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首页> 外文期刊>Molecular and Cellular Biology >Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.
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Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

机译：编码小鼠核糖核苷酸还原酶M1和M2亚基的可表达cDNA克隆的分离和鉴定。

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Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.
机译：哺乳动物核糖核苷酸还原酶由两个不同的亚基蛋白M1和M2组成，它们在细胞周期中受到差异调节。我们已经从冈山-Berg cDNA文库中分离了两个亚基的可表达cDNA克隆，这些文库是用抗羟基脲，M2蛋白过量的小鼠TA3细胞的mRNA制成的。用该质粒转染COS-7猴细胞后，可通过电子顺磁共振波谱证实M2蛋白的表达。亲本和羟基脲抗性TA3 mRNA的电泳和印迹分析显示，有两个M2转录本，一个为2.1碱基，一个为1.6碱基。相应cDNA的限制性核酸内切酶作图表明，这两个mRNA仅在3'非翻译末端的长度上不同。相比之下，只有一个与M1蛋白相对应的mRNA，其迁移率约为3.1千碱基。耐羟基脲的TA3细胞比亲代细胞多50至100倍的M2 mRNA，而比M1 mRNA多10倍。然而，对相应基因组DNA序列的Southern印迹分析显示，M2基因被扩增了五倍，但是M1基因仍然是单拷贝。 2,111个碱基对长的M2 cDNA的完整核苷酸序列揭示了一个开放阅读框，编码390个氨基酸，其分子量为45,100。小鼠M2蛋白序列与固结螺旋藻中的等效蛋白非常同源，而与爱泼斯坦-巴尔病毒，2型单纯疱疹病毒和大肠杆菌核糖核苷酸还原酶的较小亚基的同源性则不太明显。

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