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Identification of Mutations That Extend the Fission Yeast

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Identification of Mutations That Extend the Fission Yeast IDENTIFICATION OF MUTATIONS THAT EXTEND THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE CHRONOLOGICAL LIFESPAN BY A NOVEL PARALLEL SELECTION APPROACH by BO-RUEI CHEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Kurt W. Runge, Ph.D. Department of Genetics CASE WESTERN RESERVE UNIVERSITY January, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Bo-Ruei Chen a candidate for the Ph.D. degree *. (signed) Mark Adams, Ph.D. (chair of the committee) Kurt Runge, Ph.D. Steven Sanders, Ph.D. Peter Harte, Ph.D. Jo Ann Wise, Ph.D. (date) s 10/18/2010 _ *We also certify that written approval has been obtained for any proprietary material contained therein. Tables of Contents List of Tables v List of Figures vi Acknowledgements ix List of Abbreviations x Abstract 1 Chapter 1: Background, significance and specific aims 3 Aging, lifespan and longevity: a general introduction 4 Aging: an evolutionary perspective 6 The free radical theory of aging 7 Caloric restriction, stress management and lifespan 11 Mitochondrial activity, metabolism and aging 14 Down-regulation of the target of rapamycin (TOR) signaling and longevity 17 The insulin/insulin-like growth factor (IGF)-1 signaling and longevity 22 Sir proteins (sirtuins) and aging: more than just silencing transcription 26 Advantages of using yeast as a model to study evolutionarily conserved mechanisms of aging 33 The fission yeast Schizosaccharomyces pombe as an emerging model to study aging and longevity 35 Significance and specific aims of this study 37 Chapter 2: A new Schizosaccharomyces pombe chronological lifespan assay reveals that caloric restriction promotes efficient cell cycle exit and extends longevity 40 Abstract 41 Introduction 42 Materials and Methods 46 S. pombe strains 46 Chronological aging assays 48 Analysis of CLS assay data 49 FACS analysis (fixed cells) 49 i FACS analysis (live cells) 50 Stress sensitivity assays 50 Results 52 Chronological lifespan assay design 52 Cells in SD medium show the evolutionarily conserved lifespan shortening in response to over nutrition while cells in EMM medium do not 52 Caloric restriction extends lifespan in the SD medium-based assay 57 Longer CLS correlates with exhausting free glucose in the medium 63 Long-lived calorically restricted cells show increased stress resistance 64 The AKT orthologs sck1+ and sck2+ differentially affect lifespan under 66 normal and over nutrition conditions Deletions of the non-essential TOR kinase tor1+ or its potential 69 substrate gad8+ extends S. pombe chronological lifespan Discussion 71 Chapter 3: Construction and characterization of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe 75 Abstract 76 Introduction 77 Materials and Methods 83 Strains and media 83 Construction of the bacterial barcode-tagged insertion DNA vector library 84 Construction of the fission yeast barcode-tagged insertion mutant library 88 Determination of the size of S. pombe barcode-tagged insertion mutant 89 library and bacterial barcode library Phenotypic assays to assess mutation diversity 90 Identification of insertion sites by thermal asymmetric interlaced (TAIL)-PCR 91 Results 93 Design of a linear barcode-tagged DNA vector for insertion mutagenesis in the fission yeast S. pombe 93 Construction of the bacterial barcode-tagged insertion DNA library 97 ii Construction of the fission yeast barcode-tagged insertion mutant library 97 The insertion mutant library contains multiple diverse mutations 100 Identification of the insertion sites by TAIL-PCR 102 Discussion 106 Chapter 4: A novel parallel selection approach identified a cyclin/CDK complex, Clg1p/Pef1p, whose inactivation leads to lifespan extension in the fission yeast Schizosaccharomyces pombe 111 Abstract 112 Introduction 114 Materials and Methods 119 Strains and media 119 Chronological aging assays 121 A parallel selection for long-lived mutations using PCR-mediated barcode sequencing 121 Identification of insertion sites by thermal asymmetric interlaced 126 (TAIL)-PCR and splinkerette PCR Construction of fission yeast mutant strains for lifespan analysis 127 Expression plasmids construction 130 Yeast two hybrid assay 131 Protein extraction, immunoprecipitation and Western blotting 131 Analysis of CLS assay data 133 Results 134 An unbiased parallel selection for long-lived barcode-tagged insertion mutants using a novel sequencing strategy 134 CLS of clg1-, spncrna.142- and 28S rRNA mutants 141 Establishment of a working model for Clg1p-dependent lifespan regulation 144 Clg1p physically interacts with the CDK Pef1p 145 Clg1p and the CDK Pef1p control CLS through the same pathway 147 Identification of other Pcl-like cyclins in S. pombe 149 Deletion of psl1+ or pas1+ does not extend CLS 152 Identification of potential S. pombe Rim15p orthologs 152 iii Cek1p physically interacts with Pef1p 156 A