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•

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.

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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.

A guide to this thesis

The thesis is divided ioto five chapters. The tirst chapter is a literature review of

the genetics ofaging with emphasis ofthe genetics ofaging in C. elegans. In this

chapter, 1 introduce the subject ofaging trying to address what agjng is and which • organisms age. It is not known why animaIs age but many theories have been proposed vi

to explain aging. 1 briefly review sorne ofthese theories before discussing the genetics of • aging. 1 then brietly review what is know about the genetics ofsenescence in mammaIs, mammalian ceIllines, yeast and Drosophila This is followed by a more detailed review

ofthe genetics ofaging in C. elegans. 1begin this section by reviewing what is known

about aging in the wild type and then introduce the known genetic factors that influence

life span. This section ends with a discussion ofwhat bas been leamed about the

molecular and biochemical basis ofaging in C. elegans by studying genes that affect

longevity. Sorne ofthe materiaI in Chapter 1 is based on a review paper to which 1 have

collaborated (Table l, reL 1).

In Chapter 2, 1 present results to show that mutations in four Clock genes, clk-J.

clk-2. clk-3 andgro-l, lengthen life span from 20 to 30%. Most Clock double mutants

live even longer than this, and can live up to three times longer than the wild type. Clock

mutants appear to Iengthen life span by a mechanism that is distinct from that ofthe dauer

genes age-J, daf-2 and daf-28, the only other C. elegans genes that have been shown to

lengthen adult life span. The phenotype ofClock mutations is consistent with these

mutations reducing the worm's rate ofliving, which may explain why they live long.

This chapter is largely based on published results (Table l, ref. 2). To provide additional

background infonnation and experimentaI details, 1 have written an introduction and

materials and methods section. The results are presented in greater detail and the

implications ofthe results for our understanding ofaging are discussed at greater length.

Unpublished data on the life span ofclk-l(qm51) as weIl as more information on the

interaction ofClock genes and the dafgenes is aIso provided.

In Chapter 3, 1 show that mutations in Many eat genes, that reduce food intake, • lengthen life span. This effect appears to be due to caloric restriction because many eat vü

genes and multiple alleles ofthese genes lengthen life sp~ while appropriate controls do • not affect life span. 1 aIso show that the strength ofthe feeding defect correlates with life span eat-2 and eat-6 mutants, strengthening the interpretation that eat mutants live long

because they are caIorically restricted. To detennine ifcalorie restriction lengthens life

span by a similar mechanism to either the Clock genes orthe dauer genes, 1made a

number ofdouble mutant strains and examined their life span. 1 found that eat-2 mutants

lengthen life span by a mechanism that is genetically distinct from that ofthe dauer

mutants. Howp.ver c/k-l and eat-2 may lengthen life span by affecting the same process.

A manuscript based on this cbapter (in a condensed farm) is being prepared for

publication.

In Chapter 4, 1 present work 1have done to genetically map and characterize c/k-l.

clk-2, c/k-3 and gro-l. Sorne ofthese results have been published in two papers (Table 1

ref. 3-4): Many otafter results remaillunpublished.laIso demil the construction ofClock

double mutants and present an initial cbaracterization ofClock mutants and double

mutants. Table 4.2 bas been published as Table 2 in ret: 2. The results in Chapter 4

contributed to the positional cloning ofclk-l (Table l, ref4) and gro-l (unpublished) and

should facilitate the cloning ofc/k-2 and c/k-3. The molecular identity ofclk-l offers us

insights into the molecular basis ofaging.

In Chapter 5,1 summarize the main findings ofthis thesis and discuss their

implications for aging. • viü

Table 1: Sorne ofthe data presented in this thesis has been published in the following • papers ref. Paper

1 Siegfried Hekimi, Bernard Lakowski, Thomas M. Bames and Jonathan J. Ewbank

(1998). Molecular genetics ofIife span in C. e/egans: how much does it teach us?

Trends in Genetics 14, 14-20.

121 1 Bernard Lakowski and Siegfried Hekimi (1996). Determination ofLife-Span in 1 1

1 Caenorhabditis elegans by Four Clock Genes. Science, 272 1010-1013

3 Siegfried Hekimi, Paula Boutis and Bernard Lakowski (1995). Viable Matemal-

Effect Mutations That Affect the Development ofthe Nematode Caenorhabditis

elegans. Genetics 141, 1351-1364

4 Jonathan J. Ewbank, Thomas M. Bames, Bernard Lakowski, Marc Lussier, Howard

Busseyand Siegfried Hekimi (1997). Structural and Functional Conservation ofthe

Caenorhabditis elegans Timing Gene clk-l. Science 275,980-983.

• ix

Table ofContents • Title page Abstract ii Resume iii Acknowledgements iv Preface v Objectives ofthis thesis v A Guide to this thesis v Table ofcontents ix List offigures xi List oftables xii

Chapter 1: An introduction to the genetics ofaging with 1 emphasis on Caenorhabditis elegans

Abstract 2 Wbat is agiDg? 3 The genetic basis of life span 7 C eleglUls an exceUent system to study agjDg: 13 The genetics ofagiDg in C. elegllfts 19 The molecular identities oflige-l, dllf-2 and dllf-16 26 Oxidative stress and aging in C elegllfts: 31 Conclusions 32

• Chapter 2: Determination ofLife-Span in Caenorhabditis 33 • elegans by Four Cloek Genes Abstract 34 Introduction 35 Material! and Methods 37 Results 42 Discussion 54

Chapter 3: The Genetics ofCalorie Restriction in 64 Caenorhllbditis elegans Abstract 65 Introduction 66 Materials and Methods 70 Results 73 Discussion 91

Chapter 4: The Caenorhabditis elegllns Clock genes: four novel 96 genes that affect developmental and behavioral timing Abstract 97 Introduction 98 Materials and Methods 101 Results 109 Discussion 128

Chapter 5: Summary and Conclusions 135

References 143 • xi

List of Figures • Fig. 1.1. The temperature dependence oflife span in C. elegans 16 Fig 1.2. The dependence oflife span ofC. elegans on bacterial concentration in 17

liquid culture

Fig 1.3. Sorne features ofthe genetic pathway determining two alternative 28

developmental fates in Caenorhabditis elegans.

Fig. 2.1. AIl three clk-1 aIleles lengthen life span. 44

Fig 2.2 Clock mutations interact to determine life-span. 46

Fig. 2.3. The interaction ofclk-l with daf-16 and daf-2. 50

Fig. 2.4. Scatter plot ofthe length ofdevelopment versus Mean adult life span. 62

Fig. 3.1. Four alleles ofeat-2lengthen life span. 78

Fig.3.2. The long life ofeat-2(ad465) is not suppressed by daf-16(m26). 87

Fig 3.3. the interaction ofeat-2 with daf-2 and clk-l. 89

Fig. 4.1. A simplified genetic map ofLGIII in the vicinity ofclk-l and gro-l. 112

Fig. 4.2. A simplified genetic map ofLGIII in the vicinity ofclk-2. 120

Fig. 4.3. Embryonic development ofthe wild type (N2) and clk-3(qm38) at 20°C 122

Fig. 4.4. A simplified genetic map ofLGll, with a blowup ofthe vicinity of 125

clk-3.

Fig. 4.5. The 95% confidence intervals for clk-2 mapping. 132

• xii

List ofTables • Table 1. Some ofthe data presented in this thesis has been published in the viii following papers

Table 1.1. Quantitative analysis ofthe clk-l mutant phenotype 24

Table 2.1. Number ofdauers or partial dauers produced by mating different 41

males to the strain daf-16(m26). dap-l(el) daf-2(eI370) unc-32(e189)

Table 2.2 Mean life-span ofClock strains at 15, 18,20 and 25°C 43

Table 2.3. The Mean life span ± standard error for daf-16:clk-l, daf-16:clk-2, 52

daf-16:clk-3 and daf-16;gro-l.

Table 2.4. the interaction ofclk-l with daf-2. 55

Table 3.1. The pooled results oftwo experiments in which the life span ofeat 74

strains received from the Caenorhabditis Genetic Center were tested

Table 3.2. Mean life span ofa number ofeat mutant strains. 76

Table 3.3. The life span oforiginal une strains 81

Table 4.1. Genetic mapping data used to position clk-l and gro-l 110

Table 4.2. Length ofpost-embryonic development ofClock strains at 15, 18,20 114

and 25°C

Table 4.3. Genetic mapping data used to position c/k-2 119

Table 4.4. Genetic mapping data used to position c/k-3 124

• 1 • Chapter 1

An introduction to the genetics ofaging with emphasis on

Caenorhabdilis elegans

• 2

Abstract

• The genetics ofanimallife span has been studied best in the nematode worm

Caenorhabditis elegiUls. The advantages ofusing C. elegans to study aging are

manüold, including its short geDeradoD time, short life span, weU developed

geDetics, standard reference straiD and lack ofinbreeding depressioD. LiCe span in

C. elegans is dependent on botb temperatare and food concentration. Development

is also affeeted by the environment. Under stressful cODditions, worms can enter aD

alternative third larval stage, known as the dauer stage, aod arrest development and

aging. Previously, mutations in six genes, spe-26, rad-8, clk-l, age-l, dllf-2, and daf­

28 have been shown to leDgthen liCe spaD. spe-26 encodes a major sperm proteine It

is Dot c1ear bow spe-26 affects liCe span and its reported effect on life span may iD

fact be due to differences iD strain backgrounds. rad-8 lengthens life span oDly at

low temperatures aDd it does 50 solely by lengtbening development. Mutations in

clk-l increase adult life span as weil the leDgth ofdevelopment and the periods of

rhythmic adult behaviors. age-l dllf-2 and daf-28 regalate longevity and entry into

the dauer stage. These genes do so by affeetiDg the activity ofthe transcription

factor daf-16, which affects resistance to many stresses, including oxidative stress.

