Cloning and expression of mammalian DNA ligase gene

从小牛胸腺中纯化了DNA连接酶II(Mr=68 kDa)，并制备了兔抗血清。用抗(小牛胸腺DNA连接酶I)Ig G和抗(DNA连接酶II)Ig G免疫化学分析表明，哺乳动物细胞和组织中存在两种独立的DNA连接酶形式。DNA连接酶I(Mr=200 kDa)可能参与DNA复制，而DNA连接酶II可能参与DNA修复。由于没有直接证据表明体内存在这两种形式的DNA连接酶，因此通过基因克隆来回答有关这两种DNA连接酶的存在、表达和功能的问题是一个强有力的方法。为了用人工合成的探针分离哺乳动物DNA连接酶的cDNA克隆，对小牛胸腺DNA连接酶II的氨基末端序列进行了检测，发现氨基末端被封闭。然后对DNA连接酶II化学和酶切后的多肽进行了氨基酸序列测定，但由于得到的纯酶含量较少，没有得到可靠的结果。最近，从小牛胸腺cDNA文库中分离到两个独立的cDNA克隆，其合成的30聚体与酿酒酵母和庞氏酵母DNA连接酶推导的氨基酸序列中推测的活性部位的核苷酸序列互补。将获得的DNA片段亚克隆到pUC118/119中进行测序。当cDNA测序发现与酵母DNA连接酶可能的活性部位同源时，在大肠杆菌JM109中表达的cDNA产物将与抗小牛胸腺DNA连接酶的抗体进行检测。

DNA ligase II (Mr = 68 kDa) has been purified from calf thymus and the rabbit antiserum was obtained. Immunochemical analysis with anti(calf thymus DNA ligase I)IgG and anti(DNA ligase II)IgG has implied that two independent forms fo DNA ligase exist in mammalian cells and tissues. DNA ligase I (Mr = 200 kDa) seems to play a role in DNA replication, Whereas DNA ligase II may be involved in DNA repair. Since there is no direct evidence for the presence in vivo of these two forms of DNA ligase, a powerful approach to answering questions about the existence, expression and function of two DNA ligases is through gene cloning. In order to isolate cDNA clones of mammalian DNA ligase with synthetic probes, amino-terminal sequence of calf thymus DNA ligase II was examined, and the amino-terminal proved to be blocked. Then amino-acid sequence of isolated peptides after chemical and enzymatic clevages of DNA ligase II was determined, but no reliable result was obtained mainly because of the small amount of the pure enzyme obtained. Recently two independent cDNA clones have been isolated from a calf thymus cDNA library with a synthetic 30-mer complementary to the nucleotide sequence at the putative active site in the deduced amino-acid sequences of Saccharomyces cerevisiae and S. pombe DNA ligases. The DNA fragments from the cDNAs were subcloned into pUC118/119 for sequencing. When the sequencing of cDNAs reveals the site homologous to the putative active site of yeast DNA ligase, the products of cDNAs expressed in Escherichia coli JM109 will be detected with the antibodies against calf thymus DNA ligases.