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

DNA 拓扑异构酶 II 在细胞粘菌发育中的作用。

• 批准号: 02640518
• 负责人: TANAKA Yoshimasa
• 金额: $ 1.02万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02640518/
• 关键词: Cellular slime mold; DNA topoisomerase II; Development; Cloning; Fusion protein; Antibody; Expression vector; Western blot; DNAトポイソメラ-ゼII; クロ-ニング; 合成オリゴヌクレオチド

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.

为了研究基因表达与DNA拓扑结构之间的关系，对细胞黏菌盘状盘基钢菌的DNA拓扑异构酶II基因进行了克隆和测序。利用几种具有保守氨基酸序列的寡核苷酸探针，共克隆出含有一个DNA拓扑异构酶II基因的6.3 Kb区域。5.5 Kb区包含3849个核苷酸的开放阅读框，对应1282个氨基酸和146个KDa蛋白。同源性搜索显示,并有非常相似的氨基酸序列DNA拓扑异构酶II的真核细胞和DNA DNA促旋酶促旋酶a + b。使用印迹实验,基因被证明是一个复制的基因组d discoideum。Northern blot分析显示，该基因的转录本长约4100个核苷酸，由于转录本只能通过poly(A)^+ RNA片段检测到，因此转录本的数量很少。在大肠杆菌表达载体pGEX的谷胱甘肽s转移酶基因下游插入1.9 Kb的DNA片段，制备抗DNA拓扑异构酶II的抗体。从大肠杆菌包涵体中纯化变性融合蛋白，并将其注射到家兔体内。从兔免疫血清中提取抗体。Western blot结果表明，该抗体可识别盘状蝶细胞全浆蛋白140 KDa。该抗体不与mucoroides细胞的任何蛋白发生交叉反应，与Polyshondylium pallidum细胞的约220 KDa蛋白发生交叉反应。从指数生长期到固定生长期和发育过程中，酶蛋白的含量逐渐减少。