Gene expression and DNA replication triggered by growth factors and their receptors

生长因子及其受体触发的基因表达和 DNA 复制

• 批准号: 02404086
• 负责人: ARAI Ken-ichi
• 金额: $ 11.39万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 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-02404086/
• 关键词: DNA replication; G-protein; Signal transduction; primosome; kinase; Cytokine; Transcription factor; Receptor; 増殖因子; 遺伝子発現; 細胞周期; 発現クロ-ニング; 分子生物学

We intended to clarify the intracellular mechanisms of the cell cycle transition from G1 to S. 1) Control of replication in E. coli : G1 to S phase transition is represented by the initiation of chromosomal DNA replication. The new DNA strand synthesis in E. coli, the most thoroughly studied organism, is initiated by the action of primosome, a protein complex essential for priming DNA templates for DNA polymerase. We demonstrated that two types of primosomes, phiXI74 type (priA-dependent) and ABC type (dnaA-dependent), are functionally interchangeable in the ColEl replication. Furthermore, replication of F, R6K, and Rstl plasmids requires dnaA but not priA, whereas recA-dependent and dnaA-independent stable replicafion in E coli requires priA. We proposed that there are two types of replicons in E. coli, one dependrnt on the ABC primosome, and the other dependent on the phiXI74 type primosome. 2) GI to S transition in yeast : Mating pheromone is a yeast peptide that inhibits the initia … More tion of DNA replicafion, thereby arrests the cell cycle of target cells at Gl phase. We have previously demonstrated that its effect is exerted through a specific membrane receptor and a Gprotein. We further showed that a beta-gamma fusion polypeptide is as active as the natural beta/gamma subunit of the G-protein, so that, as a complex, both subunit are the key elements to introduce the mating pheromone signal. We have been looking for proteins that interact with the core sequence of putative chromosomal replication origins of yeast, which are likely to be the initiation factors for DNA replication. We have found several proteins and purified one of them, 100kD protein, to homogeneity. The CDC7 kinase is implicated to be involved in the initiation of DNA replication in yeast. We have constructed an overproducer of the CDC7 protein, and made antibodies against CDC7. Purification and characterization of these proteins are underway. 3) G1 to S transition in hematopoietic cells : Cytokines are a set of polypeptides that are mainly secreted from activated T cells, and act on various types of cells including hematopoietic cells. They not only support cell's viability and cell cycle progression from Gl to S, but also promote subsequent differentiation. Our research has been focusing on the role of IL-3 and GM-CSF on hematopoietic progenitor cells. Both act on very early progenitor cells to stimulate their proliferation and differentiation. Such similarities in the effects of these factors can be explained by our recent finding that receptors for both cytokines are composed of two polypeptides, alpha and beta, of which beta chain is shared. Analysis of the downstream events initiated upon binding of these cytokines to their receptors are underway. T cells are largely in GO/GI phase during the cell cycle. Upon the activation by antigen, they secrete a number of cytokines including IL-3 and GM-CSF, then they start to initiate DNA replication. We have been analyzing promoter Less