ID this thesis, 1 show that maDY other genes can lengthen life span. 1 also explore the

genetic interactions among the newly identified and previously characterized

"agiDg" genes• • 3

What is aging? • The process ofaging A great deal ofour knowledge about aging relates to the phenomenology ofhuman aging. Aging is a process that we are aIl weIl aware ot: yet it is

a surprisingly bard thing to define. There are obvious extemaL signs, such as thinning and

graying hair and the wrinkling ofskin. However, in humans it aIso manifests itseLfas a

slow and irreversible decline in physiologicaI function, decreased ability to respond to

stresses, increased risk ofcertain diseases, reduced levels ofcertain hormones and

impaired memory (Kirkwood 1996, Lamberts et al. 1997, Morrison and Rof 1996). The

changes that occur with age are complex and it is hard to find variables which are reliabLe

indicators ofaging rates. Chronological age is cLearly important, as with increasing age

cornes an increasing chance ofdying. In humans this leads to an exponentiaI increase in

mortaIity rate ofpopulations with time, an observation tirst made by Gompertz

(Gompertz 1825). Many other organisms aIso exhibit an exponentiaI increase in mortality

rate with time and this has been used as an indicator that a population is undergoing aging

(Finch et al. 1990, Johnson 1990, Promislow 1991). Due to the difficulty in defining

aging, what is often studied is the life span oforganisms. The assomption that is made is

that life span is an indicator ofthe rate ofagingl.

Which organisms age? For the purpose ofthis thesis, [will use the tenns aging

and senescence synonymously, to Mean the age-related graduai decline in physiologicaI

function leading to death. It is bard to study this decay in the wild because most animais

t Uniess it is explicitly stated otherwise, in this thesis 1 will use the term life span to refer to the Mean life span ofa strain. However in Most cases, Mean and maximum Iife span are strongly correlated, so tbat Most statements made about Mean life spans are also valid for maximum life spans. In particuIar, ail C. e/egans • genes the lengthen Mean life span also lengthen maximum life span. 4

do not survive long enough ta die ofold age, falling prey to predators ordisease tirst • (promislow 1991). In spite ofthese limitations we know that aging, as defined above, is not a universal feature ofaU animaIs. Sorne fish, such as Salmon do not show a gradual

decay in function but die catastrophicaUy after mating (Finch 1990). Outside ofthe

animal kingdom, the issue gets even more complexe Most plants eventually die, even

ancient trees. These trees do not aIways show a protracted decay long before death and

reproductive productivity cao even increase as the trees get aider(Finch 1990). Many

plants can reproduce asexuaIly, which blurs the definition ofwhat an organism is and

when it dies. In the microbiaI world it is even more complexe The Brewer's yeast

Saccharomyces cerevisiae, can reproduce asexuaUy by budding. New daughter cells cao

he identified by their smaller size than the mother cell. It is known that the mother cell

can only bud a finite number oftimes before stopping and eventuaIly dying (reviewed in

Jazwinski 1996). However, the culture ofcells derived from this mother (which has an

identicaI genotype, except for random mutations) is immortal. There appears to he no

limit on the number ofdivisions a bacteriaI cell can make and one can not distinguish

mother cells or daughter cells.

The evolution ofaging: As aging is not a universal phenomenon, a great deal of

interest bas been focussed on how and why it evolved. There are two weIl accepted

theories for how senescence may have evolved. Both ofthese theories are based on the

assomption that the selection pressure on an organism must decrease rapidly after the start

ofreproduction, becoming smaU or negligible at old age (reviewed in Kirkwood 1996).

One theory is aptly named "mutation accumulation" because it posits that since selection • pressure is low, or even zero, late in life, mutations which have deleterious effects only 5

very late in life would have little selective disadvantage (Medawar 1952). Without • pressure to rem.ove these deleterious mutations from the population they could accumulate. Eventually ail members ofa population could carry many (ate acting

deleterious

pleiotropy, posits that because selection for early fitness is stronger than for late fitness, a

mutation which increases fitness early in life May be selected for, even ifit reduces

survivallate in life. This means that there may be trade-offs between early fitness and

late survival (Williams 1957, reviewed in Rose 1990).

Sorne aging theories POsit that there is a central clock that times life span. In such

models, life span is limited by a mechanism that actively tenninates life after a certain

period oftime (reviewed in Wilson 1974). It bas been argued that the death oforganisms

May be ofbenefit to the population ifit removed post-reproductive members, and the

competition for scarce resources they represent, from the population (reviewed in Wilson

1974). However this argument bas been challenged by evolutionary theorists on two

grounds: that firstly, most animais in their natura! environments die from starvation,

disease or predation before they begin to significantly age (Medawar 1952), and that

secondly, tbis is an argument that invokes selection based on groups rather than

individuals and that there is little evidence that group selection actually occurs (Wilson

1992, Maynard-Smith (989). Thus, evolutionary theory argues stronglyagainst the

possibility ofsuch an aging clock.

The cellular and molecular basis ofaging? Many theories ofaging posit that

although aging is a process that takes place in the whole organism, life span May be • detennined by the activity ofone organ or tissue (Shock 1981). Such theories suggest 6

that either one process becomes rate-limiting for aging or that some organ specific process • acts as an aging clock (Shock 1981, Medvedev 1990). For example some theories posit that changes in the endocrine, ornervous system, control aging (Shock 1981, Medvedev

1990). However ifthese sorts offactors limit life span they might well be species or even

genotype specific (Medvedev 1990).

Most molecular-genetic theories try to explain why aging occurs on a cellular

level, under the assumption that it is what happens molecularly in the cell, that leads to

the aging and death ofthe organism. Most molecular theories posit that as animais age,

sorne cellular processes start to go catastrophically wroog. Different theories suggest

different systems failing, or failing for a different primary cause. One set oftheories

suggest that aging is due to the accumulation oferrors in somatic DNA (Szilard 1959).

As these errors accumulate, more and more processes are affected until sorne essential

cellular process goes wrong. Ifdifferent ceLls, different processes May be affected, but if

too Many cells do not function properly the organism May die. A variation on this theory

suggests that aging is primarily due to the accumulation oferrors in mitochondrial DNA

(mtDNA) (Miquel et al. 1980). This leads to cells containing mostly non-functional

mitochondria which are Dot able to supply the cell with its metabolic needs. Other

theories suggest that aging results from sorne ofthe other biochemical changes have been

noted in aging cells and organisms such as the cross linking ofproteins and lipid

peroxidation (Medvedev 1990).

The theories mentioned above, posit that age-related cellular changes, such as

mtDNA deietioDS, protein cross linking or lipid peroxidation cause aging. Other such • theories suggest that these cellular changes are just the symptoms and oot the causes of 1

aging. One such theory, known as the free radical theory ofaging (Rarman 1956), May • able to explain Many ofthese changes that occur in aging. This theory posits that aging rnay be the result ofdamage caused ÏDdirectly by the process ofrespiration. In the

process ofrespiration, molecular oxygen is reduced to water. However, a small

percentage ofoxygen Molecules are only partially reduced to produce highly reactive

superoxides, peroxides and hydroxyl radicals (reviewed in Tower 1996). These Reactive

Oxygen Species (ROS) can cause widespread damage by oxidizing a wide variety of

molecules. ROS can cause most ofthe molecular signs ofaging including mtDNA

deletions and the reduction ofactivity ofenzymes in the electron transport system (Feuers

et al. 1993). There are at least two lines ofdefense against ROS. First there are enzymes,

such as superoxide dismutase (SaD) and catalase, that help neutralize ROS and prevent

damage from occurring. The second line ofdefense involves enzymes that repair much of

this damage. In spite ofthese defenses, over rime ROS are thought to cause widespread

impainnent to cells. This may lead to declining cellular and organismal function (aging)

and eventually death.

