Histoplasma capsulatum is an important human pathogen, infecting an estimated 500,000 individuals per year in the United States alone. The organism exhibits the phenomenon of dimorphism during the infectious process. In the environment H. capsulatum grows in a saprophytic filamentous (mold) form with extensive mycelia and asexual and sexual spores. When spores or mycelial fragments are inhaled by humans, the organism grows in the body in a parasitic form as a simple budding yeast. Cysteine metabolism is believed to play an important role in the mold to yeast transformation. Yeast cells, but not mycelia, contain a novel cysteine oxidase activity. This enzyme may play a role in dimorphism by regulating intracellular cysteine levels and/or by providing a product necessary for formation and maintenance of the yeast form. In order to begin studies of the molecular biology of cysteine metabolism and its relationship to dimorphism, the goal of this project is to clone the cysteine oxidase (CysOx) gene and study its expression during morphogenesis. The enzyme will be purified and used to raise antibodies in rabbits. Partial protein sequence will be obtained by analysis on a gas phase protein sequencer. The gene will be isolated from a cDNA expression library by screening with antibodies or with synthetic oligonucleotides. The gene will be sequenced and the primary structure will be analyzed by computer. The identity of the gene will be confirmed by comparison of DNA sequence to protein sequence data. Expression of the gene will be studied during the mold to yeast transformation to address the following questions: (1) What is the time course of gene activation during morphogenesis, and how does it relate to enzyme levels? (2) Is the enzyme transcriptionally regulated? (3) Is the gene actually yeast specific? Future work will study the gene structure and arrangement, effect of chemical modulators of morphogenesis on expression and disruption of the cysteine oxidase gene to produce cysox- mutants. Completion of this work will represent the first cloning and expression studies of a """"""""phase specific"""""""" enzyme in Histoplasma and will provide the tools for in depth studies of the molecular biology of the cysteine metabolism - dimorphism relationship.
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