MUTATIONAL ANALYSIS OF THE BIOLOGICAL FUNCTION OF HMG PR

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date12/1/845/31/88

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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