老化是一個複雜的過程，受到許多路徑調控。根據研究發現，許多單一基因的突變株可以幫助老化研究。我們從一個正向的遺傳篩選實驗發現，EP1101, EP1130 和EP2456是具有長壽和抗壓的果蠅。為了了解這三株果蠅在整個基因體上的表現，我們利用微陣列基因晶片(cDNA microarray)的技術，找到共同被調控的基因群，這些標的基因可能調節長壽以及抗壓的表現型。晶片分析顯示，一些已被報導和老化有關的基因群亦參與在這三株突變果蠅當中，例如代謝、壓力反應和免疫系統。為確認標的基因可以調控長壽以及抗壓的假設，我們挑選幾株突變果蠅進行實驗，以檢視是否改變這些標的基因的表現，的確可以影響壽命和抗壓性。我們發現其中兩個新基因：CG4199和staufen，可能是和老化有關的基因。此外，在演化上具有相同功能的基因對於研究老化是非常重要的。因此我們找出晶片分析中的標的基因在線蟲當中的同源基因，利用RNAi的方法進行壽命和壓力測試。此結果顯示，降低C37C3.8 和 F55D10.3此兩同源基因的表現可以略為延長壽命，但無法增加壓力抗性。另一方面，為了預測三株長壽果蠅可能參與在老化過程的途徑，我們將這些果蠅和其他已知與老化有關的突變株交配，研究他們相互間長壽的加乘性。結果顯示，EP1130可能參與在賀爾蒙的途徑調控壽命，而EP1101則可能參與在未知的途徑。同時，這兩株果蠅均可提高對於氧化壓力敏感的突變株的抗壓性。這些研究提供一個方法來挑選出和老化有關的基因，以幫助我們決定三株果蠅長壽和抗壓的基因群以及研究老化共同的途徑。

Abstract--------------------------------------------------------------------------------------------I
Acknowledgment ------------------------------------------------------------------------------III
List of Figures------------------------------------------------------------------------------------V
List of Tables------------------------------------------------------------------------------------IV
Introduction---------------------------------------------------------------------------------------1
Materials and methods---------------------------------------------------------------------------8
Results--------------------------------------------------------------------------------------------13
I. Microarray data analysis-------------------------------------------------------------13
II. Validation of microarray data by in vivo experiments--------------------------20
III. Synergistic effect on lifespan between the EP longevity lines and other long-lived mutants---------------------------------------------------------------------22
Discussion---------------------------------------------------------------------------------------26
I. Genomic analysis of three EP longevity lines------------------------------------26
II. Genetic analysis of three EP longevity lines-------------------------------------31
Figures and Tables------------------------------------------------------------------------------34
Reference----------------------------------------------------------------------------------------62
Appendix: Plasmid rescue and inverse PCR------------------------------------------------68

[1] Arking R, Burde V, Graves K, et al. (2000) Forward and reverse selection for longevity in Drosophila is characterized by alteration of antioxidant gene expression and oxidative damage patterns. Exp Gerontol 35: 167-185.
[2] Baehrecke EH (2000) Steroid regulation of programmed cell death during Drosophila development. Cell Death Differ 7: 1057-1062.
[3] Barbieri M, Bonafe M, Franceschi C & Paolisso G (2003) Insulin/IGF-I-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. Am J Physiol Endocrinol Metab 285: E1064-1071.
[4] Bauer JH, Poon PC, Glatt-Deeley H, Abrams JM & Helfand SL (2005) Neuronal expression of p53 dominant-negative proteins in adult Drosophila melanogaster extends life span. Curr Biol 15: 2063-2068.
[5] Beckman KB & Ames BN (1998) The free radical theory of aging matures. Physiol Rev 78: 547-581.
[6] Breitwieser W, Markussen FH, Horstmann H & Ephrussi A (1996) Oskar protein interaction with Vasa represents an essential step in polar granule assembly. Genes Dev 10: 2179-2188.
