NOTE TO USERS This reproduction is the best copy available. ® UMI THE CATHOLIC UNIVERSITY OF AMERICA A New Genetic Pathway Mediating Multidrug Resistance in the Yeast Saccharomyces Cerevisiae A DISSERTATION Submitted to the Faculty of the Department of Biology School of Arts and Science Of The Catholic University of America In Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy By Anne E Fleckenstein Washington, D. 2006 UMI Number: 3198179 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction.
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This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, Ml 48106-1346 A New Genetic Pathway Mediating Multidrug Resistance in the Yeast Saccharomyces cerevisiae Anne E Fleckenstein Director: John Golin, Ph. Multiple drug resistance is quickly becoming an obstacle to the treatment of disease.
Bacteria, parasitic protazoa, yeast and mammalian cancer cells develop mutations that render them resistant to a wide variety of structurally and chemically different compounds. In the yeast Saccharomyces cerevisiae, one of the main mechanisms causing drug resistance is the loss of function of ABC transporters that are responsible for effluxing the drug from the cell. The main ABC transporter responsible for efflux of many different drugs is PdrSp. However, earlier work (Fleckenstein et.
1999, Shallom and Golin, 1996) shows that this is not the only pathway mediating resistance to these drugs. The global regulator Sin4p and the transcription factor YRR/ operate in a PDR5-independent pathway to confer drug resistance to the cell. While wild type YRR/ is not required for multidrug resistance, a gain-of- function mutation in this gene can restore resistance in a previously drug hypersensitive Apdr5 mutant. This resistance requires both Sin4p, a member of the RNA Polymerase Mediator complex and Snf5p, a component of the chromatin remodeling complex SWI/SNF.
Disruption of these genes can cause profound drug hypersensitivity that cannot be explained by the changes in PDR3S transcription or function observed. Furthermore, loss of function mutations in either of these genes does not cause the increase in drug accumulation that is seen in a ApdrS mutant. The SNF5, SIN4 and YRRI genes define a new, major pathway required for mediating multidrug resistance. This dissertation by Anne E Fleckenstein fulfills the dissertation requirement for the doctoral degree in Cellular and Microbial Biology approved by John Golin, Ph., as Director, and by James Greene, Ph.
and Pamela Tuma, Ph. john G nm, Ph. Director lem ZL Pamela Tuma, Ph. Reader il Table of Contents Introduction Yeast as a Model System for Studying Multidrug Resistance Drug Resistance in Yeast Mechanism of Yeast Multidrug Resistance RNA polymerase II holoenzyme and subcomplexes Materials and Methods Overview of Experiment Strains and Media General Techniques Purification of Plasmid DNA from Bacteria Yeast Transformation Electroporation of E.
coli B-galactosidase assay Genetic Analyses Complementation testing Tetrad analysis Minimum Inhibitory Concentration (MIC) Molecular Analyses Polymerase Chain Reaction General Protocol Construction of RR3 Asin4 knockouts Isolation of total RNA from yeast cells RT-PCR of yeast RNA Extraction of genomic DNA Vectorette PCR Transport Assays Measurement of rhodamine 6G efflux Efflux Assay for [°H]-tritylimidazole Results 43 Discussion 80 Bibliography 92 ill List of Figures Figure 1. A model for the transcription of the yeast HO gene 2. Use of the mTn3::LEU2 transposon to create mutants 5. Complementation test of RR mutants and Asin4 mutant 48 6.
Complementation test of RR3 and AsnfS mutant 51 7. Minimum Inhibitory Concentration of isogenic single and double 54 Asin4 and AsnfS mutants 8. RT-PCR of SIN4 and ACT] transcripts in YRR/ mutants 56 9. RT-PCR of possible targets of SNF'5 in YRRI-2 and AsnfS mutants 10.
Pdr5 transcript in AsnfS mutant 67 11. PDR5 transcript with varying concentrations of RNA in WT and Asnf5 68 strains 12A. A representative histogram plot of rhodamine 6G accumulation in 70 AsnfS and Apdr5 mutants 12B. A representative histogram plot of rhodamine 6G accumulation in 7] Asin4 and Apdr5 mutants.
The accumulation of [*H] tritylimidazole does not saturate 74 14. The accumulation of tritylimidazole is not energy dependent. PDRS transcription in wild type and Aspr20 strains. A representative histogram plot of rhodamine 6G accumulation in 79 Aspt20 and ApdrS strains iv List of Tables Table.
Yeast and Bacterial Strains. PCR Primers bo. Vectorette PCR Primers 39. Minimum Inhibitory Concentration of YRR/ mutants (uM) 49.
Minimum Inhibitory Concentration of Asin¢Asnf5 Strains 55. RT-PCR of SIN4 in YRR/ mutants 57. Minimum Inhibitory Concentration of SC4741 background (uM) 59 8. Band Densities of Potential Targets of SnfŠp 65 9, Levels of PDRS promoter activation in Asnf5 Mutant and Wild Type 66 10.
RT-PCR of PDRS in SNFS and Asnf5 Strains 68 11. Rhodamine 6G accumulation in SC mutants 72 12. H]-tritylimidazole Accumulation Assay 77 13. RT-PCR of PDR5 in SPT20 mutant 78 Acknowledgements My success in graduate school has truly been a team effort; | could never have done this alone.
While there are too many people to list separately, 1 must thank the following people: My parents, James and Marilynn Fleckenstein for their unwavering support (both emotional and financial), Rosemary and Jim Carey for always providing a place to escape the lab, Dr. Joshua Shallom and Sr. Susan Cronin, Ph. for teaching me the ropes of the Golin lab, Dr.
