? Copper-deficiency in humans has been linked to birth defects and cardiovascular disease. Metal nutrition studies have most easily been studied in microorganisms due to the availability of well-defined media that allow for the manipulation of copper content for growth. Chlamydomonas reinhardtii is an ideal model organism for the study of copper-deficiency because Chlamydomonas offers the advantage of growth in a simple, well-defined salts medium, and genetic amenability. The availability of an annotated genome coupled with classical and molecular genetics make Chlamydomonas an even more accessible model. Previous studies of copper-deficiency responses in Chlamydomonas have demonstrated 1) a hierarchy of copper utlization in Chlamydomonas with more essential proteins like cytochrome oxidase prioritized, 2) the occurrence of copper-independent """"""""back-up"""""""" metabolic routes that are expressed in -Cu cells to compensate for the loss of function of cuproenzymes, and 3) re-cycling of Cu salvaged from actively degraded non-essential cuproenzmes. These responses are controlled by a novel transcription factor CRR1 that binds to the previously-defined core of a CuRE. Conventional genetic and differential expression approaches over the last decade have revealed nearly a dozen genes in this signal transduction pathway. Since genome analysis predicts the occurrence of an order of magnitude more cuproenzymes in Chlamydomonas than documented previously, it is likely that there are many more copper-deficiency response genes remaining to be discovered. Targets of copper-deficiency will be identified via digital mRNA profiling using Illumina's Solexa sequencing platform in lieu of conventional microarrays for deeper and more quantitative sampling of the mRNA population. Three types of experiments are proposed: A) comparison of mRNA profiles from wild-type -Cu vs. +Cu acclimated cells, B) comparison of mRNAs isolated from wild-type cells as they transition from copper-replete to copper-deficient and vice-versa, and C) comparison of copper-deficient crrl mutant cells to copper-deficient wild-type cells. The data will be analyzed in the context of the pattern of expression of known CRR1 and Cu-deficiency targets, to identify the primary response genes to generate groups of responses. Based on the validation and prediction of function and location of candidate copper-responsive proteins, a subset will be analyzed functionally by RNAi knock-down techniques to deduce their participation in copper homeostasis. Copper is essential for human physiology, but in deficient and excess concentrations it causes metabolic disorders. The project's goal is to identify the responsive and adaptive mechanisms to copper-deficiency. ? ? ? ?
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