 (1989)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Morino,Y.: "Mammalian aspartate aminotransferase isozymes-From DNA to protein" Annals New York Acad.Sci.583. 32-47 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Setoyama,C.: "Regulatory regions of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle" J.Biol.Chem.265. 1293-1299 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Choudhury,B.K.: "Molecular cloning and sequence analysis of the human cytosolic aspartate aminotransferase gene" Biochem.Int.22. 583-591 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ding,S.-H.: "Two promoters in the 5' region of the mouse mitochondrial malate dehydrogenase gene"

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Shimada,K.: "Molecular structures and evolution of mouse isozyme genes functioning in the malate-aspartate shuttle In:Isozymes" Wiley-Liss Inc.New York,Ogita,Z.and Markert,C.M.eds, 139-158 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Nagashima, F.: "cDNA cloning and expression of pig cytosolic aminotransferase in E. coli : Amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function" Biochemistry. 28. 1153-1160 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nishi, T.: "Import and processing of mitochondrial aspartate aminotransferase : Structure-function relationship of the presequence" J. Biol. Chem.264. 6044-6061 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Morino, Y.: "Mammalian aspartate aminotransferase isozymes-From DNA to protein" Annals New York Acad. Sci.583. 32-47 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Setoyama, C.: "Regulatory regions of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle" J. Biol. Chem.265. 1293-1299 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Choudhury, B. K.: "Molecular cloning and sequence analysis of the human cytosolic aspartate aminotransferase gene" Biochem. Int.22. 583-591 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Ding, S. H.: "Two promoters in the 5' region of the mouse mitochondrial malate dehydrogenase gene"

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-03-16  

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