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， 高染色质相关高迁移率族(HMG)蛋白 真核生物在转录等核过程中发挥作用 和/或DNA复制。遗传方法是必要的 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NHP6A和HMG的基因 NHP6B，来源于酿酒酵母。特异性抗血清也是 针对这两种蛋白质而准备的。黄瓜缺失突变体的分析 NHP6A和NHP6B提供了这些HMG蛋白具有 真核细胞中重要但非必要的功能。我们 现在准备将遗传和生化方法结合起来 进一步揭示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NHP6蛋白的功能 致死性表型的生化表征 过表达NHP6的细胞。NHP6B的条件表达式HAS 是通过将基因置于诱导物的控制下实现的 推动者。在将这些单元格从 允许(不诱导)到不允许(诱导)增长 RNA、DNA和蛋白质合成发生变化的条件 至于染色质结构的变化。这些研究的结果 实验将有助于确定NHP6的体内功能。 本研究的一个重要方向是将遗传 用体外研究的方法来阐明分子 NHP6的作用机制。作为一步，从nhp6a中提取- Nhp6b-突变体将在体外转录反应中进行测试 以评估NHP6蛋白的可能作用。此外, NHP6A和NHP6B的DNA结合特异性(如果有的话)将是 硝化纤维素结合和凝胶迁移率变化的研究 化验。最后，确定NHP6蛋白之间的相互作用 对于其他染色质蛋白，将使用两种方法：(1) 用免疫沉淀法分离含NHP6的络合物 和凝胶电泳法鉴定的成分，以及(2)基因 对于与NHP6A/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的遗传系统 蛋白质之间的相互作用。 最后，由于NHP6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 将进行NHP6A。细胞的表型发生了突变 最高度保守的氨基酸的密码子将被评估 在上述化验中。这些实验将表明 哪些氨基酸对NHP6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.