TRANS-ACTING FACTORS REGULATING ADH GENE EXPRESSION

描述：这项修订提案的总体目标是调查 决定转录水平的蛋白质-核酸相互作用 拟南芥和玉米ADH基因的活性对 发育和无氧应激。这位调查员的目标是将 关于染色质和DNA结构的信息以及关于蛋白质的信息 核酸相互作用在分子水平上定义转录 复合体聚集在这些基因上。在前一批款期内， 这位研究人员进行的体外和体内结合研究确定了 G-box和Half G-box元件的数量，它们在 基因激活。植物提取物中GBF因子的筛选 与这些元素相关联已经重要地证明了这些 在表达ADH的根组织和不表达ADH的组织中存在因子 根本不表达adh(即，叶子)。活体足迹表明 这些GBF蛋白仅与G-box启动子元件结合 转录活跃的组织。蛋白质因子基因的克隆 发现了一个有趣的GF14家族 与G-box蛋白复合体相关但不相关的蛋白质 直接与DNA结合。费尔博士已经确定了 GF14蛋白与哺乳动物钙/钙调素蛋白激酶II依赖 色氨酸和酪氨酸羟化酶激活剂(14-3-3蛋白)以及 蛋白激酶C的KCIP抑制剂。这表明G14可能起作用。 在ADH激活中的调节作用.GBF蛋白已从 玉米和拟南芥都是。使用这些克隆的G14和GBF cDNA， 研究人员建议定义这些因素之间的相互关系 存在于不同位置的因子及其结合部位 每一位发起人。将这些因素分组到类中 DNase I和DMS足迹和迁移率变化的基础将决定 单个蛋白质是否识别一个或多个启动子序列 以及三个ADH基因中的各种元素是否真的是 被相似的蛋白质组识别。这些实验还旨在 确定GF14如何与GBF相互作用，以及其他蛋白质是否 与这个建筑群联系在一起。这项提议的第二个目标是 通过相互关联监测体内ADH启动子的激活状态 在带有体内足迹的转基因植物中表达。这些研究 将决定体外定义的关键结合位点 分析实际上改变了活体中蛋白质的结合和表达。 结合部位是半个G盒，可能与之相互作用 异二聚体的GBF，所以这个提议的最终目标是寻找 用于与GBF互动的其他配对合作伙伴。

DESCRIPTION: The overall goal of this revised proposal is to investigate the protein-nucleic acid interactions which define the transcriptional activity of the Arabidopsis and maize Adh genes in response to development and anaerobic stress. This investigator aims to integrate information on chromatin and DNA structures with information on protein- nucleic acid interactions to molecularly define the transcriptional complexes assembled on these genes. During the previous grant period, in vitro and in vivo binding studies by this investigator have defined a number of G-box and half G-box elements which bind proteins during gene activation. Screening of plant extracts for the GBF factors associated with these elements has importantly demonstrated that these factors are present in root tissues expressing Adh as well as tissues not expressing Adh at all (i.e., leaves). In vivo footprints indicate that these GBF proteins are bound to G-box promoter elements only in transcriptionally active tissues. Cloning of cDNAs for protein factors associated with G-boxes has uncovered an interesting family of GF 14 proteins which are associated with G-box protein complexes but not directly bound to the DNA. Dr. Ferl has identified homologies between GF14 proteins and mammalian Ca2+/calmodulin protein kinaseII-dependent activators of Trp and Tyr hydroxylases (14-3-3 proteins) and also the KCIP inhibitors of protein kinase C. This suggests that G14 may serve a regulatory role in Adh activation.GBF proteins have been cloned from both maize and Arabidopsis. Using these cloned G14 and GBF cDNAs, the investigator proposes defining the interrelationships between these factors and their binding sites which exist at different positions in each of the promoters. Grouping of these factors into classes on the basis of DNase I and DMS footprints and mobility shifts will determine whether individual proteins recognize one or multiple promoter sequences and whether the various elements in the three Adh genes are actually recognized by similar sets of proteins. These experiments also aim to define how GF14 interacts with GBF and whether other proteins are associated with this complex. The second goal of this proposal is to monitor the activation state of Adh promoters in vivo by correlating expression in transgenic plants with in vivo footprints. These studies will determine whether critical binding sites defined by in vitro analysis actually change protein binding and expression in vivo.Many of the binding sites are half G-boxes which potentially interact with heterodimeric GBFs and so the final goal of this proposal is to search for additional pairing partners that interact with GBF.