PHOTOREGULATED GENE EXPRESSION

这一建议涉及基因表达的机制， 由光线调节。 实验生物是植物拟南芥 thaliana，因其在分子遗传学方面的优点而闻名， 分析。 建议的研究涉及光的分析- 响应元件（LRE）的特征光调控基因和 蛋白质分子家族的表征（G-盒结合 因子或GBF）结合这些LRE的共同元件。 在 此外，光照射改变GBF的机制 将被审查。 高等真核生物的特征是遗传系统， 复杂的方式来发展和环境刺激。 一个部件 这种复杂性是基因家族的常见现象， 它们可以执行相关但不同的功能。 的一个例子 复杂性是转录因子的Fos/Jun家族， bZIP蛋白家族在哺乳动物发育中的作用， 肿瘤发生 GBF家族也是bZIp蛋白和GBF 4，与 Fos与其他家族成员形成异源二聚体，但不能 均二聚化。 这两个家庭的特点是家庭内遗传 监管制度。 有人建议利用分子遗传学的力量 在拟南芥中进行分析，以探索GBF家族的特性， proteins. 我们会透过研究 启动子-报告基因构建体在转基因植物和 瞬时测定系统。 这些构造的表达将是 与内源拟南芥基因的表达相比。 大 在细胞质中发现GBF蛋白的一部分。 的性质 GBF序列影响这些细胞的细胞内分布， 蛋白质将被确定。 GBF的DNA结合特性是 受光的影响。 显然，这种改变的本质 涉及磷酸化，将被检查：这项研究将包括一个 确定该反应的动力学，光的性质 要求，以及修改的GBF序列。 为这些 研究，与其他拟议的研究一样，单克隆抗体将 获得并用于区分单个GBF家族成员。 转录系统的特点是非常具体的蛋白质-蛋白质 交互. 为了进一步了解GBF家族的作用 光调控基因表达中的蛋白质， 将通过使用一种新的方法来选择具有该bZIP蛋白家族的蛋白质。 酵母中的遗传筛选。 将使用两种方法来评估 GBF家族成员的功能。 基于PCR的直接筛选 将用于搜索含有标记GBF基因的拟南芥植物 通过T-DNA插入。 此外，使用转基因植物表达 GBF-反义构建体正在探索中。 的初步结果 后一种方法表明复杂的家族内调节网络。 基于这些反义实验的结果， 提出了一种互补系统，从而可以探索 GBF家族成员的显著特征。

This proposal concerns the mechanism by which gene expression is regulated by light. The experimental organisms is the plant Arabidopsis thaliana, well known for its merits concerning molecular genetic analyses. The proposed studies concern both the analyses of light- response-elements (LREs) that characterize light-regulated genes and the characterization of a family of protein molecules (the G-box-binding factors or GBFs) that bind to a common element of these LREs. In addition, the mechanism by which irradiation with light modifies the GBFs will be examined. Higher eukaryotes are characterized by genetic systems that respond in complex ways to developmental and environmental stimuli. One component of this complexity is the common occurrence of gene families, the members of which may perform related but distinct functions. An example of such complexity is the Fos/Jun family of transcription factors and the role that this bZIP family of proteins plays in mammalian development and oncogenesis. The GBF family are also bZIp proteins and GBF4, as with Fos, forms heterodimers with the other family members but fails to homodimerize. Both families are characterized by intrafamily genetic regulatory systems. It is proposed to use the power of molecular genetic analyses in Arabidopsis to explore the properties of the GBF family of proteins. The essential features of LREs will be examined by studying the expression of promoter-reporter constructs in both transgenic plants and transient assay systems. The expression of these constructs will be compared with the expression of endogenous Arabidopsis genes. A large fraction of the GBF proteins are found in the cytoplasm. The nature of the GBF sequences that affect the intracellular distribution of these proteins will be determined. The DNA binding properties of GBF are influenced by light. The nature of this modification, apparently involving phosphorylation, will be examined: this study will include a determination of the kinetics of this reaction, the nature of the light requirements, and the GBF sequences that are modified. For these studies, as with other proposed studies, monoclonal antibodies will be obtained and used to distinguish the individual GBF family members. Transcription systems are characterized by very specific protein-protein interactions. In order to further understand the role of the GBF family of proteins in light-regulated gene expression, proteins that interact with this family of bZIP proteins will be selected via the use of a genetic screen in yeast. Two approaches will be used to evaluate the function of the individual GBF family members. A direct PCR-based screen will be used to search for Arabidopsis plants containing GBF genes tagged by T-DNA insertion. In addition, the use of transgenic plants expressing GBF-antisense constructs is being explored. Preliminary results from this latter approach indicate a complex intrafamily regulatory network. Based on the results from these antisense experiments, a novel complementation system is proposed whereby it may be possible to explore the distinguishing features of the GBF family members.