| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Rat liver contained two isozymes of fumarase which are localized in different intracellular compartments, the mitochondria and cytosol. These isozymes are almost equally distributed between these two subcellular compartments, and cannot be differentiated from each other except that the NH_2-terminal amino acid of the cytosolic isozyme was acetylated. In the rat liver it is suggested that the cytosolic and mitochondrial fumarases are products of the same gene. Therefore, in this study we intended to examine the mechanism how the two isozymes of fumarase are produced from the same gene and found the following observations.
cDNA clone for fumarase with an insert of about 1.7Kb was isolated from a rat liver cDNA library. Nucleotide sequence analysis of the insert revealed that the cDNA contained a 5'-noncoding region of 25 nucleotides, the coding region of 1521 nucleotides, and a 3'-nontranslated region of 43 nucleotides fallowed by a poly(A)^+ tail. The open reading frame encoded a polypeptide of 507-amino acid residues, which contained an additional sequence (so-called presequence) of 41-amino acid residues. Interestingly, there was another initiation codon AUG just upstream of GCA, which is the codon of the NH_2-terminal Ala of both mature enzymes. Therefore, if translation is initiated from this second initiation codon, the precursor of the cytosolic fumarase will be synthesized by the same mRNA as mitochondrial enzyme. Another experimental findings obtained by hybrid- selected translation, Northern and Southern blot, primer-extension analysis, and S-1 nuclease mapping analysis also supported the above possibility.
Recently we isolated a genomic clone for rat fumarase. Now we are trying to analyze the structure of the genomic DNA to obtain information on the mechanism of synthesis of two kinds of precursor polypeptides.
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