Helen Lau, Carnegie Mellon University
(Advisor: Dr. Susan Henry)
From Mpk1p to INO1 expression: the Study of the Biochemical Pathway via Characterization of Suppressor Mutants
In the yeast Saccharomyces cerevisiae, the INO1 gene encodes the enzyme inositol-1-phosphate synthase (IPS) which catalyzes the conversion of glucose-6-phosphate (Glu-6-P) into inositol-1-phosphate (I-1-P), which is then later converted into inositol. Inositol is essential for the growth and survival of yeast. The mutants used in this project are inositol auxotrophs. More specifically, they have a deletion in their MPK1 gene (mpk1D mutants) which prevents them from producing their own inositol. INO1 is highly regulated by a pathway that has yet to be determined. To determine what genes and how many genes are involved in this pathway, the mpk1D mutants will be induced to mutate so they can survive in inositol deficient media. Mutants of this kind are called suppressor mutants where one mutation counteracts that of another so that the double mutant is like that of the wild type. By characterization of these mutants and subsequent genetic analysis, the genes that are being disrupted can be identified and thereafter used to start constructing the unknown biochemical pathway that resides before, and thus regulates the expression of the INO1 gene.
Elisabeth Nadolny, Carnegie Mellon University
(Advisor: Dr. Frederick Lanni)
Fibroblast Contraction of Collagen Gels as Models of Extracellular Matrix: Force Generation and Cytoskeletal Organization
Collagen is the primary protein found in the extracellular matrix. Cells reshape their extracellular surroundings into more complex structures in developing organisms, examples are ligaments, tendons and the cornea. We are interested in understanding the biological and physical processes which lead to such development, specifically forces generated and the specific cytoskeletal proteins' roles in the process. We have approached the problem by developing a model system of fibroblasts in collagen accessible with light microscopy. The gel is held down on only two edges and is seeded with 3T3 fibroblasts. We have observed that the gels contract on the two free edges, causing up to a 40% reduction in width over a three day period. Imaging cell behavior within the gel has revealed that the cells densely populate these free edges forming cable like aggregations. Timelapse imaging allows us to determine forces exerted by utilizing a computerized tracker which senses pixel changes as displacement. The calculated displacement is then related, along with the material constants for collagen, to our mathematical model in order to determine force magnitude. The cytoskeletal role is currently being studied in two ways; fixed cells are rhodamine phalloidin labelled for actin and live cells expressing GFP alpha-actinin are also being imaged. We plan to observe the specific forces being generated by cells in the gels, the effects of fibronectin concentration on cell behavior and further classify the actin organization of the cells inhabiting the matrix.
Krista Pfaendler, Carnegie Mellon University
(Advisor: Dr. Javier Lopez)
Identification of factors that control alternative splicing of Ultrabithorax RNAs
Although alternative splicing is one of the major ways in which higher organisms control expression of their genes, current knowledge of the relevant mechanisms is rudimentary. The Lopez laboratory is investigating the mechanism of alternative splicing of the homeotic gene Ultrabithorax (Ubx) in Drosophila melanogaster. Alternative splicing of Ubx transcripts produces six mRNA isforms that differ in their inclusion of three elements (B, mI and mII) as a consequence of competition between flanking alternative 5' splice sites. Previous work in the lab has identified several genes whose products are required for inclusion of mI in Ubx mRNAs. Among these, sans fille (snf), virilizer (vir) and fl(2)d are also required for the female-specific alternative splicing pattern of RNAs from the Sex lethal (Sxl) gene. snf encodes the homolog of human U1A and is a component of U1 snRNP. Together with other data, these observations suggest that the products of snf, vir and fl(2)d are required for repression of specific 5' splice sites in Sxl and Ubx RNAs by recruiting U1 snRNP in a non-productive mode. This recruitment may be mediated by Sxl protein in Sxl RNA and by hrp48 protein in Ubx RNA, since these proteins have binding sites near the corresponding regulated 5' splice sites and are required for regulation in each case. I discovered that vir3 and fl(2)d2, but not hrp481, exhibit dominant female-specific lethal interactions with the dominant negative allele snfe8h, suggesting that VIR, FL2D and SNF proteins function together to repress the 5' splice site in SXL. I have characterized the interaction of vir3 and fl(2)d2 with different alleles of snf. I have not detected a dominant synthetic lethal interaction with other antimorphic, hypomorphic, or null alleles of snf. Since snfe8h, vir3 and fl(2)d2 had relatively weak dominant effects on Ubx splicing, I also tested for synergistic effects among them using this assay. Preliminary results confirm synergism in all pairwise combinations.