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.

为了研究基因表达与DNA拓扑之间的关系，对细胞粘膜模具dictyostelium discoideum的DNA拓扑异构酶II基因进行了克隆并克隆并进行了序列。术语中，使用几个寡核苷酸探针的几个基因构造的几个基因构造的6.3 kb区域含有DNA拓扑酶II基因的6.3 kb区域。测序的5.5 kb区域包含3,849个核苷酸的开放式阅读框，对应于1,282个氨基酸和146 kDa蛋白。同源性搜索表明，ORF具有与Eucaryote和DNA Gyrase A加DNA Gyrase b的DNA拓扑异构酶II非常相似的氨基酸序列。使用Southern印迹实验，该基因被证明是D. discoideum基因组中的单个拷贝。 Northern印迹分析表明，该基因的转录本长约为4,100个核苷酸，并且转录本的量很低，因为仅使用poly（a）^+ RNA分数才能检测到转录本。为了对DNA拓扑异构酶II产生抗体，将跨基因1.9 kb的DNA区域插入了大肠杆菌表达载体PGEX的谷胱甘肽-S-转移酶基因的下游。从大肠杆菌的包含体中纯化了变性的融合蛋白，并注入兔子。从兔子的免疫血清中回收抗体。使用抗体的蛋白质印迹表明，抗体识别出140 kDa的蛋白质蛋白质的全粘核细胞蛋白。然而，抗体与粘霉细胞的任何蛋白质均未反应，并与约220 kDa蛋白质的polyshondylium pallidum细胞交叉反应。从指数增长阶段到固定阶段和发育过程中，酶蛋白的量逐渐减少。