Encoded within the yeast genome are two sets of genes that are regulated in opposite fashion by heme, a cellular gauge of oxygen availability. One set is comprised of mitochondrial-functioning genes that are activated in the presence of heme, and the other set is comprised of genes believed to also serve some mitochondrial role tat are repressed in the presence of heme. In addition to sharing heme as the signal for oxygen, these two sets of genes share common regulatory factors. It is the goal of this study to define the components involved in the regulation of these genes and determine how the coordinate regulation is achieved. This study will focus on the CYC7 gene which encodes iso-2- cytochrome c, the minor cytochrome c species of the cell. This gene has the unusual property of possessing regulatory signals that respond both positively and negatively to heme, thereby sharing regulatory components with the two sets of oppositely regulated genes. The basepairs that comprise the cis-acting regulatory sequences of this gene will be defined in detail using the techniques of in vitro mutagenesis. The effect of the different regulatory sequences transplanted into an unrelated gene will be studied so that their individual contribution to the overall regulation of CYC7 can be assessed. Three trans-acting regulatory genes, the ROX genes, have been identified that are involved in the regulation of CYC7 and other heme regulated genes. These genes will be cloned by complementation of their mutant phenotypes, and their gene products will be identified. These gene products will be assessed for specific DNA binding activity and the ability to form complexes with other DNA binding proteins. Selection schemes for the isolation of additional regulatory mutations are proposed. Two other genes, the CYC1 gene which is positively regulated by heme, and the ANB1 gene which is negatively regulated by heme will be used as markers for the two sets of heme regulated genes. The effect of trans-acting factors which regulate CYC7 will be tested on these genes, and sequences which are found to serve as regulatory sites in CYC7 will be sought in these genes.
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