Kreb's Lab 2007

to Kreb's Lab                                                                                                                                                                                                       to Kreb's Lab Past Lab Members
Fall 2007: Undergraduate Research

Expression of Cup 1 Protein

Mutations in chromatin components which fail to shut down CUP1 transcription have been previously identified in our lab.  However, we do not know whether misregulation of CUP1 mRNA also results in inappropriate levels of Cup1 protein.  Using western blot analysis, I am characterizing the expression of Cup1 protein in various chromatin mutant strains during copper treatment.  I wish to determine if protein and mRNA expression patterns are consistent, especially in the strains which misregulate the shutdown of mRNA expression. This will determine whether these mutant strains perturb Cup1 protein levels and may uncover the roles of different post-transcriptional mechanisms in the regulation of the copper response, such as mRNA decapping, Cup1 protein stability, other other mechanisms.  These studies will help us to understand the different copper sensitivities of different mutant strains observed in our lab, and may reveal general mechanisms of copper homeostasis.


Summer 2007: UAA REU Program

Regulation of Copper Homeostasis

Because copper is required for normal cellular growth, but is also toxic at high concentrations, all cells possess mechanisms to maintain physiological levels of copper. Cup1, a copper metallothionein, plays an essential role in this regulation. Cells rapidly activate the CUP1 gene upon exposure to toxic levels of copper. Cup1 sequesters excess intracellular copper, minimizing the harmful effect of the metal ion. However, complete removal of copper is prevented by rapid shutdown of the gene, the mechanism of which is currently being investigated in our lab. Yeast mutants which fail to shutdown CUP1 have been previously identified in our lab. We tested the hypothesis that these mutants counteract their inability to shutdown CUP1 by inducing genes involved in copper uptake. Proteins expressed during copper starvation include Ctr1, a copper transporter which imports Cu(I), and Fre1, a reductase which reduces extracellular Cu(II) to Cu(I) for uptake by Ctr1. We measured the expression of CUP1, CTR1, and FRE1 mRNA in mutants exposed to excess copper using real time RT-PCR analysis. Surprisingly, we found that the mutants do not continue to overexpress CUP1 during long exposures to copper, and subsequently do not induce expression of CTR1 and FRE1. This suggests that mutant strains adapt to their initial misregulation of CUP1





UAA Research Experiences for Undergraduates Program

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