[7] Buck S, Wells RA, Dudas SP, Baker GT, 3rd & Arking R (1993) Chromosomal localization and regulation of the longevity determinant genes in a selected strain of Drosophila melanogaster. Heredity 71 ( Pt 1): 11-22.
[8] Choe KM, Werner T, Stoven S, Hultmark D & Anderson KV (2002) Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296: 359-362.
[9] Clancy DJ, Gems D, Harshman LG, et al. (2001) Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science 292: 104-106.
[10] DeVeale B, Brummel T & Seroude L (2004) Immunity and aging: the enemy within? Aging Cell 3: 195-208.
[11] Dhahbi JM, Mote PL, Wingo J, Rowley BC, Cao SX, Walford RL & Spindler SR (2001) Caloric restriction alters the feeding response of key metabolic enzyme genes. Mech Ageing Dev 122: 1033-1048.
[12] Ding D, Parkhurst SM & Lipshitz HD (1993) Different genetic requirements for anterior RNA localization revealed by the distribution of Adducin-like transcripts during Drosophila oogenesis. Proc Natl Acad Sci U S A 90: 2512-2516.
[13] Dubnau J, Chiang AS, Grady L, et al. (2003) The staufen/pumilio pathway is involved in Drosophila long-term memory. Curr Biol 13: 286-296.
[14] Esterbauer H, Schaur RJ & Zollner H (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 11: 81-128.
[15] Galvin JE & Ginsberg SD (2005) Expression profiling in the aging brain: a perspective. Ageing Res Rev 4: 529-547.
[16] Gilbert LI, Granger NA & Roe RM (2000) The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol 30: 617-644.
[17] Girardot F, Lasbleiz C, Monnier V & Tricoire H (2006) Specific age-related signatures in Drosophila body parts transcriptome. BMC Genomics 7: 69.
[18] Gottar M, Gobert V, Michel T, et al. (2002) The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416: 640-644.
[19] Guarente L & Kenyon C (2000) Genetic pathways that regulate ageing in model organisms. Nature 408: 255-262.
[20] Hamilton B, Dong Y, Shindo M, Liu W, Odell I, Ruvkun G & Lee SS (2005) A systematic RNAi screen for longevity genes in C. elegans. Genes Dev 19: 1544-1555.
[21] Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11: 298-300.
[22] Harman D (1981) The aging process. Proc Natl Acad Sci U S A 78: 7124-7128.
[23] Holzenberger M, Dupont J, Ducos B, et al. (2003) IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature 421: 182-187.
[24] Hwangbo DS, Gershman B, Tu MP, Palmer M & Tatar M (2004) Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429: 562-566.
[25] Ivy JM, Klar AJ & Hicks JB (1986) Cloning and characterization of four SIR genes of Saccharomyces cerevisiae. Mol Cell Biol 6: 688-702.
[26] Jiang JC, Wawryn J, Shantha Kumara HM & Jazwinski SM (2002) Distinct roles of processes modulated by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast. Exp Gerontol 37: 1023-1030.
[27] Johnstone O & Lasko P (2001) Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu Rev Genet 35: 365-406.
[28] Kaeberlein M, McVey M & Guarente L (1999) The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 13: 2570-2580.
[29] Kayo T, Allison DB, Weindruch R & Prolla TA (2001) Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci U S A 98: 5093-5098.
[30] Kenyon C (2005) The plasticity of aging: insights from long-lived mutants. Cell 120: 449-460.
[31] Kenyon C, Chang J, Gensch E, Rudner A & Tabtiang R (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366: 461-464.
[32] Kerscher S, Albert S, Wucherpfennig D, Heisenberg M & Schneuwly S (1995) Molecular and genetic analysis of the Drosophila mas-1 (mannosidase-1) gene which encodes a glycoprotein processing alpha 1,2-mannosidase. Dev Biol 168: 613-626.
[33] Kim SN, Rhee JH, Song YH, et al. (2005) Age-dependent changes of gene expression in the Drosophila head. Neurobiol Aging 26: 1083-1091.
[34] Kimura KD, Tissenbaum HA, Liu Y & Ruvkun G (1997) daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277: 942-946.
