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

Investigation of genes in the filamentous growth pathway for involvement in release of arginine from the vacuole in yeast
In the baker's yeast, Saccharomyces cerevisiae, arginine is stored in the lysosome-like vacuole during normal cell growth. When the cell is starved for nitrogen, arginine is released from the vacuole. Cytoplasmic arginine activates production of its own degradative pathway, allowing for its use as an alternative nitrogen source. To investigate how nitrogen starvation is recognized and a signal transduced by the cell, resulting in arginine release from the vacuole, we have constructed a reporter system in which green fluorescent protein (GFP) is under the control of a promoter that responds to cytoplasmic arginine. This strain expresses higher levels of GFP, measured using fluorescence microscopy, when grown in arginine-rich or nitrogen starvation media than when grown in media containing sufficient ammonia.

We are investigating genes involved in the filamentous growth pathway (Madhani, H.D. , Styles, C.A., and Fink, G. R. (1997) Cell 91, 673-684), by which yeast recognize nitrogen deficiency and alter their growth morphology, for possible participation in a signal transduction pathway that mobilizes stored arginine. Direct deletion of genes in the filamentous growth pathway, including STE11, STE12, and RAS2, and characterization of these deletion strains using the reporter system described above has been completed. Additionally the genes encoding, Dpp1p, a vacuolar membrane localized phosphatase, and Ste5p, a scaffolding protein involved in pheromone response, were deleted in the reporter strain. These deletions were verified using PCR.

Strains lacking functional Ste11p, Ste12p, Ste5p, Ras2p, and Dpp1p all show induction of GFP fluorescence in response to nitrogen starvation similar to the wild-type reporter strain. These results will be verified by characterization of the level of fluorescence of the population using flow cytometry. Further study into the pathway responsible for arginine release will be continued with a genomic mutagenization to isolate mutants defective in arginine release from the vacuole.

Supported by Carnegie Mellon Small Undergraduate Research Grants, the Beckman Foundation, and a grant from the National Institutes of Health (GM-29713 to E.W.J.).

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

Wood pulp bleaching: a cleanup approach

Wood pulp bleaching -- that is, the degradation of the lignin component of wood -- is a key step in industrial paper production. Currently, the most widely used method involves chlorinated oxidation agents, which are known to produce harmful pollutants such as chlorophenols. The Collins group has developed a series of activators that activate hydrogen peroxide to destroy a variety of substrates including trichlorophenol (TCP) and pentachlorophenol (PCP). TCP and PCP are completely consumed in under one hour using hydrogen peroxide as the oxidant and Et4N[Fe(H2O)DCB*] as the activator; all reactions were performed at room temperature in basic water. Analysis of the reaction mixture after one hour indicates the release of 2.5± 0.1 chlorides per TCP molecule and 3.5± 0.1 chlorides per PCP. 35± 5% of the chlorophenols have been mineralized. The analysis of products by various mass spectral and NMR methods will also be reported.