Gene Silencing Mechanisms in Drosophila

转化回原生生物体的基因通常会导致分散的同源拷贝的沉默，包括内源基因。会发生两种类型的静默。一种是转录的，涉及染色质结构的变化，证明是在沉默的转基因插入位点上积累的多梳状蛋白。另一种涉及转录后RNA周转，其方式类似于RNA干扰。新出现的证据表明，RNA干扰、转录后沉默和转录转基因沉默之间存在潜在的联系。在这个项目中，将采用遗传和分子方法来确定这些类型的沉默之间的任何关系。转基因果蝇的酒精脱氢酶(ADH)和白色(W)眼睛颜色基因将被用作模型系统。首先，将转化一系列表达或不表达基因的结构，以确定转录沉默是否需要沉默基因的表达。如果沉默只能在同源序列表达时发生，则RNA介导的过程将导致与转录沉默相关的染色质变化。其次，将研究RNAi类机制和转录沉默之间的相互关系。一种被预测为诱导ADH转录后沉默的构建体将被测试其产生这两种沉默的能力。接下来，将测试阻止转录后沉默的突变对配对敏感沉默的影响。此外，预计将产生与ADH启动子区域同源的双链RNA的转基因将被生产出来，并测试其诱导转录沉默的能力。对基因沉默的研究可能会揭示多细胞真核生物中基因表达和核动力学的新信息。这些沉默机制的一个作用是作为对病毒和转座子的保护，尽管在这一过程中已发现的许多基因也已知影响发育机制。了解基因沉默的基本过程有可能导致病毒保护和在基因治疗中的应用。

Genes transformed back into the organism of origin often cause silencing of dispersed homologous copies, including the endogenous gene. Two types of silencing occur. One is transcriptional and involves changes in chromatin structure as evidenced by the accumulation of Polycomb Group proteins on the silenced transgene insertion sites. The other involves post-transcriptional RNA turnover in a similar manner to RNA interference. Emerging evidence has indicated potential connections among RNA interference, post-transcriptional silencing and transcriptional transgene silencing. In this project, a genetic and molecular approach will be undertaken to determine any relationship among these types of silencing. Transgenes of the Alcohol dehydrogenase (Adh) and white (w) eye color genes in Drosophila will be used as a model system. First, a series of constructs will be transformed that do or do not exhibit gene expression to determine whether transcriptional silencing requires the expression of the silencing gene to occur. If silencing can only occur when the homologous sequences are expressed, an RNA mediated process would be implicated in causing the chromatin changes that are associated with the cases of transcriptional silencing. Secondly, the interrelationship between RNAi-like mechanisms and transcriptional silencing will be examined. A construct predicted to induce post-transcriptional silencing of Adh will be tested for its ability to produce either type of silencing. Next, mutations that block post-transcriptional silencing will be tested for their influence on pairing sensitive silencing. Further, a transgene predicted to generate double stranded RNA homologous to Adh promoter regions will be produced and tested for its ability to induce transcriptional silencing. Studies of gene silencing are likely to reveal new information about gene expression and nuclear dynamics in multicellular eukaryotes. One role of these silencing mechanisms is as a protection against viruses and transposons, although many of the genes that have been identified in the process are also known to affect developmental mechanisms. Understanding the basic processes of gene silencing has the potential to lead to virus protection and to have applications in gene therapy.