Hsieh T S, Brown S D, Huang P, Fostel J
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
Nucleic Acids Res. 1992 Dec 11;20(23):6177-82. doi: 10.1093/nar/20.23.6177.
We synthesized a DNA probe specific for the gene encoding eucaryotic DNA topoisomerase I by the polymerase chain reaction. The sequences of the primers for this reaction were deduced from the regions with extensive homology among the enzymes from the fission and budding yeasts, and the human. From the clones isolated by screening a Drosophila cDNA library with this DNA probe, two cDNA clones of 3.8 and 5.2 kb were characterized and completely sequenced. Both cDNA sequences contain an identical open reading frame for 972 amino acid residues. The 3.8 kb messenger RNA is likely generated by using a polyadenylation site 5' upstream to that used in generating the 5.2 kb mRNA. The predicted amino acid sequence shows that a segment of 420 amino acid residues at the amino terminus is hydrophilic, similar to the amino terminal 200 residues in the yeast and human enzymes. Furthermore, the Drosophila enzyme is unique in that the amino terminal 200 residues are enriched in serine and histidine residues; most of them are present in clusters. The rest of the Drosophila sequence is highly homologous to those from yeast and human enzymes. The evolutionarily conserved residues are identified and are likely the critical elements for the structure and function of this enzyme. A plasmid vector containing the cloned cDNA was constructed for the expression of Drosophila protein in Escherichia coli. The enzymatic and immunochemical analysis of the polypeptide produced in this heterologous expression system demonstrated that the expressed protein shares similar enzymatic properties and antigenic epitopes with DNA topoisomerase I purified from Drosophila embryos or tissue culture cells, thus establishing the bacterial expression system being useful for the future structure/function analysis of the Drosophila enzyme.
我们通过聚合酶链反应合成了一种针对真核生物DNA拓扑异构酶I编码基因的DNA探针。该反应引物的序列是从裂殖酵母、芽殖酵母和人类的酶中具有广泛同源性的区域推导出来的。用这种DNA探针筛选果蝇cDNA文库分离得到的克隆中，对两个分别为3.8 kb和5.2 kb的cDNA克隆进行了表征并完成了全序列测定。两个cDNA序列都包含一个由972个氨基酸残基组成的相同开放阅读框。3.8 kb的信使RNA可能是通过使用一个位于产生5.2 kb mRNA所用聚腺苷酸化位点上游5'处的聚腺苷酸化位点产生的。预测的氨基酸序列表明，氨基末端420个氨基酸残基的一段是亲水的，类似于酵母和人类酶中的氨基末端200个残基。此外，果蝇的这种酶的独特之处在于其氨基末端200个残基富含丝氨酸和组氨酸残基；其中大多数以簇的形式存在。果蝇序列的其余部分与酵母和人类酶的序列高度同源。确定了进化上保守的残基，它们可能是该酶结构和功能的关键元件。构建了一个包含克隆cDNA的质粒载体，用于在大肠杆菌中表达果蝇蛋白。对在这个异源表达系统中产生的多肽进行的酶学和免疫化学分析表明，所表达的蛋白与从果蝇胚胎或组织培养细胞中纯化的DNA拓扑异构酶I具有相似的酶学性质和抗原表位，从而证明该细菌表达系统对未来果蝇酶的结构/功能分析是有用的。
