Gregory Newby, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Peter Berget
The Construction of Protease Biosensors Based on ScFv Constructs
The variable fragment of single chain antibodies, or scFvs, have been isolated by yeast surface display that tightly bind the fluorogenic dye malachite green (MG). Some of these scFvs are composed of only a variable light or variable heavy chain, while others are composed of both a light and heavy chain connected by a flexible linker of amino acids. The desired product of this project is a biosensor molecule that will cause the fluorescence of MG when there are proteases active in its environment. This product could be used as a tool for cell biologists to examine protease activity inside of a living cell by use of a fluorescence microscope. A known, heavy chain only scFv that causes the fluorescence of MG has been linked to a light chain such that its interaction with the dye is 99% blocked. Recognition sequences of amino acids for the proteases Caspase 3 and HRV-3C have been inserted into the linker between these domains at the DNA level. The resulting gene has subsequently been cloned into yeast in two kinds of vectors – one will cause its secretion in liquid culture, and the other will cause the protein to be displayed on the surface. These genes must now be induced so that the protein can be examined for fluorescence both before and after cleavage by the protease. If the scFv appears to be functioning as hypothesized, its binding kinetics will be tested and optimal conditions will be found for its growth and use. If reasonable conditions and requirements for the scFv are found, the end result would be a new technology for detecting protease activity.
|Steve 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 development as well as 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 and/or accurate splicing of long introns, due to its apparent restriction to introns longer than 10 kb and occurrence 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, genetic manipulations to delete recursive splice site RP3 from within the endogenous Ultrabithorax (Ubx) gene of Drosophila are underway. These include using a homologous recombination strategy to replace an internal portion of the gene with a modified version bearing the deletion. Currently, several candidates have been isolated possibly containing the deletion. Genetic and molecular tests are being performed to verify the recombination event. The effects of the RP deletion on biological function will be assessed by analysis of homeotic transformations in homozygotes and in trans-heterozygotes with null Ubx alleles. Upon verification of the mutant, qualitative and quantitative RT-PCR analyses will be used to compare the pattern of splicing and the level of expression between the wild type and the mutant Ubx alleles. Alternatively spliced isoform ratios will also be compared at different stages of Drosophila development. Chromatin immunoprecipitation (CHIP) with antibodies against RNA Polymerase II will be used to analyze the possible effect of the RP deletion on transcript elongation through the long Ubx intron.
|Samantha Spath, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Charles Ettensohn
Localization of Maternal Components of the Wnt Signaling Pathway in the Sea Urchin Embryo
Signaling pathways are highly conserved in development and have been found to regulate various processes, including axis formation and cell differentiation. One such pathway that has been studied extensively is the canonical Wnt pathway. It involves a Wnt ligand binding to Frizzled and LRP receptors and initiating a cascade that stabilizes the transcriptional activator ß-catenin. In the sea urchin, it is already known that ß-catenin is localized to the nuclei of vegetal blastomeres in early cleavage, but not in animal blastomeres. This has been shown to be necessary for vegetal cell specification. It is not known, however, what causes this differential localization of ß-catenin. A similar asymmetry of ß-catenin is found in Xenopus embryos along the dorsal/ventral axis and it has been determined that maternal Wnt11 is responsible for ß-catenin stabilization on the dorsal side. While there is no Wnt11 in sea urchins, a similar mechanism may be in effect for the animal/vegetal axis of the urchin embryo. Thus, the purpose of this project is to look at the upstream regulators of the Wnt pathway and determine if any are involved in axis formation. Specifically, I am looking at the localization of maternal Wnt and Frizzled mRNAs in the urchin egg and early embryo using in situ hybridization and RT-PCR techniques. In situ hybridization on unfertilized eggs reveals an asymmetric localization of mRNA in approximately 30% of eggs for SpFz4, SpFz5/8, SpWnt1, SpWnt6, SpWnt7 and SpWnt16 genes. RT-PCR using isolated animal and vegetal blastomeres demonstrates that SpFz mRNA is located throughout the early embryo, while SpFz5/8 mRNA is restricted to the vegetal blastomeres. SpFz9/10 mRNA appears to be absent from the early embryo. Given these results, I have identified potential candidates in the pathway for specification of the animal/vegetal axis. This and future work will allow for comparison among organisms, thus increasing our understanding of axis formation and patterning as a whole.
|Stefanie Sydlik , Department of Chemistry, Carnegie Mellon University
Mentor: Dr. Richard McCullough
Regioregular Random Copolymers of Thiophene and 3-Alkylthiophene
Regioregular polythiophenes are an important class of conjugated polymers for practical applications. One common way to alter the properties of polythiophene is by altering the side chain in the 3 position or by making a copolymer. Here, I synthesized random copolymers of 3(alkylthiophene): thiophene in the ratios 3:1 and 1:1, where the alkyl group was hexyl, octyl, or dodecyl. All copolymers were soluble in common organic solvents and formed good films. Very high conductivities (maximum 1670 S/cm) were observed using the 4-point probe method. Previously, the highest reported conductivity for a polythiophene-based polymer was 1000 S/cm. Hole mobilities in line with the industry standard (10-1 cm2/V s) were found via testing in a transistor device. H-NMR and GPC were used to characterize the polymers. Physical characteristics of thin films were examined via AFM, GISAXS, and SEM. The GIWAXS images allowed us to investigate the morphology and it was found that better electrical properties are reported for polymers with decreasing order and increasing amorphous character. This is contrary to what previous work has reported. This work demonstrated that synthesizing random copolymers is a reliable and easy approach to incorporating unsubstituted thiophene in to a poly-3-alkylthiophene copolymer to enhance physical and electrical properties.