Roles of DNA topoisomerase II in the development of the cellular slime molds.

为了研究基因表达与DNA拓扑结构的关系，对细胞黏菌盘基网柄菌（Dictyosteeliumdiscoideum）的DNA拓扑异构酶II基因进行了克隆和序列测定，利用几种生物DNA拓扑异构酶II基因保守区的寡核苷酸探针，共克隆了6.3Kb的DNA拓扑异构酶II基因。测序的5.5 Kb区域含有3，849个核苷酸的开放阅读框，其对应于1，282个氨基酸和146 KDa蛋白质。同源性分析表明，该ORF与真核生物的DNA拓扑异构酶II和DNA促旋酶a + DNA促旋酶B的氨基酸序列非常相似。Southern杂交结果表明，该基因在D.盘状突北方印迹分析显示，该基因的转录本约为4,100个核苷酸长，并且转录本的量很低，因为转录本只能用poly（A）^+ RNA组分检测。为了制备抗DNA拓扑异构酶II的抗体，将该基因的1.9Kb的DNA片段插入到大肠杆菌谷胱甘肽-S-转移酶基因的下游。coli表达载体pGEX。从大肠杆菌包涵体中纯化出变性的融合蛋白。大肠杆菌，并注射到兔子体内。从兔免疫血清中回收抗体。Western blot结果表明，该抗体可识别D.盘状细胞但该抗体与D.结果表明，该蛋白与pallidum细胞中约220 KDa的蛋白有交叉反应。从指数生长期到稳定期和发育期，酶蛋白的含量逐渐减少。

To study the relationship between gene expression and DNA topology, the DNA topoisomerase II gene of the cellular slime mold Dictyostelium discoideum was cloned and sequenced.Totally, 6.3 Kb region containing a DNA topoisomerase II gene was cloned using several oligonucleotide probes which have conserved amino acid sequence regions among the genes of several organisms. The 5.5 Kb region sequenced contained a open reading frame of 3,849 nucleotides which correspond to 1,282 amino acids and to 146 KDa protein. Homology search showed that the ORF has very similar amino acid sequence to DNA topoisomerase II of eucaryote and DNA gyrase a plus DNA gyrase b. Using Southern blot experiment, the gene was shown to be a single copy in the genome of D. discoideum. Northern blot analysis revealed that the transcript of the gene is about 4,100 nucleotides long and the amount of the transcript is low because the transcript could be detected only using poly(A)^+ RNA fraction. To make antibody against the DNA topoisomerase II, the DNA region spanning 1.9 Kb of the gene was inserted in the downstream of glutathion-S-transferase gene of the E. coli expression vector pGEX. The denatured, fusion protein was purified from inclusion body of E. coli and injected into a rabbit. Antibody was recovered from immune serum of the rabbit. Western blot using the antibody showed that the antibody recognized a 140 KDa protein of whole homogenate of D. discoideum cell. However, the antibody did not cross-react with any proteins of D. mucoroides cell and cross-reacted with about 220 KDa protein of Polyshondylium pallidum cell. The amount of the enzyme protein gradually decreased during from exponentially growing phase to stationary phase and during development.