2003 Tobacco Settlement Fund-supported Participants-Department of Biological Sciences - Carnegie Mellon University

2003 Tobacco Settlement Fund-supported Participants

Eric BiondiEric Biondi, Dickinson College (Advisor: Dr. Javier Lopez)

Engineering DBR Mutants That Bind But Do Not Debranch RNA Lariats
The process by which pre-mRNA splicing-removing introns in the form of circular lariats-is not well understood. Tools for stabilization and purification of the intron lariats generated by the pre-mRNA splicing reaction would be very useful for studies involving genome-wide analyses of splicing patterns based on lariat characterization. This project will aim to generate an affinity purification reagent derived from lariat debranching enzyme (DBR). DBR is highly specific for the 2'-5' phosphodiester bond at the lariat branch junction. We will use PCR-based site-directed mutagenesis of yeast DBR to change specific amino acid residues in an attempt to inhibit the cutting activity of the enzyme while minimizing effects on substrate binding. The mutant DBR variants will be tested for complementation of various phenotypes associated with a dbr1 null allele in yeast, including the accumulation of lariats. The same mutants will then be tagged with hexahistidine, expressed and purified from E. Coli, and tested for lariat binding in vitro. The mutant DBR proteins will also be tested for dominant negative effects leading to lariat accumulation when overexpressed in yeast and Drosophila SL2 cells. Dominant negative behavior could be exploited for controlling lariat accumulation in multicellular organisms.

Matthew DenholtzMatthew Denholtz, Gettysburg College (Advisor: Dr. Jonathan Minden)

Modification of 2D-DIGE Techniques for the Purpose of Low Concentration Protein Identification in Bovine Serum
When a person becomes ill, there are likely to be protein changes in their serum caused by that illness. For example, tumor cells often show elevated telomerase levels, and heart attack patients show elevated myoglobin levels in their blood due to tissue damage in the heart. Serum is what is remains of the blood after clotting takes place. There are numerous methods of detecting proteins in the serum, but this project focused entirely on two dimensional difference gel electrophoresis (2D-DIGE). 2D-electrophoresis works by separating a complex mix of proteins ­ first, by their isoelectric point and second, by their size. Samples were compared by labeling each sample with one of two different fluorescent dyes. The samples were then run on the same gel. Differences were detected in the protein content of each sample by taking two pictures, one at each of the dye¹s respective wavelengths, and comparing the two pictures through a looping movie that alternately shows each picture. The major problem with this technique for its use in diagnosis is that the concentrations of the serum proteins vary by five or six orders of magnitude. Proteins that are found in high abundance, such as albumin, mask proteins that are less abundant. The goal of this project was to modify some of the aspects of the DIGE technique with hopes of accurately and reproducibly identifying proteins across a wide range of concentrations using newly synthesized, fluorescent, DIGE dyes.

Kristy Jacobus, Dickinson College (Advisor: Dr. Gordon Rule)

Structural Genomics Analysis Using PEPMORPH
The structure of a protein is what determines its function. By comparing the structure of an unknown protein to that of a known protein, it is helpful in making inferences about the function of the known protein. The original intent of this project was to do a structural analysis of proteins, with a few specific goals in mind. First, the overall objective was to use limited NMR data to look at protein structure changes as drugs are bound to the protein. The second goal was to detect homologies between unknown human proteins and known proteins, in hopes of shedding some light on the function of these unknown proteins. Both of these tasks are performed by a computer program called PEPMORPH. In addition, experimental data is required to test the capabilities of PEPMORPH. A small RNA binding protein, rho130, was used as a model system, and it had been intended that NMR data be collected on the unliganded protein for homolog detection and with the protein-nucleic acid complex to simulate drug induced change. Thus far, sufficient isotopically labeled rho130 has been purified to initiate the NMR studies.