The geoetic basis of lüe span

It is clear that genes are important in determining life span, even though the

heritability oflife span appears to be low (reviewed in Finch and Tanzi 1997, Curtsinger

et al. 1995). Work in Many organisms has shown that genetic factors influence life span,

although the story is often very complexe The general subject ofthe genetics ofaging has

been reviewed in severa! high profile joumals in the last few years (Finch and Tanzi 1997, • Jazwinski 1996, Martin et al. 1996, Curtsinger et al. 1995), so 1 willjust give a general 8

overview ofwhat is known in the four most commonly studied systems apart from C. • elegans: humans, mice, Drosophila and yeast. Genetics ofmammalian lifè span. Little is known about genetics factors that extend

the life span ofmammals. There is sorne evidence in bath mice and humans that different

aIleles ofthe major histocompatibility system are associated with increased longevity. It

is aIso known that the s2 alIele ofapolipoprotein E (apoE) is found at a higher frequency

among centenarians than the s4 allele, which promotes vascular disease (Schâchter et al.

1994). A great number ofgenes have now been identified that increase the chances of

succumbing to age associated life-shortening diseases, such as Alzheimer's and certain

fonns ofcancer. It is not clear, however, whether this provides us with infonnation about

the general process ofaging. There are aIso a number ofgenetic syndromes (progeria

syndromes) that resemble accelerated aging and lead to premature death. The best known

ofthese syndromes is Wemer's syndrome which affects about 1 in 100,000 people.

People with this sYndrome prematurely develop Many ofdiseases associated with aging

including arteriosclerosis, type fi diabetes, cataracts, osteoporosis and certain cancers

(reviewed in Kirkwood 1997). The gene for Wemer's sYndrome bas recently been cloned

and round to encode a DNA helicase important in DNA replication or repair (Yu et al.

1996 and 1997). These results suggest that the fidelity ofDNA replication May be

important in aging. However, the major criticism raised against this argument is that

Werner's syndrome is not normal aging, and results from studying this syndrome May

ooly explain this pathological condition. This is a general criticism raised against

studying genes that shorten life span (Finch 1990). It is difficult to distinguish between • whether such genes are involved in normal aging orjust in pathological conditions. Most 9

gerontologists are far more interested in genes and treatments that cao increase life span. • The argument being that anything that lengthens life span must by definition affect the normal process ofaging.

Recently one such mutation has been identified in mammals. Ames dwarfmice lac~

or have a severely redueed number of, anterior pituitary cells leading to deficiencies in

growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH) (Brown-Borg et

aL 1996). They are normal size at birth but only reaeh one third ofthe normal adult sÏZe.

Under standard laboratory conditions they live much longer (on average one year longer)

than other animals from the same strain (Brown-Borg et al. 1996). This discovery has not

generated a great deal ofenthusiasm probably due to the clear deleterious effects ofthis

mutation.

Calorie restriction: It was tirst demonstrated more than 70 years ago that

reducing the food intake ofrodents could increase life span (McCay 1935). Sïnce that

time this result has been replicated in a wide range oforganisms. Calorie restriction is the

only experimental procedure that has been shown to reproducibly lengthen mammalian

life span (reviewed in Sohal and Weindruch 1996). This work will be reviewed in more

detail in the introduction to Chapter 3.

Cellular senescence: Since it is difficult, expensive and time consuming to

study Iongevity in mammals, a great deal ofwork has been done on cell cultures. Primary

cell cultures ofnormal cells will ooly divide a few number oftimes in vitro before ceasing

division (Hayflick 1956). The average number ofcell divisions is know as the culture's

replicative capacity, or life span. It has been notoo, that the average life span of • mammalian species correlates with the replicative capacity ofcell cultures derived from 10

these species (Rohme 1981). Cell cultures derived from older individuals divide fewer • times than those from younger individuais (Martin et al. 1970). Furthennore~ the ceUs of people with the disease Wemer's syndrome~ which leads to wbat looks like premature

aging and to early death, also display a shorter replicative life span (Martin et al. 1970).

These observations bave Iead to the hypothesis that aging in multi.cellular organisms is

determined by cellular life span.

There appears to be a link between cellular life span and the length oftelomeres,

the repetitive sequences ofDNA found at the ends ofchromosomes. It bas been sbown

that most somatic ceUs lack telomerase, the enzyme necessary for lengthening telomeres~

and that their telomeres shorten with repeated divisions (reviewed in de Lange 1998).

This bas lead to the suggestion that telomeres represent the mitotic clock that times

cellular senescence (Harley 1991). Evidence now suggests that telomeres are just one

factor determining cellular life span (reviewed in Smeal and Guarente 1997~ de Lange

1998) Whatever the role oftelomeres in cellular senescence, the link between cellular

and organismallife span is tenuous, as Many ceUs ofthe adult body, sucb as the nervous

system do not divide. AIso cells whicb are removed from this control on replicative

capacity become immortalized (reviewed in Smith and Pereira-Smith 1996). In the body

this can lead to cancer, which often shortens the life ofthe individual. This bas lead to the

suggestion that cellular senescence is an antineoplastic mechanism (Newbold et al. 1982).

The genetics offife span in yeast. The Brewers yeast Saccharomyces cerevisiae

is an excellent system to study the genetics ofcellular life span. In S. cerevisiae~ mother

cell life span is defined not as the chronological age ofthe cell, but rather, as the number • oftimes a ceU can bud to produce a new daughter cella As the motber gets older its 11

budding rate decreases and it eventually stops and dies (reviewed in Jazwinski 1996). In • yeast severa! mutations are known to affect the life span ofmother cells. To find genes invoived in aging in S. cerevisiae, Jazwinski and colleagues cioned

genes that are differentially expressed during the yeast life span (Egilmez et al. 1989).

The deletion ofone such gene, LAGI, increases life span by 50% (D'Mello et al. 1994).

The deletion ofanother such gene, LAG2, decreases life span, while overexpression

lengthens Mean and maximum life span (ChiIdress et al. 1996). Two other genes that are

homologues and that are aiso differentially expressed during aging, have opposite effects

on life span. RAS} shortens life span while RAS2 extends longevity (reviewed in

Jazwinski 1996). These two genes are highly pleiotropic and it is not clear how they

regulate life span, but interestingly, RAS2 aIso regulates most stress responses in yeast

including response to starvation, UV light, heat shock and oxidative stress (reviewed in

Jazwinski 1996).

Guarente and colleagues have identified another gene, SIR4, which regulates yeast

longevity. One allele ofthis gene, SIR4-42, was identified based 00 its resistance to

starvation and was subsequently shown to increase life span (Kennedy et al. 1995). SIR4

interacts with SIR2 and SIR3 to form a complex required for transcriptional silencing

(reviewed in Kennedy and Guarente 1996). SIR4-42 is believed to leogthen life span by

redirecting the transcriptional silencing complex from its normal sites ofaction to sorne

unidentified agjng locus (Kennedy et al. 1995). Recently it bas also been shown that

mutations in the yeast gene SGSl cause premature aging in yeast (Sinclair et al. 1997).

SGSl encodes a DNA helicase with homology to the human Wemer's syndrome gene • suggesting a link between c~llular senescence and aging in multicellular organisms 12

(Sinclair et al. 1997). It bas been proposed that cellular a~g is due to enlargement and • fragmentation ofthe nucleolus (Guarente 1997). In support ofthis theory, in sgsl cells the nucleolus enlarges and fragments prematurely (Sinclair et al. 1997).

Work in yeast bas clearly demonstrated that there are molecular genetic

determinants ofcellular Iife span. However, as mentioned above, it is not clear yet if

there is a strong link between cellular and organismallife span. No gene, or gene family,

has been found that can lengthen both cellular and organismal life span.

The genetics oflift span in Drosophila: Studying aging in long lived animais

such as mammals, poses Many problems, not the least ofwhich is that experiments take a

very long time to complete. Genetic experiments, which often require that an investigator

to study the transmission ofa trait over several generations, proceed extremely slowly in

mammals. Due to the problems offinding genes affecting longevity in mammals, Many

researchers has focussed on trying to find such genes in much shorter-lived invertebrates.

The invertebrate model systems Drosophila and C. elegans have been extensively used in

this research. 1will fust briefly review the work on Drosophila before concentrating on

the work on C. elegans.

Attempts to find genes which lengthen Iife span in Drosophila have been

hampered by the fact that inbred Drosophila strains show severe inbreeding depression of

life span. Most laboratory strains live significantly shorter than new isolates and

hetcrozygous cross progeny between two strains often live longer than the two original

strains (reviewed in Rose 1991 and Tower 1996). Because ofthese problems aging • research in Drosophila bas focussed in two areas: the evolution ofaging using selection 13

procedures on outbred populations~ and the generation oftransgenic animais in a • characterized background. Many selective breeding experiments have shown that the life span ofDrosophila

is quite plastic. Selection for resistance to starvation or desiccation, increases life span

(Rose et al. 1992). Selection for late fertility a1so increases life span and leads to reduced

fecundity at young age and increased developmental tintes (reviewed in Tower 1996).

