TRANSCRIPTION OF ACANTHAMOEBA RNA POLYMERASE II GENES

阿米巴引起一种基本上无法治愈的角膜炎 在人类身上。治疗的困难与患者的 阿米巴原虫分化为耐药包囊。为了 采用合理的方法来治疗阿米巴，基于反义 因此，首先需要了解分子 水平阿米巴如何调节其生长的生命周期， 分化 我们以前在阿米巴基因转录方面的工作已经确定了 几个启动子元件和调节转录因子， TATA盒结合蛋白（TBP）基因表达所需的。TBP是 这是阿米巴所有基因转录所必需的。水平 TBP基因表达受一种独特的DNA结合蛋白调控， TPBF。TPBF又可以通过磷酸化调节。 这是一个详细描述调控蛋白的建议， TBP基因的启动子元件，并研究它们在基因表达中的作用。 在阿米巴分化过程中表达。它是假设 TPBF和其活性所需的辅活化剂在 包囊形成此外，TBP基因表达的抑制剂可能成为 在分化过程中活跃，从而关闭TBP基因表达。 具体而言，我们将构建和检测TBP基因启动子，以确定 积极和消极的调节因素的影响， 分化转录因子TFBF已被克隆， 一个完整的评价其结构和意义， 磷酸化TPBF刺激转录的机制将 通过研究其与辅活化剂APC 1的相互作用进行检查， 以及在启动期间发生的下游事件。的影响 将确定这些相互作用的差异，以便测试 上述假设。最后，共激活子APC 1和推定的 囊肿特异性阴性因子将被纯化，从长远来看， 克隆，以确定其机制。 这些研究与疾病治疗的相关性是直接的。他们将 确定控制或允许分化的生化途径。 这些途径可以通过反义方法特异性靶向， 以杀死细胞或阻止分化。在此，我们希望 积累基本的形成和必要的试剂，这或相关的 接近。

Acanthamoeba causes an essentially untreatable form of corneal keratitis in humans. Difficulty in treatment is linked to the ability of Acanthamoeba to differentiate into a drug-resistant cyst. In order to adopt a rational approach to Acanthamoeba therapy, based on antisense oligonucleotides, it is necessary to first understand at the molecular level how Acanthamoeba regulates its lifecycle of growth and differentiation. Our previous work on gene transcription in Acanthamoeba has identified several promoter elements and regulating transcription factors that are required for expression of the TATA box binding protein (TBP) gene. TBP is itself required for all gene transcription in Acanthamoeba. The level of TBP gene expression is regulated by a unique DNA binding protein called TPBF. TPBF can in turn be regulated by phosphorylation. This is a proposal to characterize in detail the regulatory proteins and promoter elements of the TBP gene, and to investigate their role in gene expression during Acanthamoeba differentiation. It is hypothesized that TPBF and a coactivator needed for its activity are inactivated during encystment. In addition, an inhibitor of TBP gene expression may become active during differentiation, thereby turning off TBP gene expression. Specifically, we will construct and assay TBP gene promoters to determine the effects of positive and negative regulatory elements during differentiation. The transcription factor TFBF has been cloned, permitting a complete evaluation of its structure and the significance of its phosphorylation. The mechanism of transcription stimulation by TPBF will be examined by investigating its interaction with a coactivator, APC1, as well as downstream events that occur during initiation. The effect of differentiation on these interactions will be determined in order to test the hypothesis stated above. Last, the coactivator APC1 and the putative cyst-specific negative factor will be purified, and in the longer term cloned in order to determine their mechanisms. The relevance of these studies to disease treatment is direct. They will identify the biochemical pathways that control or permit differentiation. These pathways can be specifically targeted by antisense approaches in order to either kill cells or to prevent differentiation. Here, we wish to accumulate the basic formation and reagents necessary for this or related approaches.