This proposal seeks to increase our knowledge of the molecular events underlying the control of gene expression and the regulation of enzyme function. The principal organism chosen for the proposed research is Saccharomyces cerevisiae (yeast); the regulatory system is the biosynthetic pathway leading to the amino acid leucine. Yeast has in recent years become a model organism for the study of intracellular regulation of eukaryotic cells, because it has many of the features of higher eukaryotes, while being almost as manageable as bacterial cells with respect to growth in different environments and genetic manipulation. The leucine pathway is attractive, because it is subject to at least four different yet interrelated regulatory mechanisms and occupies two cellular compartments. We plan to use the tools of biochemistry, genetics, and molecular biology to (i) characterize a regulatory gene and its putative protein product; (ii) analyze the induction mechanism apparently governing the expression of two unlinked genes; (iii) study a newly discovered pair of isoenzymes and their structural genes; (iv) study the mechanism by which an enzyme of the leucine pathway is imported into the mitochondria; (v) study enzyme activity modulation; and (vi) attempt to express leu genes of Neurospora crassa in yeast. We expect that our work will provide us with a coherent view of the regulation of this complex system and will, in so doing, contribute to the development of more general concepts of eukaryotic regulation. Knowledge of the strategy employed by eukaryotic cells in regulating their activities is essential if one wants to define the aberrations in cellular regulation that are at the basis of most diseases.
