Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Entitled For the degree of Is approved by the final examining committee: Chair To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________ ____________________________________ Approved by: Head of the Graduate Program Date Jennafer M. Whybrew Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces serevisiae. Master of Science Martin Bard N. Douglas Lees Brenda Blacklock Martin Bard N. Douglas Lees 07/31/2010 Graduate School Form 20 (Revised 1/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: For the degree of ________________________________________________________________ I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Teaching, Research, and Outreach Policy on Research Misconduct (VIII.3.1), October 1, 2008.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. ______________________________________ Printed Name and Signature of Candidate ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/viii_3_1.html Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae. Master of Science Jennafer M. Whybrew 07/31/2010 AEROBIC UPTAKE OF CHOLESTEROL BY ERGOSTEROL AUXOTROPHIC STRAINS IN CANDIDA GLABRATA & RANDOM AND SITE-DIRECTED MUTAGENESIS OF ERG25 IN SACCHAROMYCES CEREVISIAE. A Thesis Submitted to the Faculty of Purdue University by Jennafer Marie Whybrew In Partial Fulfillment of the Requirements for the Degree of Master of Science December 2010 Purdue University Indianapolis, Indiana ii For my family, thank you for all of your support. iii ACKNOWLEDGEMENTS I would like to thank the following people for making this work possible: Dr. N.D. Lees, Dr. Brenda Blacklock, Dr. Mark Goebl, and Dr. Martin Bard for teaching and directing me in my studies; Brett Barnes, Jacob Layer, and Ken Polezoes the undergraduate researchers that assisted me over the past two years. iv TABLE OF CONTENTS Page LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x UNITS xi ABSTRACT xii CHAPTER 1 INTRODUCTION 1 1.1 Sterols-Structure and Function 1 1.2 Sterol Biosynthesis 4 1.2.1 The Mevalonate Pathway 4 1.2.2 Sterol Biosynthesis: Farnesyl Pyrophosphate to Ergosterol 5 1.2.3 The Role of Heme in Ergosterol Biosynthesis 8 CHAPTER 2 MATERIALS AND METHODS 10 2.1 Strains, Media, and Growth Conditions 10 2.1.1 Bacterial Strains, Media, and Growth Conditions 10 2.1.2 Yeast Strains 11 2.1.3 Yeast Media and Growth Conditions 12 2.2 DNA Manipulations 13 2.2.1 Plasmids 13 2.2.2 Endonuclease Restriction Digest 14 v Page 2.2.3 Agarose Gel Electrophoresis 14 2.2.4 Ethanol Precipitation 15 2.3 Transformations 15 2.3.1 Bacterial Transformations 15 2.3.2 Yeast Transformations 16 2.3.3 Spot Plate Assays 17 2.4 Preparation of DNA 17 2.4.1 Bacterial Plasmid via Mini Prep 17 2.4.3 Yeast Plasmid Preparation 18 2.5 Gas Chromatography 19 2.5.1 Saponification 19 2.5.2 Gas Chromatography 19 2.5.3 Gas Chromatography/Mass Spectrophotometry 20 2.6 DNA Sequencing 20 2.7 DNA Manipulations 21 2.7.1 Yeast Gene Disruptions 21 2.7.2 Site-Directed Mutagenesis in Saccharomyces cerevisiae 23 2.7.3 Random Mutagenesis in Saccharomyces cerevisiae 27 CHAPTER 3 AEROBIC UPTAKE OF EXOGENOUS CHOLESTEROL IN ERGOSTEROL AUXOTROPHS IN CANDIDA GLABRATA 31 3.1 Introduction 31 3.2 Results 37 3.3 Discussion and Conclusions 59 3.4 Future work 61 CHAPTER 4 CHARACTERIZATION OF ERG25 IN SACCHAROMYCES CEREVISIAE 63 4.1 Introduction 63 vi Page 4.2 ERG25 Consensus Diagram 66 4.3 Results 67 4.3.1 Site-directed Mutagenesis Results 67 4.3.2 Random Mutagenesis Results 71 4.4 Discussion and Conclusions 75 4.5 Future Work 78 LIST OF REFERENCES 80 vii LIST OF TABLES Table Page 2.1 Yeast Strains 11 2.2 Plasmids 13 2.3 ERG25 mutagenesis sequence primers 20 2.4 PCR primers for yeast gene disruptions 22 2.5 PCR parameters for yeast gene disruptions 23 2.6 Site-directed mutagenesis PCR primers 24 2.7 PCR parameters for site-directed mutagenesis 27 2.8 Gap repair primers for insert 28 2.9 PCR parameters for Gap Repair 28 3.1 Accumulating sterol precursors for specific ergosterol genes 38 3.2 GC results for erg1, erg7, erg11, erg25, and erg27 39 4.1 Site-directed amino acid changes 67 4.2 Site-directed mutagenesis amino acid change, complementation, and GC results 69 4.3 GC sterol profile values for complementing site-directed mutagenesis strains 70 4.4 Sequence results for random mutagenesis 74 4.5 Random mutagenesis amino