2001 Beckman Scholars at Carnegie Mellon-Department of Biological Sciences - Carnegie Mellon University

2001 Beckman Scholars at Carnegie Mellon

Rebecca FrederickRebecca Frederick, Department of Biological Sciences, Carnegie Mellon University
(Mentor: Dr. Elizabeth Jones)

Regulation of Arginine Storage in the Vacuole of Yeast

An important function of cells is their ability to selectively store and use nutrients at appropriate times. In yeast, the amino acid arginine is stored in the vacuole during growth on a good nitrogen source and released during nitrogen starvation for use as an alternative nitrogen source. The goal of this work was to find proteins involved in a pathway by which yeast cells recognize the absence of a nitrogen source and signal for arginine release from the vacuole. A strain in which green fluorescent protein (GFP) and beta-galactosidase are each under the control of an arginine-responsive promoter has been constructed. GFP and beta-galactosidase can be assayed within ten hours after starvation, presumably stimulated by arginine release from the vacuole. Strains bearing mutations in genes that are involved in the pathway controlling arginine release should not show increased levels of GFP and beta-galactosidase expression upon starvation. Deletion of genes involved in one nitrogen starvation response pathway did not affect expression levels of GFP during starvation, suggesting that the filamentous growth pathway is not involved in arginine storage regulation. To identify genes that are involved, 33,000 colonies from a mutagenized cell population were screened for defects in starvation response. Five putative mutants are currently being pursued to isolate the mutations and eventually clone the genes in which mutations lie. Furthermore, preliminary evidence suggests that cells lacking vacuolar arginine pools due to mutations in Class C vacuolar protein sorting genes are not able to activate reporter gene expression during nitrogen starvation.

Lorraine HsuLorraine Hsu, Department of Chemistry, Carnegie Mellon University
(Mentor: Dr. Bruce Armitage)

The Development of a Novel Biological Sensor: Regulation of Aptamer Binding

Thermal Regulation of Aptamer Binding to Thrombin
Aptamers are double or single stranded oligonucleotides that bind to specific molecules. The means of regulating aptamer binding with its target molecule will be investigated as a potential biological sensor. Our attentions have been focused initially on disrupting the aptamer by DNA/PNA hybridization. It has been found that quadruplex structures formed by single stranded DNA exhibit aptamer properties, binding the protein thrombin with nanomolar affinity.

Because the secondary structure of the aptamer generates the specific binding affinity, disruption of the quadruplex structure will prevent thrombin binding. The original thrombin binding aptamer (TBA) was modified, having 6-mer overhangs at each end as the target site for the disrupting agent. The disrupting component used will be a complementary (12-mer) single stranded peptide nucleic acid (PNA) targeting both overhangs. A beta-Alanine spacer was added in the middle of the PNA for flexibility. By introducing the complementary PNA strand to the modified TBA structure, hybridization should disrupt the secondary structure of the aptamer, and thus prevent thrombin from binding. Since duplex hybridization is temperature dependent, the 'switch' for thrombin binding would be temperature regulated.

Thus far, binding of the PNA strand to the aptamer has been studied by spectroscopic methods. Based upon CD and UV-Vis spectra, the PNA binds without significantly altering the G-quadruplex structure. This may be partially attributed to the flexible linker in the PNA. Future work includes modifying the lengths of both aptamer and PNA, using a G-quadruplex binding dye for fluorometric measurements, removing the PNA spacer, and introducing thrombin to the system.

The development of aptamer-based sensors coincides with the need to develop reusable sensors. Unlike most biological sensors, aptamer-based sensors are recyclable which would save much time, money, and resources. This project is part of a multi-investigator initiative supported by the Keck Foundation.

Ronald MillerRonald Miller, Department of Biological Sciences, Carnegie Mellon University
(Mentor: Dr. Elizabeth Jones)

Dihybrid Screen for Pep5p and Pep3p interactors

In yeast cells, Pep5p and Pep3p are known to be present in a complex necessary for the transport of proteins to the vacuole. In this experiment, I am trying to determine which proteins encoded by yeast gene fusions show physical interactions with Pep5p and/or Pep3p. Once an interaction is identified, the gene is sequenced and compared with known interactors of Pep5p and Pep3p. Twenty-three of the Pep5p interactors have been sequenced. Of the sequenced genes, some of the encoded proteins are known interactors, for example Pep3p and Vam6p; others, including Tuf1p, have not been previously shown to interact with Pep5p. The validity of the observed interactions will be tested in future experiments.

Christopher Noser

Christopher Noser, Departments of Chemistry and Biological Sciences, Carnegie Mellon University
(Mentor: Dr. Dr. Terrence Collins)

Cleaning Up: The Destruction of Priority Organic Pollutants

Each year, more than one hundred million tons of wood pulp are oxidatively bleached, resulting in the production of hundreds of thousands of tons of chlorinated aromatics. A major component of these, the chlorophenols, are of significant environmental concern due to their recalcitrance, propensity for bioaccumulation, and adverse health effects (including suspected carcinogenicity). One well-studied representative of the chlorophenols is 2,4,6-trichlorophenol (TCP).

TetraAmidoMacrocyclicLigand (TAML®) iron complexes, developed by the Collins group at Carnegie Mellon, efficiently activate hydrogen peroxide to degrade TCP in pH 10 water at room temperature, with significant mineralization and conversion to small biodegradable acids. Various analytical techniques, including many chromatographic and mass spectral methods, were used to identify and quantitate degradation products. After one treatment of TAML® technology, the following products were identified: CO2 + CO (35±5%), formic acid (5%), oxalic acid (10%), chloromaleic acid (16%), malonic acid (8-16%), hydroxymalonic acid (1-2%), chloromalonic acid (3-5%), and some oxidative coupling products (5-6%); this accounts for 84-97% of the initial carbon. After additional treatment of TAML®, it was determined that the coupling products and the chloromaleic acid can be easily destroyed. Finally, it was determined that TAML activators are themselves non-toxic, they produce no dioxins within detectable limits during the course of oxidative degradation, and thus they present currently the most efficient system for the degradation of recalcitrant chlorophenols.