[35] Kirkwood TB (1977) Evolution of ageing. Nature 270: 301-304.
[36] Kirkwood TB (1988) The nature and causes of ageing. Ciba Found Symp 134: 193-207.
[37] Kirkwood TB (2002) Evolution of ageing. Mech Ageing Dev 123: 737-745.
[38] Lai CQ, Parnell LD, Lyman RF, Ordovas JM & Mackay TF (2007) Candidate genes affecting Drosophila life span identified by integrating microarray gene expression analysis and QTL mapping. Mech Ageing Dev 128: 237-249.
[39] Lee CK, Weindruch R & Prolla TA (2000) Gene-expression profile of the ageing brain in mice. Nat Genet 25: 294-297.
[40] Lee CK, Klopp RG, Weindruch R & Prolla TA (1999) Gene expression profile of aging and its retardation by caloric restriction. Science 285: 1390-1393.
[41] Lee CK, Allison DB, Brand J, Weindruch R & Prolla TA (2002) Transcriptional profiles associated with aging and middle age-onset caloric restriction in mouse hearts. Proc Natl Acad Sci U S A 99: 14988-14993.
[42] Lin K, Dorman JB, Rodan A & Kenyon C (1997) daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319-1322.
[43] Lin SJ, Kaeberlein M, Andalis AA, et al. (2002) Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418: 344-348.
[44] Livak KJ & Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408.
[45] Longo VD & Kennedy BK (2006) Sirtuins in aging and age-related disease. Cell 126: 257-268.
[46] Luo Y, Amin J & Voellmy R (1991) Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes. Mol Cell Biol 11: 3660-3675.
[47] Lynch M & Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 290: 1151-1155.
[48] Maier B, Gluba W, Bernier B, et al. (2004) Modulation of mammalian life span by the short isoform of p53. Genes Dev 18: 306-319.
[49] Mangel M (2001) Complex adaptive systems, aging and longevity. J Theor Biol 213: 559-571.
[50] McCarroll SA, Murphy CT, Zou S, et al. (2004) Comparing genomic expression patterns across species identifies shared transcriptional profile in aging. Nat Genet 36: 197-204.
[51] McElwee JJ, Schuster E, Blanc E, Thomas JH & Gems D (2004) Shared transcriptional signature in Caenorhabditis elegans Dauer larvae and long-lived daf-2 mutants implicates detoxification system in longevity assurance. J Biol Chem 279: 44533-44543.
[52] Park SK & Prolla TA (2005) Gene expression profiling studies of aging in cardiac and skeletal muscles. Cardiovasc Res 66: 205-212.
[53] Parkes TL, Kirby K, Phillips JP & Hilliker AJ (1998) Transgenic analysis of the cSOD-null phenotypic syndrome in Drosophila. Genome 41: 642-651.
[54] Partridge L & Gems D (2002) Mechanisms of ageing: public or private? Nat Rev Genet 3: 165-175.
[55] Pattison JS, Folk LC, Madsen RW, Childs TE & Booth FW (2003) Transcriptional profiling identifies extensive downregulation of extracellular matrix gene expression in sarcopenic rat soleus muscle. Physiol Genomics 15: 34-43.
[56] Pearl R (1928) The Rate of Living. ed.^eds.), p.^pp. University of London Press, London.
[57] Pletcher SD, Macdonald SJ, Marguerie R, Certa U, Stearns SC, Goldstein DB & Partridge L (2002) Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster. Curr Biol 12: 712-723.
[58] Porter AG & Urbano AG (2006) Does apoptosis-inducing factor (AIF) have both life and death functions in cells? Bioessays 28: 834-843.
[59] Puca AA, Chatgilialoglu C & Ferreri C (2007) Lipid metabolism and diet: Possible mechanisms of slow aging. Int J Biochem Cell Biol.
[60] Raff JW, Whitfield WG & Glover DM (1990) Two distinct mechanisms localise cyclin B transcripts in syncytial Drosophila embryos. Development 110: 1249-1261.