Sorne ofthese long lived lines sho\v increased resistance to oxidative stress but not to

starvation or desiccation (Force et al. 1995). Sorne ofthese long-lived lines also bave an

increased frequency ofa particular ailele ofCopper/Zinc superoxide dismutase

(Cu/ZoSOD) (Tyler et al. 1993). Other lines display enhanced resistance to starvatio~

desiccation and beat, as weIl as, increased lipid content (Graves et al. 1992). Sorne of

these lines only display extended longevity when raised at high larval density, which is

know ta induce the transcription ofsorne antioxidants (Buck et al. 1993). The selective

breeding approach bas oot yet identified individual genes which lengthen life span but it

bas shown that the genetic basis oflife span in Drosophila is very cornplex and polygenic

(reviewed in Tower 1996).

By the transgenic approach arr and Sohal showed that simultaneous over­

expression ofthe enzymes CulZnSOD and catalase (two ofthe main enzymes that

detoxify ROS) cao lengthen life span (Orr and SohalI994). This result generated a great

deal ofexcitement because it appeared to be strong support for the Cree radical theory of

aging. However recently arr and Sohal's results have been criticized as being equally • explicable by background effects (Tower 1996). 14

c. elegans an ex~elleDtsystem to study aging: • Introduction to the worm There are sorne distinct advantages in using the free­ living nematode C. elegans as a model system to study the genetics oflife span in a

multicellular organisms (reviewed in Johnson and Lithgow 1992). (1) It is small (the adult

is 1 mm long), and its life cycle from fertilization to the onset ofreproduction lasts only

three days (reviewed in Wood 1988). It can therefore be cultivated in large numbers with

a minimum ofpractical problems due to space or expense. (2) It is normally short lived.

For example, the wild-type reference strain (N2) lives only for 15 days on average at

200 C (K1ass 1977). This allows experiments to be concluded \vithin weeks oftheir

initiation. (3) C. elegans is an intemally self-fertilizing hermaphrodite (reviewed in Wood

1988). This means that fully isogenic lines can be studied without the problems associated

with inbreeding depression because it is the nonnal mode ofexistence ofthis species to

be homozygous at most loci most ofthe time. Furthermore, mutations that make the

animaIs very sick can nonetheless be studied because, under Iaboratory conditions, the

worms do not need to move, either to feed (they live on a Iawn ofbacteria on which they

feed), or to mate (they fertilize their oocytes intemally with their own sperm). (4) From

the start ofthe use ofC. elegans as a model system, a unique reference strain (N2) has

been used. The vast majority ofmutations known in wonns, including mutations

affecting life span, have been generated in this background. Thus, genetic background

effects are kept to a minimum. in C. elegans.

The aging worm: Worms display Many morphological changes as they age. • Aging worms accumulate pigment granules in the intestinal epithelium which eventually 15

occupy most ofthe cytoplasmic volume (Epstein et al. 1972). At great age, body wail • muscle cells, nerve complexes, and mitochondria ofthe hypodennis degenerate (Epstein et al. 1972.. Boianowski et al. 1981). In old worms, the gonad atrophies and the whole

body become highly vacuolated (Johnson et al. 1984). In the nucleus there is increased

variation in the condensation ofchromatin, as weIl as an increase in the variation in

nuclear and nucleolar volume (Goldstein and Curis 1987). There is a notable increase in

nuclear DNA damage and a decrease in transcriptional capacity (reviewed in Johnson and

Simpson 1985). Aging worms aIso display changes in behavior including decreased

movement, defecation and pharYngeaI pumping rates (reviewed in Johnson 1984, Johnson

and Simpson 1985)

Aging in the wild type: The life span ofC. elegans is clearly dependent on

culture conditions. Klass investigated the factors that affect the life span ofthe wild type

(Klass 1977). He found that like Many other poikilotherms (animaIs that do not regulate

their internaI temperature) life span is strongly dependent on temperature. As temperature

is lo\vered within the viable range, life span increases in an approximately linear fashion

(see Fig 1.1). In this temperature range the length ofdevelopment shows a similar

response to temperature (Klass 1977) so that the length ofdevelopment at adult life span

are strongly correlated.

The life span ofthe wild type has also been shown to be dependent on other

environmental conditions, including the amount offood. Worms raised in axenic liquid

media clearly live longer than those raised on monoxenic agar plates (reviewed in

Johnson and Simpson 1985). In monoxenic liquid culture, the life span ofthe worm is • dependant on bacterial concentration (see Fig. 1.2). At extremely low food 16

Figure 1.1 The temperature dependence oflife span in C. elegans. Adapted from Klass • 1977. Within the viable range of26°C to 10°C there is a inverse relation between temperature and life span.

40

30 CD -rn ->-=lU c:~ 20 m c: CD lU E~ rn 10

0 10 15 20 25 30 Temperature (C)

• 17

Figure 1.2. The dependence oflife span ofC. e/egans on bacterial concentration in liquid • culture. Adapted from Klass 1977.

35 ~------. 30 -fi» ~ 25 ~ -..c CL fi» :! ..c CD E 5 o -'------"" 1.00E+OS 1.00E+07 1.00E+OS 1.OOE+09 bacterial concentration (cells/ml)

• 18

concentrations, the worms are chronically starved and die rapidly ofstarvation. However, • as the concentration ofbacteria is increased, life span increases up until 108 bacteria/ml. At concentrations above this, average life span is reduce~ falling to about 70% ofthe

9 maximum at high concentrations ofbacteria ( > 10 ). It is also known that starved worms

arrest both development and aging, while severely restricting nutritional conditions by

switching worms from bacterial growth to axenic medi~ drastically slows both aging and

development (Johnson et al. 1985). In the laboratory, worms are normally cultured under

rich conditions (on agar plates with lawns ofE. coli bacteri~ strain OP50). The work of

Klass and others suggests that reducing food intake should lengthen life span by the

process ofcaloric restriction. This was confirmed by the work ofHosono and colleagues

who showed that reducing bacterial concentrations on agar plates lengthens life span

(Rosono et al. 1989).

Aging in C. elegans is also affected by sex and mating. There are two sexes in C.

elegans, males and hermaphrodites. The hermaphrodites normally reproduce by internai

self-fertilization, but can also accept sperm from males. Mating substantially shortens

hermaphrodite life span by a mechanism that is independent ofegg production or receipt

ofsperm (Gems and Riddle (996). There are conflicting reports about the cost ofmating

on males, with one report showing a significant reduction oflife span by mating (Van

Voohries (992), while another found 00 efIect ofmating on male life span (Gems and

Riddle 1996). Unpublished data may explain this discrepancy. Gems has found that male

life span is depeodent on the number ofmales on a plate (D. Gems personal

communication). The more males on a plate the shorter the average life span, suggesting • that males will expend energy trying to mate males when no hermaphrodites are present. 19

Agingin Dauers. Under good conditions for gro~ worms normally go • through four rapid larval stages (known as Li-U) completing post-embryonic development in two and a halfdays. However, ifthe worm encounters adverse growth

conditions it can use an alternative developmental strategy to the one outlined above. If

LI or L2 larvae encounter harsh conditions they can develop into a dauer larva instead of

the normal L3 larva (reviewed in Riddle 1988 and Riddle and Alberts 1997). The dauer

larva is a developmentally arrested survival and dispersal stage, in which the worm does

not feed and has an altered energy metabolism. Dauer larvae have sealed mouths and

anuses, as weil as an a1tered body structure, which makes them resistant to desiccatioD.

Worms constitutively produce a pheromone, which is the primary determinant ofthe

dauer fate. When worms become crowded on a plate, the local concentration ofthe

pheromone increases and induces young larvae to become dauers. The dauer response is

reinforced by high temperatures and low food concentration. When dauer larvae

encounter food and low pheromone concentrations they molt into L4 larvae and continue

normal development. Dauers can survive for up to 6 months instead ofthe four week

maximum. for worms which do not enter the dauer state (Riddle 1988). Dauers appear to

be non-aging; that is, no matter how long worms have remained dauers after they come

out ofthe dauer stage they have a similar Mean post dauer life-span, and their total non­

dauer life span is comparable to N2 (Klass and Hirsh 1976).

The geDetics ofaging in C e/egllns

Recombinant inbred fines. Wild type strains ofC. elegans have been isolated

from a diverse set oflocations which span a wide portion ofthe globe (Hodgkin and • Doniach 1997). Individual wild isolates have very similar life spans (Johnson and Wood 20

1982, Johnson 1984, Johnson 1987, Ebert et al. 1996). To detennine how Many genes • might affect life span in the wonn and how life span can evolve in C. elegans, two groups have looked at recombinant inbred lines generated in crosses between two such wild

isolates ofC. elegans (Johnson and Wood 1982, Johnson 1987, Ebert et al. 1993 and

1996, Shook et al. 1996). In these experiments two different wild isolates with many

polymorphisms are crossed. The FI cross progenyare allowed to self-fertilize and

individual F2 progeny are placed singly on plates to derive multiple independent lines.

