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。 一类非组蛋白染色质相关蛋白 高迁移率族（HMG）蛋白。 虽然HMG蛋白具有 虽然已经深入研究，但其功能仍不清楚。 然而，在这方面， 它们与染色质结构的改变有关， 出现在细胞过程中，如转录和DNA 复制的 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结合 酵母中HMG蛋白的研究方法 啤酒。 酵母HMG蛋白S2、S3和S4的功能将在下文中详细描述。 通过诱变编码每种蛋白质的基因并测定 这些变化对各种细胞过程的影响。 没有HMG基因已克隆从任何生物体，所以最初我们将克隆 编码酵母HMG蛋白的基因。 由于酵母HMG蛋白 已经纯化，我们打算用蛋白质克隆基因。 一 这种方法需要对每个HMG蛋白的一部分进行测序， 根据氨基酸序列预测的适当寡核苷酸， 使用寡核苷酸作为杂交探针来鉴定HMG 基因组酵母DNA文库中的基因。 作为第二种方法，我们将提出 针对每种纯化的HMG蛋白的抗体，并使用它们来筛选 插入细菌表达的酵母DNA序列文库 vector. 一旦基因被克隆，它们将被测序。 然后 每个HMG基因将在体外诱变并转化回酵母中， 替换同源野生型HMG基因。 因此，只有突变的HMG 蛋白质将在细胞内合成。 分析一系列 HMG蛋白改变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，本发明还提供了一种方法， 将分离和分析HMG突变体的基因外抑制子， 建立HMG蛋白与其他染色质蛋白的相互作用。 这些实验将为今后的研究提供基础和方向。 HMG功能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.