MOLECULAR-CLONING AND EXPRESSION OF THE GENE ENCODING THE KINETOPLAST-ASSOCIATED TYPE-II DNA TOPOISOMERASE OF CRITHIDIA-FASCICULATA
MOLECULAR-CLONING AND EXPRESSION OF THE GENE ENCODING THE KINETOPLAST-ASSOCIATED TYPE-II DNA TOPOISOMERASE OF CRITHIDIA-FASCICULATA
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DOI：
10.1016/0166-6851(92)90244-e
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发表时间：
1992-01-01
影响因子：
1.5
通讯作者：
RAY, DS
中科院分区：
文献类型：
--
作者：
PASION, SG;HINES, JC;RAY, DS
一个II型DNA拓扑异构酶，拓扑IImt，以前被证明是与动基体DNA的锥虫Crithidia束状。 编码这种动质体相关的拓扑异构酶的基因已被克隆通过免疫学筛选的Crithidia基因组表达文库的单克隆抗体提出对纯化的酶。 基因CfaTOP 2是单拷贝基因，并表达为4.8-kb聚腺苷酸化转录物。 CfaTOP 2的核苷酸序列已被确定，并编码一个预测的多肽的1239个氨基酸的分子量为138445。 通过对大肠杆菌中表达的β-半乳糖苷酶-CfaTOP 2融合蛋白的免疫印迹分析和对来自纯化的topoIImt的胰蛋白酶肽序列的分析，支持克隆基因的鉴定。 CfaTOP 2与其他真核生物的核II型DNA拓扑异构酶具有显着的同源性，这表明在Crithidia中，核和线粒体形式的拓扑异构酶II由相同的基因编码。
A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.