proposed model of chronological lifespan control in S. pombe by 157 Clg1p, Pef1p and Cek1p Discussion 159 Chapter 5: General discussion and future directions 165 Using yeast as a model to study aging: advantages and limitations 166 The fission yeast Schizosaccharomyces pombe as an emerging yeast aging 169 model Current approaches for large-scale genetic screen for genes determining 170 yeast lifespan Developing a S. pombe CLS assay and a novel genetic screen for long-lived 174 fission yeast mutants Limitations of the current barcoded insertion mutant library and probable 177 alternatives A proof-of-principle parallel genetic screen identified the Clg1p-Pef1p- 179 Cek1p lifespan-regulating pathway Cell cycle, senescence and aging 183 Future directions 185 Bibliography 188 iv List of Tables Chapter 1 Table 1.1. Non-genetic interventions that extend model organism lifespan 5 Table 1.2. Evolutionarily conserved genes mentioned in this chapter whose orthologs have been implicated in the regulation of aging and longevity 5 Chapter 2 Table 2.1. Oligonucleotides used in this study 47 Table 2.2. Cells grown in caloric restriction (0.1% glucose) medium exhaust their glucose supply as they reach maximum density 64 Chapter 3 Table 3.1. The supplements and their amounts in the YC - uracil mixture used in the selection of insertion mutants 83 Table 3.2. Oligonucleotides used in this study 85 Table 3.3. Characterization of the diversity of mutations in the barcode- tagged insertion mutants by 4 phenotypic analyses. 102 Table 3.4. Insertion sites, detailed structures of the integrated insertion vector and adjacent chromosome sequences determined by TAIL-PCR and sequencing 105 Chapter 4 Table 4.1. Fission yeast strains used in this study 120 Table 4.2. Oligonucleotides used in this study 123 Table 4.3. Barcode sequencing of surviving mutants after selection for 14 days in stationary phase and insertion mutations identified from the overrepresented mutants 138 Table 4.4. Barcode sequencing of pool#1 and #2 insertion mutants in the initial culture (day0) for long-lived mutant selection 138 Table 4.5. Identification of S. cerevisiae orthologs of S. pombe Spbc20f10.10p by BLASTP search 150 Table 4.6. Comparison of S. cerevisiae Rim15p and its probable S. pombe orthologs Cek1p and Ppk18p identified by BLASTP search 154 v List of Figures Chapter 1 Figure 1.1. The effects of oxidative stress on aging 8 Figure 1.2. Mitochondrial metabolism and aging 9 Figure 1.3. TORC1 signaling positively regulates cell growth and negatively 19 regulates longevity Figure 1.4. The insulin/IGF-1 signaling pathway negatively regulates stress resistance and longevity 23 Figure 1.5. Transcriptional silencing regulation by the budding yeast Sir proteins 27 Figure 1.6. Loss of Sir2p results in extrachromosomal rDNA circles (ERCs) formation and aging in budding yeast 28 Figure 1.7. A simplified schematic of the effects of SIR2/sirtuin on lifespan and health 30 Chapter 2 Figure 2.1. Over nutrition shortens S. pombe chronological lifespan in SD medium but lengthens lifespan in EMM medium 54 Figure 2.2. CLS curves in SMM-based medium are multiphasic and show an unusual response to caloric restriction 54 Figure 2.3. Cell density remains constant during the CLS assays in the SD and EMM media 55 Figure 2.4. EMM, but not SD medium, results in the accumulation of a large fraction of cells with sub-1N DNA content 56 Figure 2.5. Cell viability determined by propidium iodide staining and 58 FACS analysis parallels the decline in CFU/ml Figure 2.6. Caloric restriction extends lifespan in S. pombe 59 Figure 2.7. Long-lived S. pombe cells grown in 0.1% glucose maintain a constant DNA FACS profile while aging 62 Figure 2.8. Long-lived calorically restricted S. pombe show increased stress resistance 65 Figure 2.9. Deletion of the AKT kinase gene sck2+ extends lifespan under normal conditions while deletion of the gene for the paralogous kinase sck1+ does not 67 Figure 2.10. Deletion of either AKT kinase sck1+ or sck2+ extends lifespan in over nutrition conditions 68 Figure 2.11. Deletion of tor1+ or gad8+ kinase extends lifespan 70 vi Chapter 3 Figure 3.1. The deletion and tagging strategy used in S. cerevisiae and S. pombe gene deletion mutant collections 80 Figure 3.2. Amplification of insertion vector-genomic DNA junction by TAIL-PCR 92 Figure 3.3. The schematic of the insertion vector used to construct the barcode-tagged insertion mutant library 96 Figure 3.4. Discrimination of different products of linear insertion DNA with an ura4+ marker after transformation into fission yeast 98 Figure 3.5. A selection strategy for cells containing a single copy and multiple copies of ura4+ genes based on the hypothetical metabolic outcome of altered Ura4p levels and low concentrations of 5-FOA on cell survival. 99 Figure 3.6. The flowchart of fission yeast barcode-tagged insertion mutant library construction 100 Figure 3.7. Schematics of insertion mutations identified in this study 104 Chapter 4 Figure 4.1.
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