MUTATIONAL ANALYSIS OF THE FUNCTIONS OF HMG PROTEINS

所有真核生物的DNA都被包装成核蛋白结构 叫做染色质。 体外研究表明， 来自高等植物的染色质相关高迁移率族（HMG）蛋白 真核生物在核过程中发挥作用， 和/或DNA复制。 遗传学方法是必要的， 直接阐明HMG蛋白在细胞中的功能。 因此，我们克隆了两种HMG蛋白NHP 6A和NHP 6B的基因。 NHP 6B，来自酿酒酵母。 还检测了特异性抗血清。 针对这两种蛋白质制备。 缺失突变体的分析 NHP6A和NHP6B提供了这些HMG蛋白具有以下功能的证据： 在真核细胞中重要但非必需的功能。 我们 现在正准备将联合收割机遗传学和生物化学方法结合起来， 进一步揭示了这些蛋白质在细胞中的作用。 我们建议通过以下方法来破译NHP 6蛋白的功能： 生物化学特征的致命表型的原因， 过表达NHP 6的细胞。 NHP 6B的条件表达式具有 通过将基因置于诱导型 启动子 这些细胞将被分析，在将它们从 允许（非诱导）到非允许（诱导）生长 条件下，RNA，DNA和蛋白质合成的变化以及 至于染色质结构的变化。 的结果予以 这些实验将有助于确定NHP 6的体内功能。 联合收割机是本研究的一个重要方向， 体外研究方法来阐明 NHP6的功能。 作为一个步骤，从nhp6a- 将在体外转录反应中测试nhp6b-突变体 以评估NHP 6蛋白的可能作用。 此外，本发明还提供了一种方法， NHP 6A和NHP 6B的DNA结合特异性，如果有的话， 通过硝酸纤维素结合和凝胶迁移率变化研究 测定。 最后，为了确定NHP 6蛋白的相互作用， 对于其他染色质蛋白，将使用两种方法：（1） 通过免疫沉淀分离含NHP 6的复合物 以及通过凝胶电泳鉴定的组分，和（2）基因 与NHP 6A/6B相互作用的蛋白质将使用一种新的 β-半乳糖苷酶合成依赖于 蛋白质相互作用 最后，由于NHP 6蛋白是一种新型DNA的原型， 结合基序称为HMG盒，结构/功能分析， NHP6A将被执行。 细胞突变的表型， 将评估最高度保守的氨基酸的密码子 在上述测定中。 这些实验将表明 哪些氨基酸对NHP 6的功能至关重要。

All eukaryotic DNA is packaged into a nucleoprotein structure called chromatin. In vitro studies have suggested that the chromatin-associated High Mobility Group (HMG) proteins from higher eukaryotes play a role in nuclear processes such as transcription and/or DNA replication. A genetic approach is necessary to elucidate directly the functions of HMG proteins in the cell. Therefore, we have cloned the genes for two HMG proteins, NHP6A and NHP6B, from Saccharomyces cerevisiae. Specific antisera were also prepared against both proteins. Analysis of deletion mutants in NHP6A and NHP6B provided evidence that these HMG proteins have important, but nonessential, functions in the eukaryotic cell. We are now poised to combine genetic and biochemical approaches to reveal further the roles of these proteins in the cell. We propose to decipher the functions of the NHP6 proteins by biochemically characterizing the cause of the lethal phenotype of cells overexpressing NHP6. Conditional expression of NHP6B has been achieved by placing the gene under the control of an inducible promoter. These cells will be analyzed, after shifting them from permissive (noninducing) to nonpermissive (inducing) growth conditions, for changes in RNA, DNA and protein synthesis as well as for changes in chromatin structure. The results of these experiments will help define the in vivo functions of NHP6. An important direction of this research is to combine the genetic approaches with in vitro studies to elucidate the molecular mechanisms of NHP6 function. As one step, extracts from nhp6a- nhp6b- mutants will be tested in in vitro transcription reactions to assess the possible roles of the NHP6 proteins. In addition, the DNA-binding specificity, if any, of NHP6A and NHP6B will be investigated by nitrocellulose binding and gel mobility shift assays. Lastly, to determine the interactions of the NHP6 proteins with other chromatin proteins two approaches will be used: (1) NHP6-containing complexes will be isolated by immunoprecipitation and the components identified by gel electrophoresis, and (2) genes for proteins interacting with NHP6A/6B will be cloned using a novel genetic system in which beta-galactosidase synthesis depends upon protein-protein interactions. Finally, since the NHP6 proteins are prototypes for a novel DNA- binding motif called the HMG Box, a structure/function analysis of NHP6A will be undertaken. The phenotypes of cells mutated in the condons for the most highly conserved amino acids will be assessed in the assays described above. These experiments will indicate which amino acids are critical for NHP6 function.