INFORMAnON TO USERS This manuscript has been reprodueed from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, sorne thesis and dissertation copies are in typewriter face. while oIhers may be from any type of computer printer. The quality of thla ntproduction .. dependent upon the q..11ty of the copy submitted. Broken or indisti1ct print, coIored or poor quality illustrations and photographs. print bleedthrough. s~ndard margïns. and improper alignment can adversely affect reproduction. ln the unlikely event that the author did not send UMI a complete manuscript and there are missing pages. these will be noted. Also. if unaulhorized copyright material had to be ntmoved. a note will indicate the defetion. Oversize materials (e.g.• map5. drawings. charts) are reproduced by sectioning the original. begiming al the upper Ieft-hand corner and continuing from Ieft to right in equal sections with small overlaps. Photographs induded in the original rnanuscript have been reproduced xerographically in this copy. Higher quality 6- x 9"' black and white photographie prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI direclly to ORter. Bell & HoweIllntormation and Leaming 300 Nor1h leeb Road, Ann Arbor, MI 48106-1346 USA e UMI800-521..Q600 • Genetic factors affecting life span in the nematode Caenorhabditis elegans Bernard C. Lakowski, Department ofBiology, McGill University. March 1988 A thesis submitted to the Faculty ofGraduate Studies and Researcb in partial fulf"t1lment ofthe requirements ofthe degree ofPh. D. © Bernard C. Lakowski 1998 • National Ubrary Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie services services bibliographiques 395 Wellington Street 395. rue We8ington Ottawa ON K1 A ON4 Ottawa ON K1A 0N4 Canada canada The author has granted a 000­ L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies ofthis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership ofthe L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts frOID it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permISSIon. autorisation. 0-612-44483-X Canadl ü • Abstract The nematode worm Caellorlrllbditis elegalls bas become a model system for the analysis ofthe geneties ofaging. Previously, mutations in four genes, age-l, clk-l, daf-2 and daf-28 bad been sbown to lengthen adult life span. Based on the molecular genetic analysis ofthese genes, the sole function ofthe dauer genes age-l,daf-2 and possibly daf-28 is to regalate the activity ofthe forkhead-like transcription factor daf-16. dtlf-16 May determiDe ure span by regulatïDg the transcription ofgenes that are necessary for resistance to stresses, especiaUy oxidative stress. ~Iutations in clk­ 1 affect bebavioral and developmental timing as weU as increasÏDg Mean and maximum life span. 1 sbow that mutations in the genes clk-2, clk-3 and gro-l affect many ofthe same processes as clk-l and that tbese four genes interact to determine tbe lengtb ofdevelopment and adult life span. Tbese four Clock genes lengthen lüe span in a manner that is distinct from that ofthe dauer geBes. clk-l bas been c10ned and bas been impUcated in the regalation ofmetaboUsm. This SDggests that Clock mutants May live long becaDse they bave reduced metabolic rates. 1 also sbow that mutations in 7 geBes tbat affect feeding bebavior, eat-l, etlt-2, eat-3, eat-6, eat-I3, eat­ 18 and ullc-26 lengthen lüe span. This effect is presumably due to reduced calorie intake (calorie restriction) which has been shown to lengthen the ure span ofa wide variety ofanimais. etlt-2 lengthens Iüe span by a mecbanism that is distinct from that ofthe dauer mutaDts but may be similar to that ofthe Clock mutants. Tbis suggests tbat caloric restrictioB may also redDce metaboUc rates, possibly througb down-regalation ofthe Clock genes. These results indicate tbat life span in C. elegtlns is a polygenic trait, inftuenced by Many different pbysiological processes. The study ofgenes that affect aging in C. elegalls provides support for tbe antagonistic pleiotropy and free radical theories ofaging• • üi Résumé • Le ver nématode Caenorhabditis elegans est devenu un organisme modèle pour étudier la génétique du vieillissement. L'effet de mutations dans les quatre gènes age-J: clk-l, daf­ 2, et daf-28 a auparavant été décrit comme pouvant augmenter la durée de vie du ver. L'analyse génétique et moléculaire de ces gènes suggère que la seule fonction des gènes dauer age-l, daf-2 et éventuellement daf-28 serait de réguler l'activité du facteur de transcription semblable à forkhead codé par le gène daf-16. daf-16 pourrait déterminer la durée de vie en régulant la transcription de