MUTATIONAL ANALYSIS OF THE FUNCTIONS OF HMG PROTEINS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date5/1/924/30/97

Funding

  • National Institutes of Health
  • National Institutes of Health: $178,250.00
  • National Institutes of Health
  • National Institutes of Health: $164,801.00

ASJC

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

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