MUTATIONAL ANALYSIS OF THE BIOLOGICAL FUNCTION OF HMG PR

所有真核生物的核DNA都被包装成核蛋白结构 叫做染色质。一组非组蛋白染色质相关蛋白 是高迁移率族(HMG)蛋白。尽管HMG蛋白有 经过深入调查，它们的功能尚不清楚。然而， 它们与染色质结构的改变有关， 在转录和DNA等细胞过程中出现 复制。为了确定HMG的生物学功能 蛋白质，我们建议使用遗传和生化的组合 酵母菌HMG蛋白的研究方法 酿酒。酵母HMG蛋白S2、S3和S4的功能将是 通过突变编码每种蛋白质的基因并检测 这种变化对各种细胞过程的影响。 还没有从任何生物体中克隆到HMG基因，所以我们首先要克隆 酵母HMG蛋白的编码基因。由于酵母中的HMG蛋白 我们打算用这些蛋白质来克隆基因。一 这种方法将需要对每个HMG蛋白的部分进行测序，合成 根据氨基酸序列预测的适当寡核苷酸和 以寡核苷酸为杂交探针鉴定HMG 基因组酵母DNA文库中的基因。作为第二种方法，我们将提高 针对每个纯化的HMG蛋白的抗体，并用它们来筛选 插入细菌表达的酵母DNA序列文库 向量。一旦基因被克隆，它们将被测序。然后 每个HMG基因都会在体外进行诱变，然后再转化回酵母中， 替换同源野生型HMG基因。因此，只有突变的HMG 蛋白质将在转化子中合成。分析了一系列 具有改变的HMG蛋白的细胞中的生化过程将定义 第一次，HMG蛋白的细胞功能。此外, Hmg突变体的基因外抑制物将被分离和分析 建立HMG蛋白与其他染色质蛋白的相互作用。 拟议的实验将为以下方面提供基础和方向 未来HMG作用的分子机制的体外研究。

All eukaryotic nuclear DNA is packaged into a nucleoprotein structure called chromatin. One group of non-histone chromatin-associated proteins is the high mobility group (HMG) proteins. Although the HMG proteins have been intensively investigated, their function remains unclear. However, they have been implicated in the alteration of chromatin structures which appear during cellular processes such as transcription and DNA replication. In order to determine the biological functions of the HMG proteins, we propose to use a combination of genetic and biochemical approaches to study the HMG proteins in the yeast Saccharomyces cerevisiae. The functions of the yeast HMG proteins S2, S3 and S4 will be analyzed by mutagenizing the gene encoding each protein and assaying the effects of such alterations on various cellular processes. No HMG genes have been cloned from any organism so initially we will clone the genes encoding the yeast HMG proteins. Since the yeast HMG proteins have been purified, we intend to use the proteins to clone the genes. One approach will entail sequencing part of each HMG protein, synthesizing appropriate oligonucleotides as predicted from the amino acid sequences and using the oligonucleotides as hybridization probes to identify the HMG genes in a genomic-yeast-DNA library. As a second approach, we will raise antibodies against each purified HMG protein and use them to screen a library of yeast DNA sequences inserted into a bacterial expression vector. Once the genes have been cloned, they will be sequenced. Then each HMG gene will be mutagenized in vitro and transformed back into yeast, replacing the homologous wild type HMG gene. Thus, only the mutant HMG protein will be synthesized in the transformant. Analysis of a range of biochemical processes in cells with altered HMG proteins will define, for the first time, the cellular function of an HMG protein. In addition, extragenic suppressors of the HMG mutants will be isolated and analyzed to establish the interactions of HMG proteins with other chromatin proteins. The proposed experiments will provide the foundation and direction for future in vitro studies on the molecular mechanisms of HMG function.