gènes nécessaires à la résistance à divers stress, en particulier au stress oxidatif. Des mutations dans le gène clk-l affectent le déroulement temporel de nombreuses étapes du développement et du comportement chez le ver; elles augmentent également sadurée de vie moyenne et maximale. Dans ce travail, je démontre que ces mêmes processus sont affectés par des mutations dans les gènes clk­ 2, clk-3 et gro-l. De plus, je démontre que ces quatre gènes interagissent pour déterminer la durée du développement et la durée de vie chez l'adulte. Ces quatre gènes Clock augmentent la durée de vie d'une manière distincte de celle des gènes dauer. clk-I a été cloné et semble être impliqué dans la régulation du métabolisme. Ceci suggère que le taux métabolique réduit pourrait être à l'origine de l'augmentation de la durée de vie des mutants Clock. Par ailleurs, je démontre que des mutations dans sept gènes impliqués dans le comportement alimentaire, eat-l, eat-2, eat-3, eat-6, eat-/3, eat-/8 et unc-26, augmentent la durée de vie. Cet effet est probablement dû à une réduction de la consommation calorique (appelé restriction calorique), qui a été montré chez de nombreux animaux comme responsable de l'augmentation la durée de vie. Le mécanisme par lequel eat-2 augmente la durée de vie est distinct de celui des gènes dauer, mais pourrait être similaire à celui des gènes Clock. Ceci suggère que la restriction calorique peut réduire les taux métaboliques, vraissemblablement par une régulation négative des gènes Clock. Ces résultats indiquent que chez C. elegans la durée de vie est un trait polygénique, influencé par plusieurs processus physiologiques différents. L'étude de ces gènes qui affectent la durée de vie chez C. elegans supportent la pléiotropie opposée et la radicaux libres théories du veillissement. • Transduction: C. Bénard IV • Acknowledgements: l would like to thank all past and present members ofthe Hekimi lab for their help and support ofthe years. In particular 1would like to tbank Wendy Lai who helped with sorne ofthe early Clock aging experiments. 1 also thank: Tom Barnes and Jonathan Ewbank for their advice on writing papers, and for Many hours ofdiscussion on C. elegans genetics and biology and Stephanie Felkai, Claire Bénard and Anne Wong for 100king after sorne aging experiments for a few days, when 1 needed the odd weekend off Mattieu Lupien, Jonathan Ewbank and Jason Lemieux communicated sorne unpublished results and Claire Bénard translated the abstracto And ofcourse 1 thank my supervisor Siegfried Hekimi, who bas aIso taught me a great deal about how science really works and what one needs to succeed. l thank Bob Horvitz, Ken Kemphues, Leon Avery, Victor Ambrose and Jonathan Hodgkin for a few strains and Theresa Stiemagle at the Caenorhabditis Genetics Center for the hundreds ofstrains she has sent me over the years. Without these strains, this thesis \vould not have been possible. 1would also like to thank the J.W. McConnell Foundation and Fonds pour la Fonnation de Chercheurs et l'Aide à la Recherche (FCAR) Québec for fellowships that allowed me to devote ail ofmy time to research. l aIso thank the members ofthe Copaholics who helped make Montréal winters bearable, and my friends and family who helped to keep me going. Finally, 1 thank • Alexandra for aIl ber support and for making it ail worthwhile. v Preface • Objectives ofthis thesis In this thesis, 1 present work 1have done to determine the genetic factors that influence the life span ofthe nematode Caenorhabditis elegans. To do this~ 1studied the life span ofvarious mutant strains, trying to identify mutations that lengthen life span. Such mutations must affect the normal process ofaging. Thus, by analyzing such mutants, we May gain insight ioto those factors that affect aging rates, and perbaps even the ultimate causes ofaging. 1aIso try to determine how these factors interact genetically. To do this, 1constructed Many double mutant strains, determined their life spans, and examined their phenotypes. Dy such analysis, 1hoped to detennine ifthere are different mechanisms that affect life span, and ifso bow these mechanisms interrelate. Finally, 1 aIso sougbt to help clone some ofthe genes that affect life span, to better understand the molecular basis ofaging. To facilitate the cloning ofthe genes clk-I, clk-2. clk-3 and gro-I, 1mapped these genes to their closest flanking genetic markers. When the genetic position ofa gene is weil known in C. elegans, this can be used to help detennine the physical position ofthe gene and to select suitable clones to test transformational rescue ofthe mutant phenotype.