Das A, Dasgupta A, Sharma S, Ghosh M, Sengupta T, Bandopadhyay S, Majumder H K
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India.
Nucleic Acids Res. 2001 May 1;29(9):1844-51. doi: 10.1093/nar/29.9.1844.
The gene encoding type II DNA topoisomerase from the kinetoplastid hemoflagellated protozoan parasite Leishmania donovani (LdTOP2) was isolated from a genomic DNA library of this parasite. DNA sequence analysis revealed an ORF of 3711 bp encoding a putative protein of 1236 amino acids with no introns. The deduced amino acid sequence of LdTOP2 showed strong homologies to TOP2 sequences from other kinetoplastids, namely Crithidia and Trypanosoma spp. with estimated identities of 86 and 68%, respectively. LdTOP2 shares a much lower identity of 32% with its human homologue. LdTOP2 is located as a single copy on a chromosome in the 0.7 Mb region in the L.donovani genome and is expressed as a 5 kb transcript. 5'-Mapping studies indicate that the LdTOP2 gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon occurring at -639 from the predicted initiation site. Antiserum raised in rabbit against glutathione S-transferase fusion protein containing the major catalytic portion of the recombinant L.donovani topoisomerase II protein could detect a band on western blots at approximately 132 kDa, the expected size of the entire protein. Use of the same antiserum for immunolocalisation analysis led to the identification of nuclear, as well as kinetoplast, antigens for L.donovani topoisomerase II. The in vitro biochemical properties of the full-length recombinant LdTOP2 when overexpressed in E.coli were similar to the Mg(II) and ATP-dependent activity found in cell extracts of L.donovani.
从动基体血鞭毛虫原生动物寄生虫杜氏利什曼原虫（LdTOP2）的基因组DNA文库中分离出编码II型DNA拓扑异构酶的基因。DNA序列分析显示一个3711 bp的开放阅读框，编码一个1236个氨基酸的推定蛋白质，无内含子。LdTOP2推导的氨基酸序列与其他动基体（即克氏锥虫和锥虫属）的TOP2序列有很强的同源性，估计同源性分别为86%和68%。LdTOP2与其人类同源物的同源性低得多，为32%。LdTOP2在杜氏利什曼原虫基因组0.7 Mb区域的一条染色体上以单拷贝形式存在，并作为一个5 kb的转录本表达。5'-定位研究表明，LdTOP2基因转录本在转录后成熟，微型外显子的反式剪接发生在预测起始位点下游-639处。用兔抗含有重组杜氏利什曼原虫拓扑异构酶II蛋白主要催化部分的谷胱甘肽S-转移酶融合蛋白产生的抗血清，可在蛋白质印迹上检测到一条约132 kDa的条带，这是整个蛋白质的预期大小。使用相同的抗血清进行免疫定位分析，鉴定出杜氏利什曼原虫拓扑异构酶II的核抗原以及动基体抗原。全长重组LdTOP2在大肠杆菌中过表达时的体外生化特性与杜氏利什曼原虫细胞提取物中发现的Mg(II)和ATP依赖性活性相似。
