权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Cloning and expression of mammalian DNA ligase gene

哺乳动物DNA连接酶基因的克隆与表达

基本信息

批准号：
61580164
负责人：
TERAOKA Hirobumi
金额：
$ 1.15万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61580164/
关键词：
DNA ligase DNA replication DNA repair nene expression cDNA cloning cDNAクローニング

项目摘要

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.

从小牛胸腺中分离纯化了分子量为68 kDa的DNA连接酶Ⅱ，并制备了兔抗血清。用抗小牛胸腺DNA连接酶I和抗DNA连接酶II IgG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。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这两种形式的DNA连接酶在体内的存在，一个强有力的方法来回答有关两种DNA连接酶的存在，表达和功能的问题是通过基因克隆。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的氨基端序列进行了测定，发现氨基端被封闭。用DNA连接酶Ⅱ进行化学和酶解后，对分离的肽段进行了氨基酸序列测定，但主要由于所得纯酶量少，结果不可靠。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-mer与酿酒酵母和S.粟酒酵母DNA连接酶。将来自cDNA的DNA片段亚克隆到pUC 118/119中用于测序。当cDNA的测序显示与酵母DNA连接酶的推定活性位点同源的位点时，将用抗小牛胸腺DNA连接酶的抗体检测在大肠杆菌JM 109中表达的cDNA的产物。