Regulatory analysis of squalene synthase (ERG9) from Saccharomyces cerevisiae and the cloning and characterization of the C-4 sterol methyloxidase gene (ERG25) from Candida albicans
Abstract
Sterols are important molecules in regulating membrane fluidity and permeability in eukaryotic cells. The ergosterol biosynthetic pathway is a specific branch of the mevalonate pathway. The biosynthetic pathway leading to the formation of ergosterol in yeast and cholesterol in animals has been well conserved making yeast an excellent model system in which to study sterol biosynthesis and regulation. This research has focused on two separate aspects of sterol synthesis in yeast. In the first part, the transcriptional regulation of squalene synthase (ERG9) in Saccharomyces cerevisiae, the first enzyme dedicated to the synthesis of sterols was investigated. ERG9 has been shown to be regulated by heme and oxygen through the yeast heme activator protein transcription factors HAP1 and HAP2/3/4. Interruptions in ergosterol biosynthesis either upstream and downstream of the squalene synthase step by either mutation or pharmacological methods increased ERG9 expression. Two other transcription factors, the yeast activator protein transcription factor yAP-1, and the phospholipid transcription factor complex INO2/4 also regulate ERG9 expression. Deletion analysis, site-directed mutagenesis, and electromobility shift assays of the ERG9 promoter revealed two novel cis-elements that regulate ERG9 expression. Genetic screens to identify genes that regulated squalene synthase expression were performed. In addition to sterol and heme biosynthetic mutants identified in the screen, three non-ergosterol biosynthetic genes were identified, two of which (YOR195W and YLL028W) were shown to interact through the cis-elements identified in this study. Furthermore, deletion analysis of these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities. All of these data demonstrate that squalene synthase is subject to multiple forms of regulation at the level of transcription by many different mechanisms. In the second part of this study, the C-4 sterol methyloxidase gene (ERG25) of Candida albicans, a common human pathogen, was investigated as a potential antifungal target. Since ERG25 is essential in yeast, it may be a good antifungal target. This study reports the cloning and characterization of the C. albicans ERG25 gene, and the creation of C. albicans ERG25 conditional lethal alleles.
Degree
Ph.D.
Advisors
Bard, Purdue University.
Subject Area
Molecular biology|Genetics
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