Tsugeki R, Mori H, Nishimura M
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Eur J Biochem. 1992 Oct 1;209(1):453-8. doi: 10.1111/j.1432-1033.1992.tb17309.x.
Two different cDNA clones, pMCPN60-1 and pMCPN60-2, encoding the mitochondrial homologues of chaperonin 60 (Cpn60) were isolated from a cDNA library of germinating pumpkin cotyledons by use of mixtures of synthetic oligonucleotides based on the N-terminal amino acid sequence of the protein. Determination of the complete nucleotide sequences of the two cDNA revealed that pMCPN60-1 and pMCPN60-2 each contain one open reading frame that encodes a protein of 575 amino acids with molecular masses of 61052 Da and 61127 Da, respectively. The deduced amino acid sequences of the two polypeptides include a 32-residue N-terminal putative mitochondrial presequence attached to the mature polypeptides, and they are 95.3% identical. From a comparison of deduced amino acid sequences with other Cpn60, it appears that the mature polypeptides of pumpkin mitochondrial Cpn60 are 44-59% identical to the other Cpn60, namely, GroEL of Escherichia coli, the 60-kDa heat-shock protein (Hsp60) of mitochondria in the yeast Saccharomyces cerevisiae, P1 protein of mammalian mitochondria and the Ribulose-1,5-bisphosphate carboxylase/oxygenase subunit-binding proteins alpha and beta of plastids in higher plants. Genomic Southern-blot analysis identified at least two copies of the gene for mitochondrial Cpn60 in the pumpkin genome. The levels of mRNA for mitochondrial Cpn60 in cotyledons, hooks and hypocotyls of pumpkin seedlings increased in response to heat stress, as deduced from Northern-blot analysis, indicating that pumpkin mitochondrial Cpn60 is a heat-induced stress protein.
利用基于该蛋白质N端氨基酸序列的合成寡核苷酸混合物，从发芽南瓜子叶的cDNA文库中分离出两个不同的cDNA克隆，pMCPN60 - 1和pMCPN60 - 2，它们编码伴侣蛋白60（Cpn60）的线粒体同源物。对这两个cDNA的完整核苷酸序列测定表明，pMCPN60 - 1和pMCPN60 - 2各自包含一个开放阅读框，分别编码一个由575个氨基酸组成的蛋白质，分子量分别为61052 Da和61127 Da。这两种多肽的推导氨基酸序列包括一个连接到成熟多肽上的32个残基的N端推测线粒体前序列，它们的同源性为95.3%。通过将推导氨基酸序列与其他Cpn60进行比较，发现南瓜线粒体Cpn60的成熟多肽与其他Cpn60的同源性为44% - 59%，即大肠杆菌的GroEL、酿酒酵母线粒体中的60 kDa热休克蛋白（Hsp60）、哺乳动物线粒体的P1蛋白以及高等植物质体中的1,5 - 二磷酸核酮糖羧化酶/加氧酶亚基结合蛋白α和β。基因组Southern杂交分析确定南瓜基因组中线粒体Cpn60基因至少有两个拷贝。从Northern杂交分析推断，南瓜幼苗子叶、弯钩和下胚轴中线粒体Cpn60的mRNA水平在热胁迫下升高，表明南瓜线粒体Cpn60是一种热诱导应激蛋白。
