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

我们试图阐明细胞周期从G1期向S期转变的细胞内机制：1)在大肠杆菌中复制的控制：G1期到S期的转变表现为染色体复制的启动。在研究最深入的生物体--大肠杆菌中，新的DNA链合成是由原始体的作用启动的，原始体是一种蛋白质复合体，对启动DNA聚合酶的DNA模板是必不可少的。我们证明了在ColEL复制中，两种类型的原始体，phiXI74型(依赖于PriA)和ABC型(依赖Dna A)在功能上是可互换的。此外，F、R6K和rst1质粒的复制需要Dna A而不需要PriA，而在大肠杆菌中依赖于recA和不依赖Dna A的稳定复制需要PriA。我们提出在大肠杆菌中存在两种类型的复制子，一种依赖于ABC原始体，另一种依赖于phiXI74型原始体。2)酵母中GI向S的转变：交配信息素是一种抑制初始…的酵母肽更多的DNA复制，从而使靶细胞的细胞周期停滞在G1期。我们以前已经证明，它的作用是通过特定的膜受体和G蛋白发挥作用的。我们进一步证明，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，作为一个复合体，这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假定的染色体复制起点的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。我们已经发现了几种蛋白质，并纯化了其中的一种，100kD蛋白，以获得均一。CDC7激酶参与了酵母DNA复制的启动。我们构建了CDC7蛋白的高产表达系统，并制备了抗CDC7的抗体。对这些蛋白质的纯化和鉴定正在进行中。3)造血细胞的G1向S转变：细胞因子是一组主要由激活的T细胞分泌的多肽，作用于包括造血细胞在内的各种细胞。它们不仅支持细胞从G1到S的活性和细胞周期进程，而且还促进后续的分化。我们的研究主要集中在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。这些因素的影响如此相似，可以用我们最近的发现来解释，这两种细胞因子的受体都由两个多肽组成，即α和β，其中的β链是共享的。对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。在细胞周期中，T细胞主要处于Go/Gi期。当被抗原激活后，它们会分泌包括IL-3和GM-CSF在内的多种细胞因子，然后开始DNA复制。我们一直在较少地分析发起人

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