[61] Ramet M, Manfruelli P, Pearson A, Mathey-Prevot B & Ezekowitz RA (2002) Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644-648.
[62] Riehle MA & Brown MR (1999) Insulin stimulates ecdysteroid production through a conserved signaling cascade in the mosquito Aedes aegypti. Insect Biochem Mol Biol 29: 855-860.
[63] Rine J & Herskowitz I (1987) Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics 116: 9-22.
[64] Rogina B & Helfand SL (2004) Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc Natl Acad Sci U S A 101: 15998-16003.
[65] Rogina B, Helfand SL & Frankel S (2002) Longevity regulation by Drosophila Rpd3 deacetylase and caloric restriction. Science 298: 1745.
[66] Rogina B, Reenan RA, Nilsen SP & Helfand SL (2000) Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 290: 2137-2140.
[67] Rorth P (1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci U S A 93: 12418-12422.
[68] Sanchez D, Lopez-Arias B, Torroja L, Canal I, Wang X, Bastiani MJ & Ganfornina MD (2006) Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila. Curr Biol 16: 680-686.
[69] Sawicki R, Singh SP, Mondal AK, Benes H & Zimniak P (2003) Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. Biochem J 370: 661-669.
[70] Schwarze SR, Weindruch R & Aiken JM (1998) Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila. Free Radic Biol Med 25: 740-747.
[71] Seroude L, Brummel T, Kapahi P & Benzer S (2002) Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster. Aging Cell 1: 47-56.
[72] Shore D (2000) The Sir2 protein family: A novel deacetylase for gene silencing and more. Proc Natl Acad Sci U S A 97: 14030-14032.
[73] Simmer F, Moorman C, van der Linden AM, et al. (2003) Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol 1: E12.
[74] Simon AF, Shih C, Mack A & Benzer S (2003) Steroid control of longevity in Drosophila melanogaster. Science 299: 1407-1410.
[75] Tatar M, Bartke A & Antebi A (2003) The endocrine regulation of aging by insulin-like signals. Science 299: 1346-1351.
[76] Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM & Garofalo RS (2001) A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292: 107-110.
[77] Thellin O, Zorzi W, Lakaye B, et al. (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75: 291-295.
[78] Tu MP, Yin CM & Tatar M (2002) Impaired ovarian ecdysone synthesis of Drosophila melanogaster insulin receptor mutants. Aging Cell 1: 158-160.
[79] Tyner SD, Venkatachalam S, Choi J, et al. (2002) p53 mutant mice that display early ageing-associated phenotypes. Nature 415: 45-53.
[80] van Eeden F & St Johnston D (1999) The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr Opin Genet Dev 9: 396-404.
[81] Van Voorhies WA & Ward S (1999) Genetic and environmental conditions that increase longevity in Caenorhabditis elegans decrease metabolic rate. Proc Natl Acad Sci U S A 96: 11399-11403.
[82] Walker DW, Muffat J, Rundel C & Benzer S (2006) Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan. Curr Biol 16: 674-679.
[83] Weindruch R, Kayo T, Lee CK & Prolla TA (2001) Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice. J Nutr 131: 918S-923S.
[84] Welle S, Brooks AI, Delehanty JM, Needler N & Thornton CA (2003) Gene expression profile of aging in human muscle. Physiol Genomics 14: 149-159.
[85] Williams GC (1966) Natural selection, the costs of reproduction, and a refinement
of Lack’s principle. Am. Nat 100: 687–690.
[86] Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J & Speed TP (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15.
[87] Yin D (1995) Studies on age pigments evolving into a new theory of biological aging. Gerontology 41 Suppl 2: 159-172.
[88] Zheng J, Edelman SW, Tharmarajah G, Walker DW, Pletcher SD & Seroude L (2005) Differential patterns of apoptosis in response to aging in Drosophila. Proc Natl Acad Sci U S A 102: 12083-12088.
[89] Zou S, Meadows S, Sharp L, Jan LY & Jan YN (2000) Genome-wide study of aging and oxidative stress response in Drosophila melanogaster. Proc Natl Acad Sci U S A 97: 13726-13731.