acid change, complementation, and GC results 75 viii LIST OF FIGURES Figure Page 1.1 Sterol structures and IUPAC numbering system 2 1.2 Isoprenoid Biosynthetic Pathway 4 1.3 Ergosterol biosynthetic pathway flow chart 6 1.4 Ergosterol Biosynthetic Pathway 8 2.1 Diagrammatic scheme of random mutagenesis “insert” creation 27 2.2 Diagrammatic scheme of random mutagenesis “vector” creation 29 3.1 GC profile of wild type Candida glabrata 39 3.2 GC profile of ERG1 in Candida glabrata 40 3.3 GC profile of ERG7 in Candida glabrata 41 3.4 GC profile of ERG11 in Candida glabrata 42 3.5 GC profile of ERG25 in Candida glabrata 43 3.6 GC profile of ERG27 in Candida glabrata 44 3.7 Spot plate analysis of erg1 in Candida glabrata 46 3.8 Spot plate analysis of erg7 in Candida glabrata 47 3.9 Spot plate analysis of erg11 in Candida glabrata 48 3.10 Spot plate analysis of erg25 in Candida glabrata 49 3.11 Spot plate analysis of erg27 in Candida glabrata 50 [...]... of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and SiteDirected Mutagenesis of ERG25 in Saccharomyces cerevisiae Major Professor: Dr Martin Bard Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals C glabrata in. .. on the sterol A ring In S cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif We have conducted site-directed and random mutagenesis in the S cerevisiae wild-type strain SCY876 Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among... with a starting temperature of 100˚C for one minute increasing to 300˚C in 7˚C/min increments and then held for 15 minutes at the final temperature of 300˚C The injection volume per sample was 3 µl 2.6 DNA sequencing DNA sequencing reactions were performed at the Biochemistry Biotechnology Facility (BBF) Indiana University School of Medicine Primers (Invitrogen) used for sequencing are listed in Table... various plant, animal, and fungal species Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for sitedirected mutagenesis The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required... different kingdom sterols to be substituted in the membrane in place of a sterol end product For example, if a deleterious event occurs in the ergosterol biosynthetic pathway preventing the production of the ergosterol end product, cells can utilize cholesterol (the animal kingdom end product sterol) in place of ergosterol Studies exploring this phenomenon of sterols have lead to a greater understanding of. .. Bacterial strains were grown in Luria-Bertani (LB) media with the addition of 60 µg/ml of ampicillin for selection LB media consisted of 10 g of pancreatic digest of casein, 5 g of yeast extract, and 10 g/L of sodium chloride (46) dissolved in milli-Q water and autoclaved for 25 minutes Solid media required the addition of 2% (w/v) granulated Difco agar (Becton Dickinson, Sparks, MD) prior to autoclaving... exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically Two groups of sterol auxotrophic C glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol xiii precursor Sterol auxotrophs have been created in C glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol. .. participates in transforming lanosterol to ergosterol (49) Lewis and colleagues reported that a heme mutation is required for aerobic rescue of an erg mutation (64) Of significant concern for this study are the recent reports indicating some strains of C glabrata (an opportunistic human pathogen) are capable of aerobic sterol uptake (51) 10 CHAPTER 2 MATERIALS AND METHODS 2.1 Strains, Media, and Growth Conditions... freshly grown in appropriate media for each strain For example, strains with essential gene disruptions were grown anaerobically on YPD plates with sterol supplementation and strains not containing essential gene disruptions were grown aerobically on YPD Either pelleting liquid cultures or scrapping cells from plates and dissolving cells in sterile water created cell solutions The concentration of the cell... ERG11, ERG25, and ERG27 gene disruptions were made in C glabrata wild type strain 2001HT and the ERG25 gene disruption was made in S cerevisiae wild type strain SCY876 using histidine as the selectable marker The pRS303 plasmid was used for PCR amplification of the HIS3 gene with specifically designed primers (listed in Table 2.4) containing 60 base pairs of homology at the ends for the specific gene being