2008 Beckman Scholars at Carnegie Mellon-Department of Biological Sciences - Carnegie Mellon University

2008 Beckman Scholars at Carnegie Mellon

Kathleen McCannKathleen McCann, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Javier López

Effect of Genetic Polymorphisms on Alternative Splicing of RNA for the Dopamine Reuptake Transporter

The dopamine reuptake transporter (DAT) plays a role in termination of dopaminergic signaling in the brain. Defects in DAT expression or function have been proposed to play roles in Parkinson’s disease and schizophrenia. Common sequence variations within the human SLC6A3 gene, which encodes DAT, have been associated with increased risk for schizophrenia. Most of these single-nucleotide polymorphisms (SNPs) are located within introns. A cluster of risk-associated SNPs is located deep within intron 3, surrounding a predicted recursive splice site. My results indicate that this recursive splice site defines a novel alternatively spliced exon (E3b), whose inclusion is influenced by the risk-associated SNPs. The inclusion of E3b truncates the DAT open reading frame and this may function as a regulatory mechanism to fine-tune dopaminergic signaling. We hypothesized that the identified SNPs influence schizophrenia risk by changing the sequence of splicing regulatory elements, thus altering the frequency of inclusion of E3b in DAT mRNAs. My project is designed to determine whether the risk-associated SNPs alter the frequency of retention of E3b in mRNAs. To test the effects of the SNPs on the splicing of E3b, I cloned the relevant fragment of SLC6A3 from 1 non-risk haplotype and 5 risk haplotypes into pCMV. These constructs contain all of intron 3 and portions of the flanking exons E3 and E4, and are transcribed from a constitutive promoter in the plasmid. The constructs were transfected into HeLa cells. RNA samples were harvested from the transfected cells and RT-PCR was used to determine the effects on E3b inclusion. RT-PCR data showed that the overwhelming splicing isoform of the non-risk construct showed E3b exclusion, while the splicing patterns of the risk haplotype constructs displayed two splicing isoforms: E3-E4 and E3-E3b-E4, where the isoform including E3b occurred approximately 25% of the time. This suggests that the risk-associated SNPs do alter the normal splicing patterns of SLC6A3.

Gregory NewbyGregory Newby, Department of Biological Sciences, Carnegie Mellon University

Mentor: Dr. Peter Berget

Development of Fluorescent Protease Biosensors Using a Single Chain Variable Fragment Platform

My project deals with single chain variable fragment antibodies (scFvs), which are proteins that are synthetically derived from the variable portions of IgG antibodies. scFvs are smaller than the full-sized antibodies they are derived from but still capable of having high affinity interactions with a specific ligand. I am developing a protease biosensor using an scFv that binds to and quenches the natural fluorescence of fluorescein dye. My protease biosensor should cause an observable increase in fluorescence in the presence of an active protease. I have produced derivatives of this fluorescein-binding scFv that should act as a substrate for three different proteases, depending on which specific amino acid recognition sequence is inserted into the scFv. The idea is that fluorescein can be bound to the scFv and then injected into the cellular system of interest. I should be able to observe an increase in fluorescence if the scFv is in the presence of active protease, because it will be cleaved by the protease and fluorescein should fall out of the binding pocket to give a fluorescent signal. The goals for this project were to observe the capabilities of the secreted protein to bind and quench the fluorescence of fluorescein, and to demonstrate a significant increase in fluorescence after cleavage by the intended protease in vitro. The scFvs were produced in E. coli and purified using NTA-Ni+2 resin column chromatography. The fluorescence of biosensor-fluorescein complexes were analyzed on fluorimeters. When a solution containing the biosensor-fluorescein complex was treated with the appropriate active protease, the fluorescence signal was observed to increase significantly. Modifications that are intended to further increase the fluorescence signal released by the biosensor will be examined by additional research in the fall semester. Because proteases are so commonly used by cells and viruses as mechanisms to alter or destroy proteins, I hope that this biosensor will be useful in subsequent research done to further our knowledge about how proteases are utilized in biological systems.

Steve ReillySteve Reilly, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Javier López

Functional Analysis of Recursive Splicing by Gene Replacement

Genes containing long introns tend to have complex expression and play important roles in normal development and in human disease. Nevertheless little is known about the mechanisms that promote efficient transcription and processing of RNAs from such genes. Recursive splicing has been proposed to play a role in facilitating transcript elongation or accurate splicing of long introns, because it appears to be restricted to introns longer than 10 kb and occurs at highly conserved sites. In recursive splicing, special signals (RPs) function first as 3’ splice sites and regenerate 5’ splice sites, leading to removal of long introns as a series of smaller fragments that can be spliced cotranscriptionally without leaving a trace in the final RNA. The goal of this project is to test the hypothesis that recursive splicing is critical for the correct and or efficient expression of genes with long introns. For this purpose, recursive splice site RP3 has been deleted from within the endogenous Ultrabithorax (Ubx) gene of Drosophila. This was achieved using a homologous recombination strategy to replace an internal portion of the gene with a modified version carrying the deletion. Genetic and molecular tests were used to verify the recombination event and reduction to mutant copy in multiple independent isolates. The effects of the RP deletion on biological function are currently being assessed by analysis of homeotic transformations in homozygotes and in trans-heterozygotes with null Ubx alleles.  The mutant allele is associated with a loss of function Ubx phenotype. An unexpected result was a strong but unstable silencing of the white gene, which was included as a marker for identification of the homologous recombination event as it becomes inserted between the duplicated elements. This silencing has not been observed for marker insertions in other regions of the Ubx locus, suggesting that a local sequence or chromatin feature near RP3 is responsible. Aberrant reduction events that retain and activate the white marker gene were recovered occasionally. These presumably resulted from non-homologous end-joining events and resulted in deletions that can be used to map the silencing elements of this region of the genome.

Natalie WeirNatalie Weir, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Chen Ho

Auto-oxidation of Human Hemoglobin and the Roles of Amino Acid Residues in the Distal Heme Pocket

This study explores the structural and functional effects of amino acid substitutions at helical position E11, located 3-6 Å from bound O2 in the distal heme pocket of both α- and β-chains of human normal adult hemoglobin (Hb A). Four recombinant hemoglobins (rHbs), rHb (αV62L), rHb (αV62I), rHb (βV67L), and rHb (βV67I), have been expressed in Escherichia coli and purified. O2 affinity, Bohr effect, cooperativity of the oxygenation process, auto-oxidation, and 1H-NMR spectra have been measured for each mutant. Substitution of Leu in either subunit, or Ile in the β-subunit does not result in significant functional deviations in the O2 affinity, Bohr effect, and cooperativity from those of Hb A. However, rHb (βV67I) shows a two-fold decrease in O2 affinity while maintaining the cooperativity. As expected, these mutations perturb the ligand-binding site of their respective subunits, as evidenced by 1H-NMR. rHb (αV62L) and rHb (αV62I) exhibit a small perturbation of the α1β2 interface in the deoxy state, showing that the quaternary structure of the tetramer is more sensitive to added volume at position E11 in the α-chain than in the β-chain. Auto-oxidation studies have shown that these mutants undergo faster oxidation than Hb A. In comparison to the other three mutants, rHb (βV67I) is most resistant to oxidation. Additional studies incorporating the strengths of rational mutagenesis with structural and functional studies will be carried out in order to gain a deeper understanding of the auto-oxidation reaction of human hemoglobin and the role of the distal heme pocket, P50, and α1β2 interface on the mechanism of auto-oxidation. Our studies will also provide new insights into the roles of amino acid residues in the distal heme pockets of both α- and β-chains on the structure-functional relationship in hemoglobin.