MECHANISMS OF TISSUE-SPECIFIC GENE EXPRESSION

组织特异性基因表达机制

基本信息

批准号：
2175494
负责人：
THOMAS P MANIATIS
金额：
$ 25.78万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1981
资助国家：
美国
起止时间：
1981-07-01 至 1995-06-30
项目状态：
已结题

项目摘要

The long term objective of the proposed research is to understand the mechanisms involved in temporal and tissue-specific gene expression during Drosophila development. We are approaching this problem by studying alcohol dehydrogenase (Adh) gene expression, and the function and expression of nautilus (nau), a presumptive myogenic regulatory gene. The Adh studies will involve characterization of cis-acting regulatory sequences involved in tissue-specific Adh expression in larvae and adults, and the identification of proteins that regulate Adh gene expression. We propose to study the structure, function and expression of proteins that bind specifically to DNA sequences required for normal Adh gene expression in larvae (BBF-1, BBF-2, ABF-1) and adults (AEF-1). The amino acid sequences of these proteins will be deduced from the DNA sequences of the corresponding cDNA clones, and their pattern of expression studied by in situ hybridization and RNAse protection methods. The function of these proteins will be determined by genetic studies, cotransfection experiments in cultured Drosophila cells, and by in vitro transcription analyses. A binding site for the mammalian transcription factor C/EBP has been implicated in the positive control of Adh gene expression in adults. We therefore plan to clone and characterize Drosophila genes which encode proteins that bind to this sequence to determine whether Adh gene regulation involves a functional analog of the mammalian C/EBP gene. If successful, the Adh studies should provide a detailed understanding of the protein-DNA and protein-protein interactions involved in regulating the complex pattern of temporal and tissue-specific expression of Adh. The primary goal of the nau gene project is to understand the regulatory mechanisms involved In the earliest stages of myogenesis in Drosophila. The DNA sequences required for nau expression in muscle precursor cells will be identified in P element transformation experiments, and these sequences will be used to identify genes that control nau gene expression. The latter will be accomplished by screening a lambda gt 11 cDNA expression library for clones that express proteins that bind specifically lo the regulatory sequences. A variety of molecular approaches will be used to determine whether the nau gene is directly involved in the regulation of gene expression in muscle precursor cells. Finally, we plan to identify the targets of nau gene function using the polymerase chain reaction in conjunction with methods to isolate specific DNA-protein complexes. The ultimate objective of the Adh and nau gene studies is to establish a link between the regulatory events involved in the terminal differentiation of specific cell types, and the well-characterized regulatory hierarchy involved in the establishment of specific segments in the early embryo.

拟议研究的长期目标是了解涉及时间和组织特异性基因表达的机制在果蝇发育过程中。我们正在通过以下方式解决这个问题研究乙醇脱氢酶（Adh）基因表达及其功能以及鹦鹉螺（nau）的表达，这是一种推定的生肌调节基因。 Adh 研究将涉及顺式作用调节的表征参与幼虫组织特异性 Adh 表达的序列成人以及调节 Adh 基因的蛋白质的鉴定表达。我们建议研究其结构、功能和表达与正常细胞所需的 DNA 序列特异性结合的蛋白质 Adh 基因在幼虫（BBF-1、BBF-2、ABF-1）和成虫（AEF-1）中的表达。这些蛋白质的氨基酸序列将从DNA中推导出来相应 cDNA 克隆的序列及其模式通过原位杂交和RNA酶保护方法研究表达。这些蛋白质的功能将由遗传学研究确定，在培养的果蝇细胞中进行共转染实验，并通过体外转录分析。哺乳动物转录的结合位点因子C/EBP与Adh基因的阳性控制有关成人的表达。因此，我们计划克隆并表征果蝇基因编码与该序列结合的蛋白质确定 Adh 基因调控是否涉及 Adh 的功能类似物哺乳动物 C/EBP 基因。如果成功，Adh 研究应该提供详细了解蛋白质-DNA和蛋白质-蛋白质涉及调节时间和时间的复杂模式的相互作用 Adh 的组织特异性表达。 nau 基因项目的主要目标是了解参与肌生成最早阶段的调节机制果蝇。肌肉中 nau 表达所需的 DNA 序列前体细胞将在P元素转化中被识别实验，这些序列将用于识别基因控制 nau 基因表达。后者将通过以下方式完成筛选 lambda gt 11 cDNA 表达文库，寻找表达的克隆特异性结合调节序列的蛋白质。品种齐全分子方法将用于确定 nau 基因是否直接参与肌肉基因表达的调节前体细胞。最后，我们计划确定nau基因的靶点使用聚合酶链反应与方法结合使用的功能分离特定的 DNA-蛋白质复合物。该项目的最终目标是 Adh 和 nau 基因研究的目的是建立调控之间的联系涉及特定细胞类型的终末分化的事件，以及涉及的特征明确的监管层级在早期胚胎中建立特定片段。