我们的目的是阐明细胞周期从G1期到S期转变的细胞内机制。1)在E.大肠杆菌：G1到S期的转变是由染色体DNA复制的开始。在E.大肠杆菌是研究最彻底的生物体，它是通过启动DNA聚合酶的DNA模板所必需的蛋白质复合物--启动体的作用而启动的。我们证明了两种类型的primosomes，phiXI74型（priA依赖）和ABC型（dnaA依赖），在ColEl复制功能上是可互换的。此外，F、R6K和Rstl质粒的复制需要dnaA而不需要priA，而大肠杆菌中依赖于recA和不依赖于dnaA的稳定复制需要priA。我们认为，在大肠杆菌中存在两种类型的复制子。大肠杆菌中，一个依赖于ABC型的启动子，另一个依赖于phiXI 74型的启动子。2)酵母中的GI到S转变：交配信息素是一种酵母肽，可抑制酵母中的GI到S转变。 ...更多信息 抑制DNA复制，从而将靶细胞的细胞周期阻滞在G1期。我们以前已经证明，它的作用是通过一个特定的膜受体和G蛋白。我们进一步表明，β-γ融合多肽与G蛋白的天然β/γ亚基一样具有活性，因此，作为复合物，两个亚基都是引入交配信息素信号的关键元件。我们一直在寻找与酵母染色体复制起点核心序列相互作用的蛋白质，这些蛋白质可能是DNA复制的起始因子。我们发现了几种蛋白质，并将其中一种100 kD蛋白质纯化至同质。CDC7激酶被认为参与酵母中DNA复制的起始。我们已经构建了一个过度生产的CDC7蛋白，并针对CDC7的抗体。这些蛋白质的纯化和表征正在进行中。3)造血细胞中的G1到S转变：细胞因子是一组主要由活化的T细胞分泌的多肽，作用于包括造血细胞在内的各种类型的细胞。它们不仅支持细胞的活力和细胞周期从G1到S的进展，而且促进随后的分化。我们的研究一直集中在IL-3和GM-CSF对造血祖细胞的作用。两者都作用于非常早期的祖细胞以刺激其增殖和分化。这些因子的作用的这种相似性可以通过我们最近的发现来解释，即两种细胞因子的受体由两种多肽α和β组成，其中β链是共享的。这些细胞因子与其受体结合后引发的下游事件的分析正在进行中。T细胞在细胞周期中主要处于G0/GI期。当被抗原激活后，它们分泌包括IL-3和GM-CSF在内的多种细胞因子，然后它们开始启动DNA复制。We have been analyzing promoter收起

项目成果

Tsuji, K., Nakahata, T., Takagi, M., Kobayashi, T., Ishiguro, A., Kikuchi, T., Naganuma, K., Koike, K., Miyajima, A., Arai, K., Akabane, T.: "Effects of interleukin-3 and interleukin-4 on the development of connective tissue-type mast cells : interleukin

Tsuji, K.、Nakahata, T.、Takagi, M.、Kobayashi, T.、Ishiguro, A.、Kikuchi, T.、Naganuma, K.、Koike, K.、Miyajima, A.、Arai, K.、

Matsumoto, K., Miyajima, I., Nomoto, S., Nakayama, N., Arai, K.: "Mating pheromone signal transduction in yeast." "Developmental Biology : Molecular and cellular biology of yeast and filametous funji"UCLA Symposia. Wiley-Liss Inc. 289-306 (1990)

Matsumoto, K.、Miyajima, I.、Nomoto, S.、Nakayama, N.、Arai, K.：“酵母中的交配信息素信号转导。”

Yonehara, S., Ishii, A., Yonehara, M., Koyasu, S., Miyajima, A., Schreurs, J., Arai, K. and Yahara, I.: "Identification of a cell surface 105 Kd protein which binds interleukin 3" Int. Immunol.2. 143-150 (1990)

Yonehara, S.、Ishii, A.、Yonehara, M.、Koyasu, S.、Miyajima, A.、Schreurs, J.、Arai, K. 和 Yahara, I.：“细胞表面 105 Kd 蛋白的鉴定

Yokota, Y., Arai, N., Arai, K. and Zlotnik, A.: "Interleukin-4." "Handbook of Experimental Pharmacology. ". Springer-Verlag. 577-607 (1990)

Yokota, Y.、Arai, N.、Arai, K. 和 Zlotnik, A.：“Interleukin-4”。

Nishida, J., Yoshida, M., Arai, K., and Yokota, T.: "Definition of GC rich motif as regulatory sequence of the human IL-3 gene : Coordinate regulation of the IL-3 gene by CLE2/GC box of the GM-CSF gene in T cell activation" Int. Immunol.3. 245-254 (1991)

Nishida, J.、Yoshida, M.、Arai, K. 和 Yokota, T.：“富含 GC 基序作为人类 IL-3 基因调控序列的定义：CLE2/GC 对 IL-3 基因的协调调控