TRANS-ACTING FACTORS REGULATING ADH GENE EXPRESSION

描述：本修订提案的总体目标是调查 蛋白质-核酸相互作用决定了转录 拟南芥和玉米Adh基因响应于 发育和厌氧应激。 本研究旨在整合 染色质和DNA结构的信息，以及蛋白质的信息- 核酸相互作用，以分子方式定义转录 在这些基因上组装的复合物。 在上一个赠款期间， 本研究者进行的体外和体内结合研究已确定 许多G-盒和半G-盒元件，其在蛋白质合成过程中结合蛋白质。 基因激活 GBF因子植物提取物的筛选 与这些元素相关的重要证据表明， 因子存在于表达Adh的根组织以及不表达Adh的组织中。 完全表达Adh（即，叶子）。 体内足迹表明， 这些GBF蛋白仅与G-box启动子元件结合， 转录活性组织。 蛋白质因子cDNA的克隆 与G盒相关的研究发现了一个有趣的GF 14家族 与G盒蛋白复合物相关但不相关的蛋白质 直接与DNA结合。 费尔博士发现了 GF 14蛋白与哺乳动物钙/钙调蛋白蛋白激酶II依赖性 Trp和Tyr羟化酶（14-3-3蛋白）的激活剂，以及 蛋白激酶C的KCIP抑制剂。 这表明，G14可能有助于 在Adh活化中的调节作用。GBF蛋白已从 玉米和拟南芥。 利用这些克隆的G14和GBF cDNA， 研究人员建议确定这些之间的相互关系， 因子及其结合位点存在于不同的位置， 每一个发起人。 将这些因素分类， DNA酶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 被相似的蛋白质识别。 这些实验还旨在 确定GF 14如何与GBF相互作用，以及其他蛋白质是否 与这个复杂的。 该提案的第二个目标是 监测体内Adh启动子的激活状态， 在具有体内足迹的转基因植物中表达。 这些研究 将确定是否关键结合位点定义的体外 分析实际上改变了体内蛋白质的结合和表达。 结合位点是半个G盒， 异二聚体GBF，因此本提案的最终目标是寻找 与GBF相互作用的其他配对伙伴。

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.