The life span ofthese recombinant inbred lines is then determined and the segregation of

certain polymorphie loci in this strain is determined. By complex statistical procedures

the genetic linkage between these detectable polymorphisms and the longevity affecting

loci which differ between the two isolates, cao he determined. By this process, the two

groups have detennined that alleles of3-6 loci that affect life span, are polymorphie

between the C. elegans isolates studied (Ebert et al. 1993 and 1996, Shook et al. 1996).

The approximate genetic map positions ofthese loci have been determined, but as yet, the

identity ofthe genes is unknown.

Mutant screens. At least two direct screens for loci that affect life span have

been carried out in the worm (Klass 1977, Dohon et al. 1996). In the fust screen Klass

found 8 long lived strains from 8000 lines derived from mutagenized wonns (Klass

1983). For technical reasons the screen as carried out in afer-15(b26) background, and

based on the way the screen way perfonned, the mutations isolated could not be

guaranteed to be independent. Klass round that there was a strong negative correlation

between the rate ofpharyngeal pumping (the feeding rate) and the life spans oflong lived • mutants, suggesting that caloric restriction could explain their long life (Klass 1983). In a 21

subsequent reappraisal ofthese strains done onder different environmental conditions, • Johnson could find no such relationship between food intake and life span and also found that ooly four ofKlass's mutants reproducibly lengthened life span (Friedman and

Johnson 1988a,b). Three ofthese strains contained a life-extending mutation and a

background une-3I mutation (Friedman and Johnson 1988a), indicating that they are

likely to be three isolations ofthe same mutational event. This mutation has been given

the allele name hx546 and the gene was called age-Jo age-J(h:c546) bas a profound and

robust effect on life span, lengthening mean life span by 60% and maximaIlife span by

110% (Friedman and Johnson 1988a,b).

InitiaIlyage-I could not be separated tromfer-J5 and it was thought that age-J

reduced fertility, in keeping with the antagonistic pleiotropy theory ofaging (Friedman

and Johnson 1988a,b). Subsequentlyage-I was separated fromfer-I5 and found not to

affect fertility. It was then believed that age-l had no phenotype except for its effect on

life span. age-J was promoted as an example ofa gerontogene, that is a gene tbat causes

aging as a result ofits normal function (Johnson and Lithgow 1992). However,

evolutionary theory suggests that the effect ofgenes on aging should be indirect, and

recent evidence suggests that this is aIso true for age-J (see dauer mutants).

Recently a new screen has been carried out for mutations that affect life span

(Duhon et al. 1996). The results ofthis screen have not been fully presented but we know

that three additionaI age-J mutations were identified in 3000 diploid genomes. Several

other mutations were isolated but ooly one ofthese bas been anaIyzed in a any detail, and • this mutation bas not yet been given a gene name. 22

spe-26: The antagonistic pleiotropy theory ofaging posits that there is a trade • offbetween reproductive success and longevity. To test this, mutations which limit, or eliminate, reproduction in C. elegans were examined for their efIect on life span. Ifsucb

trade-ofIs exist then one would expect that limiting reproduction sbould increase life

span. This expectation seemed to bave been met by mutations in the gene spe-26 that

drastically reduce sperm. production. Van Voorhies found that two spe-26 alleles

appeared to profoundly increase \vorm life span CVan Voohries 1992). However the

significance ofthese results bas recently been called into question. It bas been found that

the life span ofwild type (N2) worms from a range ofdifferent laboratories can be quite

different (G.ems and RiddleI996). The N2 control strain Van Voohries used appears to be

a short lived variant (Gems and RiddleI996). Recent tests ofthe life span ofspe-26

mutants with more appropriate controls, have given equivocal results and it is not clear

whether spe-26 actually lengthens life span (Vanfleteren and de Vreese 1995). spe-26 bas

been cloned and found to encode a major sperm protein which may forro part ofthe

cytoskeleton and it is unclear how this could affect life span (Varkey et al. 1995). Many

other mutations that affect fertility in different ways have also been examined, including

alleles offer-J5.fem-3 and her-J, and all ofthese mutations do not ta affect life span

(Klass 1977, Kenyon et al. 1993, Johnson 1984). It has also been shown that laser

ablation ofboth gonads, which eliminates reproduction, bas no effect on wormlife SPan

(Kenyon et al. 1993) making any link between reproduction and longevity very tenuous in

thewonn.

rad-8: A mutation in the rad-8 gene leads ta enhanced sensitivity to radiation • and oxygene Surprisingly, this mutation bas aIso been shawn ta lengthen life span, but 23

only at low temperatures (lshü et al. 1994). This effects has been shown to be solely due • to a lengthening ofdevelopment, without any change in adu[t Iife span (lshii et al. 1994). Consequently this gene is not thought to affect aging.

clk-l: Mutations in the gene, clk-l, have a[so been shown to lengthen life span

(Wong et al. 1995). Mutations in this gene lead to a pleiotropic alteration of

developmental and behavioral timing. clk-l worms display an increase in the length of

embryonic and post-embryonic development, as weIl as a lengthening ofthe period of

rhythmic adult behaviors such as the defa:ation cycle. The mean and maximal life span

ofclk-l worms is a1so greater than wild type (Wong et al. 1995). Table 1.1 illustrates this

for a number ofdeve[opmental and behavioral traits for a weak (e2519) and a more-severe

mutation (qm30). In addition, it was found that the ceU cycle ofyoung embryos (other

cell-cycle lengths were not measured) was slowed down in the same proportion as the

other features. The overall slow-down ofthe cell cycle was due entirely to a lengthening

ofinterphase without a concomitant slowing ofmitosis (Wang et al. 1995). Several other

phenomena were documented, including abnonnal reaction ofclk-l mutants to changes in

temperature and the full rescue ofall phenotypes by a maternai effect (Wong et al. 1995).

This last observation, in particular, suggests that clk-l affects a regulatory process

involved in setting, somehow, the rate at which the organism lives its life. It has been

hypothesized that the fact that mutations in the clk-l gene affect such a wide range of

timing events suggests that a general mechanism oftiming control, or a clock, is present

in C. elegans, and that clk-l affects the fonction ofthis clock (Wong et al. 1995). Studies

on clk-l mutants and other mutants with sunilar phenotypes are a major part ofthis thesis • and are discussed at length in the remaining chapters and especially in Chapters 2 and 4. 24

Table 1.1. Quantitative ana1ysis ofthe c/k-l mutant phenotype. The wild type is the • standard reference strain, N2. e2519 is a relatively weak clk-l mutation, while qm30 is a partial deletion ofthe gene and is believed to be nul!. Means and standard deviations of

the means are given. Adapted from Wong 1994 with permission.

Developmental Wild type e2519 qm30 phenotypes

Embryonic development 13.3: 1.0 17.1 ±3.9 22.8:5.0 (hours) Post-embryoDÎc 46.6 ±3.0 70.7 :4.7 99.2:6.2 developmeDt (hours) Self-brood size 302:l: 31 191 ±33 87±37 (number ofprogeny)

Lire spaD (days) 18.6:5.2 26.0 :9.0 22.8 :9.8 (max: 27) (max: 45) (max: 46)

Behavioural Wild type e2519 qm30 phenotypes

Defecation 50.8:5.6 69.4±9.9 92.4: 15.0 (meaD ofOve cycle iD seconds) Pumping(cycles 1 minute) 259.0:1:23.7 156.0:29.0 170.3:1: 26.9

SwïmmiDg 120.7: 6.5 91.7:1:5.7 75.6:1: 4.9 (cycles 1 miDute)

• 2S

Dauer mutants: A large array ofmutations have been found that affect dauer formation • (the Dafphenotype) (reviewed in Riddle and Alberts 1997). dafmutants can be grouped into two general classes: dauer defective (daf-d) mutants prevent entry into the dauer

stage even in the presence ofhigh levels ofpheromone, while the dauer constitutive (daf­

e) mutants enter the dauer stage even in the absence ofpberomone. Most daf-c mutations

were found as temperature sensitive conditional mutants that always form dauers at

25.5°C but develop nonnally at 15°C. By complementation tests the dafmutations were

put into complementation groups, and by looking at the pattern ofepistasis in a range of

daf-e daf-d double mutants, a complex genetic pathway was elucidated.