Dottie Hutter, Ms. Sherry Supernavage, Ms. Donia Palomo, Mrs. Leanne Hanson, Dr.
Michael Mitchell and Lt. Lynette Hamilton, Ph. whose friendship, support and willingness to join me at bars will always make me remember graduate school fondly, The faculty and students of the Biology Department, And especially Dr. John Golin, whose genuine enthusiasm for research and teaching kept me going more times than I can count! VI Introduction Multiple drug resistance is quickly becoming an obstacle to the treatment of disease.
Drugs that are used for the treatment of infectious disease as well as those required for cancer chemotherapy are no longer effective because the organisms or cell that they target have developed a way to combat the drug. Many bacteria have become resistant to commonly used drugs such as penicillin and other antibiotics (Lewis e?. New drugs are being developed, but organisms can become resistant to those quickly. The CDC reports that the first bacteria resistant to the drug fluorquinolone appeared after that drug had only been in use for only 6 months (www.
The emergence of drug resistant tuberculosis is caused by the incomplete treatment or lack of treatment for infection by a drug susceptible strain (Cohen and Murray 2004) and is a growing threat in many developing countries (www. Not only are bacterial infections becoming more difficult to treat, but cancer cells can become resistant to chemotherapeutics as well (Bradley et. Itis estimated that 40% of operable cancers and 80% of inoperable cancers develop drug resistance (www. Mutations in the gene p53 are not only associated with the development of cancer, but Zhan et.
(2005) demonstrated that deletion of p53 is also associated with the development of multidrug resistance. Broad based resistance is usually due to overexpression of members of the ATP-binding cassette (ABC) superfamily of membrane transporters (Gottesmann et. In breast cancer, the ABC transporter BCRP (breast cancer resistance protein) is required for resistance to 2 mitoxantrone and anthracycline (Sarkadi et. Over-expression of this protein causes hyper-resistance to anti-tumor agents and reduces the accumulation of the drugs.
This over-expression of BCRP also enhances the efflux of rhodamine 123 (Doyle and Ross 2003). Multidrug resistance in human cancers is primarily due to two proteins P- glycoprotein (P-gp) encoded by the MDRI (multidrug resistance 1) gene and the multidrug resistance associated protein, MRP1 (Gottesman, Fojo and Bates 2002; Cole et. There are 30 ABC transporters in yeast and 47 in humans. The general structure of the ABC transporter consists of four domains: two hydrophobic transmembrane regions and two soluble ATP-binding cassettes.
The hydrophobic domains are thought to form a pore through which the substrate is moved. These domains are not well conserved among the different transporters and may be the determinants of substrate specificity. The nucleotide binding domain is the most conserved part of the protein. There are several consensus motifs including the Walker A and Walker B motifs and a region called the signature region.
Although other proteins that bind ATP contain the Walker A and B motifs, the presence of the signature region defines an ABC transporter. ABC transporters use the energy of ATP hydrolysis to efflux or import substrates (Gottesman et. 1995, Horio, Golttesman and Pastan 1998) and have many functions in cells besides multidrug resistance. For example, the protein Ste6 (sterile 6) is responsible for transporting the steroid mating hormone across the cell membrane (www.
Mutations in proteins other than transporters can alter drug resistance. For example, loss of the chromatin remodeling complex SWI/SNF (mating type 3 switch/sucrose non-fermenting) may render tumor cells resistant to the lethal effects of cis-platin (Strobeck et. Not only can mutations in this complex render a cancer cell resistant to a substrate, but the lack of this complex can cause cancer. In humans, the SWI/SNF ATPase subunits, BRG1 and BRM, are lost in a subset of human cancer cell lines and human primary cancers (Reisman et.
These proteins, the human homologs of yeast SNF2 (sucrose non-fermenting 2), are tumor suppressors. Another member of the human SWI/SNF complex is associated with the development of tumors in soft tissue and the nervous system. INI1 (integrase interactor 1) is the human homolog of the yeast SNFS5 and was first identified as a protein required to bind the Human Immunodeficiency Virus (HIV) protein integrase and stimulate its DNA joining activity (Kalpana et. Homozygous loss of function mutations in mice result in death between embryonic 3.
Fifteen percent of the mice that were heterozygous for the JNI/ mutation developed a loss of heterozygocity at this locus, which resulted in undifferentiated or poorly differentiated sarcomas (Guidi et. This mutation causes aggressively malignant tumors that often arise in children less than two years old (Kufe et. Because the only treatment is surgery and relapses generally occur after six months, the current survival rate for these cancers is less than 10%. Analysis of the sequence found a frameshift or nonsense mutation that caused a truncation of the INI1 protein.
In addition, the truncation of one allele was associated with the loss of the other allele. 4 Cancers caused by mutation in JN// are probably so aggressive because IN//, in addition to being part of a complex that modifies histone, also plays a role in the regulation of the cell cycle. INI1 is directly recruited to the cycling D1 promoter. If INI1 is not present, cyclin D1 expression is not repressed and the cell continues to progress through the cell cycle.
Clearly more research is required to understand the roles these chromatin remodeling proteins are playing in the progression of cancer. Our studies suggest the role may be in a regulator of multidrug resistance. Yeast as a Model System for Studying Multidrug Resistance Yeast are an excellent model system for studying multidrug resistance. Saccharomyces cerevisiae is a eukaryote that has all the major organelles found in higher eukaryotes.
The genome is completely sequenced and encodes nearly 6,000 genes (Goffeau et. Yeast grow quickly with a doubling time of approximately 2 hours minutes and most importantly for genetic studies, the organism can exist as either haploid or diploid. There are two mating types (a and a).