Fountain Research Group

MOLECULAR BIOLOGY AND BIOINFORMATIC STUDIES ON HEAT SHOCK PROTEIN REGULATION

Joel Moore, Katie De Rosa, Andrew Slater and Chris Van Vranken are working on the regulatory system for Class I heat shock proteins in prokaryotic organisms. Their focus is on the binding of the regulatory protein HrcA to its target DNA sequence. The target sequence is a DNA inverted repeat called CIRCE that lies in the promoter region of the heat shock protein operon. The HrcA binds to this inverted repeat and inhibits transcription. Abrupt changes in temperature, pH, and salt concentration results in the dissociation of the HrcA protein thus allowing expression of heat shock proteins that aid in the refolding of proteins.

The HrcA and CIRCE regulatory mechanism is highly conserved in prokaryotic organisms and lends itself to a bioinformatics study. Chris Van Vranken has done extensive sequence alignments and phylogenetic analysis on a large number of HrcA proteins and has identified a highly conserved region near the N-terminus that contains a helix-turn-helix motif. Chris is currently working on docking these helices into the conserved inverted repeat DNA sequence (CIRCE) to try to identify possible structures that would show sequence specific interactions. If successful this will be one of a few examples of the use of in silico identification and structure generation of a DNA protein complex. Chris is also populating a database with the locations of inverted repeat sequences within sequenced bacterial genomes using the Perl programming language and the BioPerl Modules. He intends to identify functionally significant inverted repeats based on prevalence, and compare these to inverted repeats of known function.

Joel Moore and Katie De Rosa are working on a dual plasmid system to study the heat shock response in vivo. They are currently constructing a reporter plasmid that contains a CIRCE sequence in the promoter region of a beta-galactosidase gene. The plasmid is constructed so that different CIRCE sequences can be inserted and their response to heat shock studied. The second plasmid contains the HrcA gene from Bacillus subtilis. In this experiment the HrcA gene is expressed producing HrcA protein that inhibits the transcription of the beta-galactosidase gene on the reporter plasmid. When exposed to heat shock, the HrcA:CIRCE complex dissociates allowing the transcription and then translation of beta-galactosidase gene. Measuring the beta-galactosidase activity will provide and estimate for the stability of the HrcA:CIRCE complex and the temperature at which heat shock is initiated.

Andrew Slater is working on the expression and purification of the HrcA protein. HrcA protein forms an aggregate when over expressed making it difficult to study via in vitro methods. Andrew will be attempting several techniques to disrupt the HrcA aggregates and renature the HrcA protein into an active form for DNA binding studies.

SYNTHESIS OF MODIFIED DNA NUCLEOTIDES TO CREATE METAL BINDING HELICES.

David Critton will be continuing the research started by Nathan Genung, Dayle Acquilano, and Joel Moore. He is currently synthesizing a catechol nucleoside. When two catechols nucleotides are positioned opposite one another in a duplex they can form a pocket that is capable of binding metal ions. This will place a metal ion directly in the helix. Lanthanide metals like europium have fluorescence properties and can be placed in the DNA duplex using this method. The fluorescent properties of europium can be studied in the DNA duplex. This system can be utilized to study charge transfer in DNA and can also be used to label DNA fragments for detection using a phosphorimager.

CHARACTERIZATION OF THE SESQUITERPENES IN PATCHOULI OIL.

Michael Valerio is working on the separation and identification of sesquiterpenes in patchouli oil using chromatographic techniques. He is taking crude patchouli oil and separating fractions based on their polarity using column chromatography and thin layer chromatography. The various fractions will be run on a capillary gas chromatograph to assess their purity and then on a capillary gas chromatograph- mass spectrometer to identify individual components in each fraction. Using chemical modification reactions he should be able to obtain pure sesquiterpene compounds. These samples can then be used as substrates to study the biochemical pathways for the synthesis of other sesquiterpenes.

DETECTION OF HEAVY METALS IN MOUNT SAINT HELEN’S VOLCANIC SOIL.

Natalie Dart’s research project uses the recently acquired x-ray fluorescence instrument to detect the presence of heavy metals in sediments and soils. This project is a collaboration with Dr. Jonathan Titus in the Biology Department. Dr. Titus is looking at the amount of organic material in the soil and its relation to the growth of specific plant species. Natalie will be identifying the elemental composition of Mount Saint Helen’s volcanic soil. She will be attempting to use x-ray fluorescence to identify changes in metal ion concentration in different regions around Mount Saint Helen’s. Her project will try to correlate metal ion concentrations and plant growth.