Recently it has been found that sorne daf-c mutations can not ooly affect the

ability ofworms to become dauers, but also the adult life span ofworms. This bas been

shown by using two experimental paradigms: worms are either raised continuously at the

permissive temperature, or are allowed to develop past the L3 stage at the permissive

temperature (to prevent entry ioto the dauer stage) and then shifted up to the restrictive

temperature (Kenyon et al. 1993). By using either ofthese paradigms it bas been shown

that daf-2 mutants can live substantially longer that the \vild type similarly treated

(Kenyon et al. 1993). With the temperature shift paradigm, adult life span cao be more

than doubled (Kenyon et al. 1993). The daf-c phenotype, and the increased life span of

daf-2 mutants, is completely suppressed by mutations in the daf-d gene daf-16 (Kenyon et

al. 1993, Gottlieb and Ruvkun 1993, Ogg et al. 1997, Lin et al. 1997). Subsequent

analysis ofthe life span ofa number ofdafmutants bas shown that mutations in the daf-c

gene formally known as daf-23. also increased life span and that this effect could be aIso • he suppressed by daf-16 (Larsen et al. 1995). It was then shown that the long life induced 26

by mutations in age-1 could also be suppressed by daf-16, and that age-1 mutants are in • fact da.fc at 27°C (Dorman et al. 1995, Malone et al. 1996). Due to the close genetic proximity ofage-I and daf-23, their similar phenotypes, and there similar interactions

with da.f16, a complementation test was done. It was found that age-1(hx546) fails to

complement daf-23 a11eles for both the dauer formation defect and life span (Malone et al.

1996, Morris et al. 1996, Tissenbaum and Ruvkun 1998). daf-23 alleles are now

generally believed to be allelic to hx546. and daf-23 has been renamed age-I (Malone et

al. 1996, Morris et al. 1996, Tissenbaum and Ruvkun 1998).

Three other dauer genes affect life span. A dominant mutation in the gene da.f28

has also been shown to increase life span (Malone et al. 1996). The effect ofda.f28 00

life span is much weaker than that ofage-I or daf-2, and it is not clear how da.f28

interacts with the other dauer genes (Malone et al. 1996). Two other genes modulate the

long life span ofage-l and daf-2 mutants in complex ways. Mutations in the da.fd gene

daf-18 do not suppress the long life ofdaf-2 mutants, but do suppress the long life of

sorne, but not aU, age-I aUeles (Larsen et al. 1995, Dorman et al. 1995). Mutations in the

gene da.f12 have been shown not to affect life span on their own, but to have an allele

specifie interaction with daf-2 (Larsen et al. 1995). Sorne da.f2; da.f12 double mutant

strains can increase adult life span up to four foid (Larsen et al. 1995).

The molecular identities oflige.}, daf.2 and daf-16

age-l has been cloned and was found to encode a oematode homologue ofthe

p1l0 subunit ofphosphatidylinositol3-kinase (pI 3-kinase) (Morris et al. 1996). piiO is

the catalytic subunit that toms the lipid phosphatidylinositol (4,5)-bisphosphate into • phosphatidylinositol (3,4,5)-trisphosphate. PI 3-kinase is part ofa very well-studied 27

signal transduction pathway and acts downstream ofdimeric growth-factor-receptor • tyrosine kinases (reviewed in KapeUar and Cantley 1994). Among the types ofreceptors that has been round upstream ofPI 3-kinase, are those ofthe insulin receptor family.

Recently it has been shown that daf-2 encodes a nematode member ofthat family

(Kimura et al. 1997). Together, these molecular findings suggest very strongly that age-l

and daf-2 participate in a signal transduction cascade similar to that described in

vertebrates. This is fully consistent with its place and role in the signaling pathway that

goes from the secretion ofpheromone to the implementation ofthe dauer larval fate in

wonns (Fig. 1.3).

Based on genetic data, the primary, and perhaps ooly, function ofthe DAF-

2/AGE-l signal is to regulate the activity ofdaf-16. In screens for mutations that can

suppress the Daf-c phenotype ofdaf-2 mutants, daf-16 alleles are easily recovered

(Gottlieb and Ruvkun 1993, Ogg et al. 1997, Lin et al. 1997). Ooly one other mutation,

the only ailele ofdaf-18, bas been shown to suppress daf-2. Molecular nuU aileles ofda/­

16 fully suppress all aspects ofthe daf-2 and age-l phenotypes (Ogg et al. 1997, Lin et al.

1997). These daf-16 mutants are viable and have no obvious morpbologjcal or

developmental abnormalities suggesting that the DAF-21AGE-l signal is dispensable for

nonnaI growth (Ogg et al. 1997, Lin et al. 1997). daf-16 encodes three members ofthe

hepatocyte nuclear factor 3 (HNF-3)/forkhead family oftranscriptional regulators (Ogg et

al. 1997, Lin et al. 1997). This suggests that DAF-16 might directly regulate the

transcription ofgenes necessary for the increased longevity seen in age-l and daf-2

mutants. As ofyel, no targets ofdaf-16 regulation have been identified genetically or • mo1ecularly. 28

Figure 1.3 Some features ofthe genetic pathway determining two alternative • developmental fates in Caenorhabditis elegans. The pheromone is produced constitutively and promotes the dauer rate. Mutational inactivation ofdaf-2 or age-I

mimics this effect aod leads to constitutive dauer formation at high temperature~

suggesting that these genes are normally inhibited by pheromone. daf-16 also promotes

the dauer fate, as mutational inactivation ofdaf-J6 prevents dauer formation even in the

presence ofpheromone or mutational inactivation ofdaf-2 or age-Jo Many daf-2 and

age-J mutations are temperature-sensitive mutations that increase the life span ofthe

worms at the permissive temperature. This effect is completely suppressed by mutations

in daf-J6. Worms nonnally recover from the dauer state when they encounter

environments where the pheromone concentration is low. Strains harboring temperature

sensitive daf-2 or age-J mutations cao recover from temperature induced dauer formation

by shifting worms to the permissive temperature.

•' U- • z U- 0 0 ~ E (]) 0 ~• C> CD ~ .g CI) E Q) ~ 0 lb - ~ ~ I~ 0 ©D ~ ~ ....> ~ 0 [Qk - ~ et) ~ --1 .:=d

LL. LL. Z 0 0 ID (]) C C> 0 ~ 0 ~ E @d) (Q 0 ©3» ~ §2 .... I~ ~ .... ID .. 0 .r:. ~ ~ -.... c.... ~ Q) .r:. ~ 0 • 0 30

What does this moiecular information tell us about the mechanisms underlying the • increase in life span ofage-J and daf-2 mutant wonns? Presumably, at the permissive temperature for age-J and daf-2, the signal nonnally acting via these two genes to tell the

developing worm not to become a dauer larva is oot entirely shut down nor fully 'on' as in

the wild-typellow pheromone situation (Fig. 1.3). daf-J6 is partially activated and the

worm is presumably in a physiologicaI state that bas sorne ofthe characteristics ofthe

physiology ofdauer larvae, including sorne ofthose that promote long life. However, it is

not clear how the physiology oflong lived age-J or daf-2 mutants is altered. We know

that dauer larvae are different from nonnal adult worms in Many ways, including body

structure, behavior and physiology, so it will take sorne effort to catalogue and interpret

the differences in gene expression between these two very different developmental stages.

Entry into the dauer stage is aIso regulated by the action ofMany genes other than daf-2

and age-Jo Sorne oftheses genes have been clooed and implicate at least one other type

ofsignal transduction cascade involving TGF-13 signaling (reviewed in Riddie 1997).

Interestingly, dauer-constitutive mutants in TGF-13 signaling components do not increase

life span (Kenyon et al. 1993, Larsen et al. 1995). This suggests that the morphological,

and possibly many other, differences between dauers and the wild-type worms might not

he necessary for long life. One such difference is probably lipid storage. Dauer

constitutive mutations, that have no effect on life span (daf-7), and thase that clearly

lengthen life span (daf-2), both Iead to the accumulation ofhigh levels oflipids (Kimura

et al. 1997) showing that lipid accumulation is Dot sufficient for long lire.

•' 31

Oxidalive stress and agiDg in C elegans: • To try to understand further the molecular basis ofIongevity in C. elegans, a number ofresearchers have compared the biochemistry ofthe wild type and strains with

altered life spans. From this work it is clear that resistance to oxidative stress is

correlated with long life, in support ofthe free radical theory ofaging. Life span in C.

elegans is dependent on oxygen concentrations (reviewed in Matsuo 1993). Under high

oxygen concentrations worm lire span is decreased as compared to atmospheric oxygen

concentrations (Honda and Matsuo 1992). Consistent with this observation, under very

low oxygen concentrations worm life span is increased (Honda et al. 1993). These results

suggest that an oxygen-dependent process, presumably respiration, affects life span. mev­

l(knl) mutants, which have reduced cytoplasmic superoxide dismutase activity, show a

similar response to the wild type, but live shorter than the wild type at all oxygen

concentrations ( Honda et al. 1993, reviewed in Matsuo 1993). Thus, a reduced ability to

resist oxidative stress correlates with reduced life span. Furthermore, like Many other

organisms (reviewed in Lee et al. 1997), as worms age the frequency ofmitochondrial

DNA (mtDNA) deletions increases (Melov et al. 1995). However, these deletions

accumulate at a slower rate in age-l mutants, than in the wild type (Melov et al. 1995).

