PATRICK FRANK DENOR, Marquette University


Although several modes of genetic regulation have been thoroughly studied in procaryotic organisms, the occurence of very few analgous regulatory mechanisms in eucaryotic systems suggests that regulation of gene expression in higher organisms is achieved via mechanisms unique to eucaryotes. In an attempt to elucidate what these mechanisms are, an analysis of the glycerol phosphate (Glp) system Neurospora crassa was initiated. This system is unique in that it possesses both a mitochondrial (glycerol-3-phosphate dehydrogenase) and a cytosolic (glycerol kinase) component, and thus presents an opportunity to examine nuclear-mitochondrial interactions as well as nuclear regulatory mechanisms. Examination of the Glp system required the isolation, and genetic and biochemical characterization of strains of N. crassa which were unable to dissimilate glycerol. Nineteen such mutants were isolated. Genetic analysis of these mutants revealed that they comprised three distinct complementation classes. The first class consisted of three strains which failed to complement each other as well as a previously described Glp1 mutant. Subsequent genetic mapping revealed that the affected locus in these new mutants was allelic to the lesion in the original Glp1 strain. Another nine of the mutants comprising the second complementation class, failed to complement with the previously isolated Glp2 strain. Mapping of the affected gene in these mutants established allelism with glp2. The last complementation class consisted of seven strains which possessed a mutation at a locus (glp4) mapping near the centromere of linkage group VI. Genetic analysis of the three loci was completed with the construction of a fine structure map of each. Enzymatic analysis of glycerol induced Glp1 mutants revealed that they had depresses levels of both glycerol-3-phosphate dehydrogenase and glycerol kinase, but no enzymatically inactive glycerol kinase cross reacting material (CRM) was detected in these extracts. These strains were however fully induced to wild-type levels when subjected to either deoxyribose or cold induction. In addition, the Glp1 mutants were found to take up ('14)C-glycerol at a rate directly proportional to the level of glycerol kinase present in each. These data suggest that the function of glp1 may be to regulate the expression of glycerol kinase. Glp2 strains were found to have no detectable glycerol-3-phosphate dehydrogenase when glycerol induced. Polyacrylamide gel electrophoresis of solubilized mitochondria from these mutants revealed an absence of a protein peak corresponding to glycerol-3-phosphate dehydrogenase, accompanied by an increase in the concentration of a fast migrating peptide. This evidence provides support for the previously proposed hypotheses that glp2 is the structural gene for glycerol-3-phosphate dehydrogenase. The last class of mutants, Glp4 were found to lack glycerol kinase enzymatic activity when subjected to induction by glycerol, deoxyribose or cold. However, glycerol kinase CRM material was detected in some of the glycerol induced mutant extracts. In addition, a Glp4 revertant strain which grew at one-half the wild-type rate on glycerol, was found to have glycerol kinase with an altered K(,m) for ATP. These observations suggest that glp4 is the structural gene for glycerol kinase.

Recommended Citation

DENOR, PATRICK FRANK, "STUDIES ON THE INDUCIBLE GLYCEROL DISSIMILATORY SYSTEM OF NEUROSPORA CRASSA" (1981). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI8111851.