MUTATIONAL ANALYSIS OF THE BIOLOGICAL FUNCTION OF HMG PR

所有真核生物的细胞核DNA都被包装成核蛋白结构

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.