But why do age-l mutants accumulate mtDNA deletions more slowly that the

wild type? One possibility is that age-l mutants may have slower metabolic rates than

the wild type, so they accumulate free radical damage more slowly. However based on an

indirect measure ofmetabolic rates, Vanfleteren and de Vreese, concluded that respiration

in daf-2 and age-l mutants is not decreased at anyage and is even elevated relative to the • wild type at old age (Vantleteren and de Vreese 1995). Rather age-l and daf-2 mutants 32

may live long because they bave elevated defenses against oxidative damage late in life. • Bath daf-2 and age-l mutants bave elevated activities ofSOO and catalase relative to contraIs at old age (Vanfleteren and de Vreese 1995, Larsen et al. 1995) and age-l

mutants are hyper-resistant to oxidative stress (Vanfleteren 1993, Larsen et al. 1995).

age-l and daf-2 mutants appear to be resistant to Many other stresses including heat shock

and UV damage (Lithgow et al. 1994, 1995, Murakami and Johnson 1996). The UV

resistance phenotype ofdaf-2 and age-l is also suppressed by daf-/6 (Murakami and

Johnson 1996). This has lead Johnson and colleagues to suggest that in the worm there is

a common mechanism oflife span extension and stress resistance that requires DAF-16

(Murakami and Johnson 1996). They suggest further, that life span can be extended by

inducing stress responses (Johnson et al. 1996).

Conclusions

Life span is clearly affected by genetic factors and these factors are beginning ta

be discovered in a number oforganisms. It is in C. elegans, however, that genes the

lengthen animallife span have been studied best. Mutations in at least four genes clearly

lengthen C. elegans life span and delay aging. Three ofthese genes, age-l, daf-2 and daf­

28, are involved in the control ofdauer formation and appear to act by modulating the

activity ofthe transcription factor daf-16. daf-16 May regulate life span by affecting the

transcription ofgenes necessary for stress response including the response to oxidative

stress. The fourth gene, clk-l, regulates the timing ofa wide variety ofdevelopmental

and behavioral traits and work on this gene, and related genes, comprises a large part of • the rest ofthis thesis. 33 • Chapter 2

Determination ofLife Span in Caenorhabditis

elegans by Four Clock Genes

• 34 • Abstract

The nematode worm Caenorlrtlbditis elegans is a model system for the study

ofthe genetic basis ofagiDg. Maternal-effect mutations iD four genes, clk-l, clk-2,

clk-3, and gro-l, interact genetically to determine both the duration ofdevelopment

and life-span.. ADalysis ofthe phenotype ofthese mutants suggests the existence ofa

general physiological dock in the worm. Mutations in certain genes involved in

dauer formation (aD alternative larva. stage induced by advene conditions in which

development is arrested) can also extend life-span, but the life extension ofClock

mutants appears to be indepeDdent oftbese genes. daf-2(e1370) clk-l(e2519) worms,

wbich carry life-span extending mutations from two different pathways, cao live

nearly live limes as long as wild type worms.

• 35 • Introduction

One way to investigate the nature ofaging is to study long-lived mutants. The

nematode worm Caenorhabditis elegans has become a model system to investigate the

genetic basis oflife span. Several genes are known to affect tife span in C. elegans and

among the best characterized ofthese are a class ofgenes that also affect dauer fonnation,

an alternative developmental pathway (reviewed in Chapter 1, Riddle 1988~ Riddle and

Alberts 1997). Three ofthe genes that affect the ability to form dauers, daf-2. age-l. and

daf-28. can lengthen a worm's life span without the worms becoming dauers (Friedman

and Johnson 1987, Kenyon et al. 1993~ Larsen et al. 1995, Malone et al. 1996). daf-28

has a very mild effect on life span and relatively tittle is known about this gene (Malone

et al. 1996). However, much more is known about age-l and daf-2 and both ofthese

genes have recently been cloned. age-l encodes a phosphatidylinositol-3-0H kinase

(Morris et al. 1996) and daf-2 encodes an insulin-like receptor (Kimura et al. 1997).

These genes are iovolved in an insulin-like signaling cascade that regulates the activity of

the forkhead-like transcription factor DAF-16 (Kimura et al. 1997, Morris et al. 1996, Lin

et al. 1997~ Ogg et al. 1997). Loss-of-functioo mutations in daf-16 strongly suppress the

extreme long life ofthe dauer mutants age-l and daf-2 (as weil as aU other phenotypes of

daf-2 and age-I) (Gottlieb and Ruvkun 1994, Larsen et al. 1995, Kenyon et al. 1993,

Dorman et al. 1995, Lin et al. 1997, Ogg et al. 1997). Consistent with age-l and daf-2

acting in the same pathway, age-I(hx546); daf-2(eI370) double mutants do oot live • longer than daf-2(eI370). 36

Mutations in at least one other gene not implicated in control ofdauer formation, • clk-l. increase Mean and maximum life-span (Wong et al. 1995). Mutations in c/k-l also lengthen early embryonic cell cycles, embryonic and post-embryonic development, as

weil as the period ofrhythmic adult behaviors, such as swimming, pharyngeal pumping,

and defecation (Wong 1994, Wong et al. 1995). In a screen for matemally-rescued viable

mutations in C. elegans, we recovered mutations in two other genes, clk-2 and clk-3.

which show the Clock (CIk) phenotype: a pleiotropic alteration ofdevelopmental and

behavioral timing ( Boutis 1995, Hekimi et al. 1995). A mutation in a fourth, previously

identified gene, gro-l(e2400). (Hodgkin and Doniach 1997) also leads to a Clock

phenotype (Wong et al. 1995, and Chaptc=r 4). The phenotypes ofthe other Clock mutants

strongly resemble clk-l mutants (Hekimi et al. 1995 and Chapte(4), however, their effect

on life span was either not known or not well studied.

In Chapter 4, l describe the effeet ofthe Clock genes on developmental and

behavioral timing and show that these genes internet genetieally to determine the duration

ofpost-embryonic development. Here 1 deseribe the effect ofthese four Clock genes on

life-span and show that they also interact genetically to determine adult life span ofC.

elegalls. The interaction ofthe Clock genes with sorne ofthe dauer genes is also

examined, to determine ifa common mechanism May underlie the extended life span seen

in the Clock mutants and in sorne ofthe dauer mutants. The implications ofthese results

for our understanding ofaging are also discussed. • 37 • Materials and methods

General metbods and straiDS: C. e/egans strains were cultured as described by

BRENNER (1974). Animais were cultured at various temperatures for aging

experiments9however strain constructions were performed at 20°C unless otherwise

noted. Wild type was the N2 Bristol strain. Mutations used are:

LGI daf-16(m26)

LGII unc-4(eI20), rol-6(eI87), age-l(hx546),fer-15(b26), clk-3(qm38)

LGIII dpy-l(el), daf-2(eI370), dpy-17(e164), gro-l(e2400), clk-l(e2519, qm30. qm51),

clk-2(qm37), unc-32(eI89)

Construction and maintenance ofClock double mutants: The construction of

Clock double mutants is described in Chapter 4. Except for clk-3(qm38);clk-l(e2519) and

clk-3(qm38);clk-2(qm37), aU Clock double mutants were maintained as lines with LG ID

mutations balanced overdpy-17(eI64), because the double homozygous mutants did not

produce viable lines. For analysis, double mutants were picked from the second

homozygous generation

Construction ordaf-2 clk-l: daf-2(eI370) clk-l(e25/9) and daf-2(eI370) clk-

1(qm30) strains were constructed in the same manner, which is described for the case of

daf-2(eI370) clk-/(e25/9). Daf-c-non-Unc recombinants ofdaj-2(e1370) unc­

32(eI89)/clk-l(e2519) hermaphrodites were picked at 25°C and then transferred to 15°C

to recover from the dauer stage. After the recombinants recovered9developed to adults • and started to produce progenY9 they were transferred to 20°C. Gravid adult non-Unc FI 38

progeny ofthe recombinants were placed singly on plates (singled). The F2 generation • was seored for growth rate and the presence of ~ Une progeny. From one plate which developed slowIy and segregated no Une progeny the daf-2(eI370) clk-l(e2519) strain

was derived. The presence ofdaf-2 in this strain was then reconfirmed by srifting some

worms to 25°C.

