TRANSCRIPTION FACTORS REGULATING CHORION GENE PROMOTERS

拟议研究的目标是阐明机制 确定转录的组织和时间特异性， activation. 所使用的模型系统是昆虫绒毛膜发生，这是一个过程 包括对一些基因表达的严格发育控制， 相关基因，其中每一个都在Eh 转录水平。 这些研究将集中在分子生物学上， 调控绒毛膜基因表达的DNA-蛋白质相互作用及其对细胞增殖的影响 分析这种调节相互作用的保守程度 物种之间。 具体目标是： 家蚕绒毛膜基因cis-101的定位及功能分析 监管要素。 体外识别绒毛膜基因启动子的区域 通过果蝇滤泡细胞核蛋白和克隆果蝇 将通过DNA酶I保护鉴定转录因子， 甲基化干扰研究。 这些地区将成为 体外诱变和这些突变对组织的影响， 将在转基因中分析启动子激活时间特异性 果蝇 家蚕绒膜调控基因的克隆与分析 表情 家蚕滤泡细胞cDNA表达文库的构建 筛选与合成寡核苷酸的结合，所述合成寡核苷酸代表 滤泡核蛋白的识别位点。 此外， 与果蝇绒毛膜基因转录相关的转录因子 将用于序列同源性筛选。 代表以下cDNA的克隆： 推定的调控基因将在结构上表征并用于 蛋白质的生产在细菌或组织培养中表达 系统. 在无细胞环境中重建转录控制相互作用 系统. 来自卵泡的具有转录活性的核提取物 组织培养细胞系将补充果蝇， 家蚕的调控蛋白及其对体外细胞增殖的影响 分析绒毛膜和非绒毛膜基因启动子的转录。 物种间补充研究将特别揭示 转录调控中的功能保守程度 交互. 上述拟议的研究将提供有关分子机制的信息 参与一类基因的转录调控， 发展控制 调节基因的种间保守性 相互作用，如在这种昆虫系统中观察到的，也是明显的， 哺乳动物系统，尽管仅在少数情况下广泛表征。 转录水平保守程度的基本信息 物种之间的调节相互作用可能与 人类疾病的动物模型的发展。

The goal of the proposed research is the elucidation of mechanisms determining the tissue and temporal specificity of transcriptional activation. The model system to be used is insect choriogenesis, a process involving stringent development controls on the expression of a number of related genes, each of which is differentially regulated at eh transcriptional level. The studies will focus on the molecular biology of DNA-protein interactions regulating the expression of chorion genes and the analysis of the extent of conservation of such regulatory interactions between species. Specific aims will be: Localization and functional analysis of silkmoth chorion gene cis- regulatory elements. Regions on chorion gene promoters recognized in vitro by Drosophila follicular cell nuclear proteins and cloned Drosophila transcription factors will be identified through DNase I protection and methylation interference studies. These regions will be the targets of in vitro mutagenesis and the effect of these mutations on the tissue and temporal specificity of promoter activation will be analyzed in transgenic Drosophila. Cloning and characterization of silkmoth genes regulating chorion gene expression. Silkmoth follicular cell cDNA expression libraries will be screened for binding to synthetic oligonucleotides representing the recognition sites of follicular nuclear proteins. In addition, cDNAs of Drosophila transcription factors associated with chorion gene transcription will be used in sequence homology screening. Clones representing cDNAs of putative regulatory genes will be structurally characterized and used for the production of proteins in bacterial or tissue culture expression systems. Reconstruction of transcriptional control interactions in cell-free systems. Transcriptionally active nuclear extracts derived from follicles and tissue culture cell lines will be supplemented with Drosophila and silkmoth putative regulatory proteins and the effect on the in vitro transcription of chorion and non-chorion gene promoters will be analyzed. The interspecies supplementation studies will be especially revealing on the extent of functional conservation in transcriptional regulatory interactions. The above proposed studies will provide information on molecular mechanisms involved in transcriptional regulation of a class of genes under stringent developmental control. Interspecies conservation of regulatory interactions, such as observed in this insect system, are also evident in mammalian systems, although extensively characterized in only a few cases. Basic information on the extent of conservation of transcriptional regulatory interactions between species could be relevant in the development of animal models for human disease.