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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。

基本信息

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

项目摘要

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DNA拓扑结构与基因表达的关系，克隆了盘基网柄菌的DNA拓扑异构酶II基因，并对其进行了序列分析，利用多种生物基因中氨基酸序列保守的寡核苷酸探针，克隆了含有DNA拓扑异构酶II基因的6.3kb区域。测序的5.5kb区域包含一个3849个核苷酸的开放阅读框，对应于1,282个氨基酸和146 KDa的蛋白质。同源性搜索表明，该基因与真核生物的DNA拓扑异构酶II和DNA旋转酶a和DNA旋转酶b的氨基酸序列非常相似。Southern印迹实验表明，该基因在盘藻基因组中为单拷贝。Northern印迹分析表明，该基因的转录本全长约4100个核苷酸，转录本的量很少，因为只能用Poly(A)^+RNA组分检测到转录本。为了制备针对拓扑异构酶II的抗体，将1.9kb的DNA片段插入到大肠杆菌表达载体pGEX的谷胱甘肽-S-转移酶基因下游。将变性后的融合蛋白从大肠杆菌包涵体中纯化并注射到兔体内。从兔免疫血清中回收抗体。用该抗体进行Western印迹分析表明，该抗体可识别盘状螺旋体细胞全匀浆的140 KDa蛋白。但该抗体不与粘虫细胞的任何蛋白质发生交叉反应，而与梅毒多菌体细胞约220 KDa的蛋白质发生交叉反应。从指数生长期到稳定期和发育过程中，酶蛋白的含量逐渐减少。