Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.
Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.
复制标题
大鼠鸟氨酸氨基甲酰转移酶前体 cDNA 的分子克隆和核苷酸序列。
DOI：
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发表时间：
1984
影响因子：
11.1
通讯作者：
Yoshito Kaziro
中科院分区：
文献类型：
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作者：
Masaki Takiguchi;Satoshi Miura;M. Mori;Masamiti Tatibana;Shigekazu Nagata;Yoshito Kaziro
通过免疫沉淀法富集大鼠肝游离多聚核糖体中的线粒体基质酶--大鼠鸟氨酸氨甲酰转移酶（EC 2.1.3.3）的信使RNA，并通过载体引物法从富集的mRNA制备重组质粒。通过杂交停滞翻译和杂交选择翻译鉴定了鸟氨酸氨甲酰转移酶的cDNA克隆。其中一个命名为pOTC-1的克隆含有1.6-β-内酰胺酶插入片段，并与大鼠肝脏中约等于1.8 β-内酰胺酶的mRNA杂交。对cDNA克隆进行核苷酸序列分析。推导的氨基酸序列表明，鸟氨酸氨甲酰基转移酶前体由322个氨基酸残基的成熟酶和32个氨基酸残基的NH 2-末端肽延伸（前序列）组成。前序列含有8个碱性氨基酸残基，没有酸性残基，也没有疏水性氨基酸延伸。将大鼠鸟氨酸氨甲酰基转移酶的氨基酸序列与最近报道的人酶的序列进行比较[Horwich，A. L.，芬顿，W.一、威廉姆斯，K. R.，Kalousek，F.，克劳斯，J. P.，杜利特尔河F.、Konigsberg，W. & Rosenberg，L. E.（1984）Science 224，1068-1074]。成熟酶部分的序列是93%相同的，而前序列的序列是69%相同的。在大鼠和人类酶的前序列中有两个高度保守的片段。其中一个保守片段与酵母线粒体延伸因子EF-Tu的前序列片段显著相似。这些结果表明，同源片段是重要的蛋白质，在细胞质中合成的运输到线粒体基质。
Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.