Mechanism of the production of two isozymes of fumarase encoded from a single gene

大鼠肝脏含有两种延胡索酸酶同工酶，它们定位于不同的细胞内，即线粒体和胞浆。这些同工酶几乎平均分布在这两个亚细胞室中，除了胞质同工酶的NH_2末端氨基酸被乙酰化外，它们不能相互区分。在大鼠肝脏中，胞浆延胡索酶和线粒体延胡索酶是同一基因的产物。因此，在本研究中，我们试图研究富马酸酶的两种同工酶是如何从同一个基因中产生的，并发现了以下观察结果。从大鼠肝脏的cDNA文库中克隆了约1.7Kb的富马酸酶的cDNA克隆。核苷酸序列分析表明，该基因包含25个核苷酸的5‘非编码区、1521个核苷酸的编码区和43个核苷酸的3’非编码区。开放阅读框编码507个氨基酸残基的多肽，包含一个41个氨基酸残基的额外序列(所谓的前序列)。有趣的是，在GCA的上游还有另一个起始密码子AUG，它是两个成熟酶的NH_2末端ALA的密码子。因此，如果从这个第二起始密码子开始翻译，胞质富马酸酶的前体将由与线粒体酶相同的mRNA合成。通过杂交选择翻译、Northern和Southern杂交、引物延伸分析和S-1核酸酶图谱分析获得的另一个实验结果也支持上述可能性。最近，我们分离到了大鼠延胡索酸酶的基因组克隆。现在，我们正试图分析基因组DNA的结构，以获得关于两种前体多肽合成机制的信息。

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.