Molecular analysis of Transcription Factors Regulating phyA Gene Expression

光敏色素是一种调节主开关，控制基因表达对光的反应。这项研究的长期目标是确定这种调控的分子机制。光敏色素对其自身phyA基因的快速转录抑制为这一目的提供了一个极好的模型系统。本提案的具体目标是：(a)鉴定和分离编码dna结合蛋白的重组克隆，这些蛋白可以识别phyA启动子中功能定义的顺式作用元件；(b)描述这些克隆因子的功能域；(c)确定最近发现的抑制因子RF1在光敏色素激活时破坏phyA转录的分子机制。实验方法将包括：(a)用代表功能定义的调控元件的寡核苷酸探针筛选表达文库，用于分离编码同源因子的cDNA克隆；(b)通过体外结合试验确定这些因子的dna识别结构域；(c)基因转移测定以确定核定位序列；(d)酵母转染和嵌合融合蛋白体外转录测定，以确定这些因子的转录激活或抑制域。这项研究的意义在于，它将有助于确定一个中心调控分子控制基因表达的机制。由于光敏色素在其他真核生物中似乎没有分子对应物，阐明这一机制将拓宽我们对真核细胞如何调节基因表达以响应环境刺激的理解。%%%%植物通过开启与生长、光合作用和其他功能有关的基因来响应光。植物细胞中负责这种作用的关键调节分子是光敏色素。这种分子与蛋白质和基因相互作用，激活遗传信息，促进生长等。为了了解分子机制，有必要确定受影响的基因区域，以及参与激活过程的蛋白质。这些研究将使我们更好地了解如何调节植物生长。

Phytochrome is a regulatory master switch that controls gene expression in response to light. The long term goal of the proposed research is to define the molecular mechanism underlying this regulation. The rapid transcriptional repression that phytochrome imposes on its own phyA genes provides an excellent model system for this purpose. The specific objectives of this proposal are: (a) to identify and isolate recombinant clones encoding DNA-binding proteins that recognize functionally defined cis-acting elements in phyA promoters; (b) to delineate the functional domains of such cloned factors; (c) to determine the molecular mechanism by which a recently identified repressor factor, RF1, disrupts phyA transcription in response to phytochrome activation. Experimental approaches will include: (a) screening expression libraries with oligonucleotide probes representing functionally defined regulatory elements for isolation of cDNA clones encoding the cognate factors; (b) in vitro binding assays to define the DNA-recognition domains of these factors; (c) gene transfer assays to delineate nuclear localization sequences; (d) yeast transfection and in vitro transcription assays with chimeric fusion proteins to define transcriptional activation or repression domains in these factors. The significance of this research is that it will help define the mechanism by which a central regulatory molecule controls gene expression. Because phytochrome appears to have no molecular counterpart in other eukaryotes, elucidation of this mechanism will broaden our understanding of how eukaryotic cells regulate gene expression in response to environmental stimuli. %%%% Plant respond to light by turning on genes that are involved with growth, photosynthesis, and other functions. A key regulatory molecule in plant cells that is responsible for this effect is phytochrome. This molecule interacts with proteins and genes to activate genetic messages for growth, etc. To understand the molecular mechanism it is necessary to identify the regions of the gene which are affected, and the proteins which are involved in the activation process. These studies will lead to greater understanding of how to regulate plant growth.