Construction ofage-l(hx546)fer-15(b26);gro-l(e2400): Because the age-l

gene has been best studied in the strain TJ401 age-l(hx546)fer-15(b26) and thefer-15

mutation has no effeet on life-span (Friedman and Johnson 1988), we used this strain to

generate a age-l fer-15;gro-1 triple mutant strain. MQ230 gro-l(e2400) males were

mated to unc-4(eI20) rol-6(eI87) hermaphrodites and wild type cross progeny were

pieked. RolUne F2 progeny were singIed, and a unc-4(eI20) rol-6(eI87); gro-/(e2400)

strain was derived from one slow developing F3 brood. TJ401 males were mated to unc­

4(eI20) rol-6(eI87); gro-/(e2400) hermaphrodites and wild type cross progeny were

picked to new plates. Thirty morphologjcally wild type F2 progeny \Vere singled and

from a plate with slow developing progeny, ~ ofwhich were RolUne (a putative to unc­

4(eI20) rol-6(eI87)/age-1(hx546) fer-15(b26); gro-l(e2400) strain), 15 morphologically

wild type F3 progeny were singled. A putative age-l(hx546) fer-15(b26);gro-1(e2400)

strain was derived from a plate whieh produced no Roi, Une or RolUne progeny. This

strain develops slowly and is sterile at 25°C confirming the presence ofgro-l andfer-15

respeetively. Since this strain lives considerably longer than gro-l (see results). 1

eonc1ude that age-l is also present in this strain.

Construction ofdaf-16; Clock doubles: To construct a daf-16(m26); clk­ • 3(qm38) double mutant strain, daf-16 males were mated to qm38 hermaphrodites. Fast 39

developing FI cross progeny were picked, and their F2 progeny were singled. Those F3 • broods which developed slowly were allowed to starve and were then screened for dauers. From a starved plate with no dauers, a putative daf-16(m26); clk-3(qm38) strain was

identified.

daf-16(m26);clk-l(e25l9). daf-16(m26);clk-2(qm37) and daf-16(m26);gro­

l(e2400) double mutant strains were constructed by mating e25l9. qm37 or e2400 males

ta daf-16(m26); dpy-l(el) daf-2(eI370} unc-32(eI89) hennaphrodites at 20°C for one

day. Mated hennaphrodites were then transferred ta 25°C. Wild type FI cross progeny

were singled. Morphologically wild type F2 progeny were singled and maintained at

25°C. From the F3 broods which segregated 1/4 DpyUnc-non-Daf-e progeny, 20

morphologically wild type progeny were singled and put at 20°C to develop. From one

slow developing F4 brood that did not segregate any DpyUnc, Dpy or Une progeny a

strain was derived.

Confirming the genotype ofdaf-16; cil doubles: During the construction ofthe

daf-16(m26}; c/k-l(e2519}. daf-16(m26); clk-2(qm37} and daf-16(m26); gro-l(e2400)

double mutant strains the presence ofdaf-16(m26) in these strains was indicated by the

ability to suppress the Daf-c phenotype ofdaf-2(eI370}. However, starved plates ofdaf­

l6(m26); c/k-l(e2519) worms produce a very low frequency ofdauers or partial dauers at

25°C. To confirm the presence ofdaf-16(m26} in the putative daf-16;clk-l. daf-16;clk-3

and daf-16;gro-1 double mutants, these strains were tested for their ability to complement

the daf-16(m26) suppression ofdaf-2(eI370). Spontaneous males from daf-16; clk-l.

daf-16;clk-3 and daf-16;gro-1 double mutant plates were allowed to mate daf-16(m26); • dpy-l(el) daf-2(e1370} unc-32(eI89) hermaphrodites at 200C for 24 hours and the mated 40

hermaphrodites were then transferred to 25°C. Twenty morphologically wild type FI • heterozygotes were singled and allowed to lay progeny for approximately 36 hours before the mothers were removed. Two days later the plates were examined for the dauer

progeny. As controls, N2 males and daf-16(m26) males were also mated to mate daf­

16(m26); dpy-l(el) daf-2(eI370) unc-32(eI89) hermaphrodites and treated like the

putative daf-/6 doubles. Ali plates derived from N2 matings segregated many dauer

progeny (see Table 2.1). Only sorne ofthe plates derived from daf-16 mating segregated

partial dauers and only a very few per plate ( Table 2.1). AlI three daf-16 Clock double

mutants segregated fewer partial dauers than the daf-16 control plates (Table 2.1). l also

did a straight outcross ofeach ofthe three strains with N2 and daf-16 males and allowed

the three seriai mating plates to starve. For the N2 matings aIl three strains produced

many dauers, while for the daf-16 matings no dauers were visible.

The genotype ofthe daf-16(m26); clk-2(qm37) strain was not confmned because

this strain displayed a clearly shorter life span than clk-2(qm37), consistent with the

detennined genotype.

Scoring life span: The aging study was done as described by Wong (Wong et al.

1995, Wong 1994), except that eggs were alIowed to hatch for up to 6 hours (for N2 and

the single mutants) or up to 32 hours (for Clock double mutants). Briefly, for each strain

at each temperature, approxirnately 200 eggs were picked ooto new plates and allowed to

hatch. At the end ofthis time aIl un-hatched eggs were removed and the LI larvae were

allowed to develop into adults. Then 50 worms were picked (or less ifthere were not

enough worms), 10 to a plate, onto fresh plates and were monitored once a day, every • day, until death. Wonns were transferred to new plates when oeeded. On the dayof 41

Table 2.1. Number ofdauers or partial dauers produced by mating different males to the • strain daf-16(m26); dpy-l(el) daf-2(e1370) unc-32(e189). Twenty plates ofF2 progeny derived from heterozygous cross progeny were examined for dauers at 25°C. The number

ofplates with dauers and the average number ofdauers per plate are given.

Males derived from strain plates with dauers # dauers/plate

N2+ 20 13.6*

DR26 daf-/6(m26) 16 2.1

MQ413 daf-16(m26); clk-l(e2519) 7 0.6

MQ393 daf-16(m26); gro-l(e2400) Il 1.1

MQ422 daf-16(m26); clk-3(qm38) 8 0.8

*Many dauers crawl offthe plate so this number is very likely an underestimate.

daf-16(m26); clk-2(qm37) not tested

• 42

death, dead worms were removed and their death was noted. For aU experiments done al • 15, 18 and 20°C the grandparents, parents and the experimental worms were maintained through out their lives at that constant temperature. Strains carrying gro-1, clk-2, orfer-15

mutations are not fertile at 25°C, so for most experiments at this temperature, wonns were

allowed to hatch al 20°C and were then transferred to 25°C. As daf-2 larvae become

dauers at 25°C, in experiments with daf-2, wonns raised at 20°C until they were young

adults and were then transferred to 25°C. Most strains were examined multiple times at

each temperature and the results for separate experiments were pooled. AU Clock single

mutant and double mutants were examined except gro-/(e2400) clk-l(e2519). The life

span ofthe gro-l(e2400) clk-l(e2519) strain was not detennined because these double

mutants are only viable in the tirst homozygous (maternally rescued) generation and

could not have been studied in the manner that the other Clock double mutants were.

Results

The lire span ofClock mutants: Mutations in the Clock genes c/k-I. 2. 3 and

gro-l result in a pleiotropic alteration ofdevelopmental and behavioral timing (Wong et

al. 1995, Chapter 4). Mutations in c/k-I and gro-l genes \Vere known to lengthen mean

and maximum life span (Wong et al. 1995, Wong 1994, Ewbank et al. 1997 and P. Larsen

personal communication). To detennine ifa11 Clock mutations lengthen life span, l

examined the life span ofthe reference strain ofthese genes at 15, 18, 20, and 25°C. AlI • four mutants strains have longer mean and maximum life spans than the wild type at ail 43

Table 2.2. Mean life-span ofClock strains at 15, 18,20, and 25°C. At each temperature the mean life-span and standard error are given. The numbers given in brackets are: first the number ofseparatc experiments in which the life-span ofa cohort (usually 50 wonns) was examined, and second the total pooled sample size. Repeated experiments gave very similar results.

Strain Genotype Mean life-span (days) 15°C 18°C 20°C 25°C· N2 + 22.0 ±0.3 (5, 236) 14.9 ±0.3 (5,250) 16.1 ±0.2 (5, 242) 9.2 ±0.3 (2, 100) CB4876 c/k-l(e2519) 29.3 ±0.5 (5, 212) 18.4 ±0.4 (5, 250) 17.3 ±0.4 (5,239) 11.6 ± 0.5 (2, 87) MQl30 c/k-l(qm30) 31.0 ±0.9 (2, 100) 19.8 ±0.5 (3, 149) 19.3 ±0.6 (3, 147) 11.1 ± 0.2 (1, 43) MQI25 clk-2(qm37) 24.6 ±0.8 (3, 139) 18.7 ±0.5 (3, 150) 18.0 ±0.5 (4, 191) Il.7 ±0.7 (l, 50) MQ131 clk-3(qm38) 25.7 ±0.7 (2, 100) 20.4 ±0.4 (5, 250) 19.9±0.6(3, 147) 13.0 ± 0.8 (l,50) MQ230 gro-l(e2400) 26.0 ±0.7 (3, 125) 19.2 ±0.6 (4, 200) 19.7 ±0.6 (3, 147) 15.6 ±0.5 (1, 50) MQI41 clk-l(e2519) c/k-2(qm37) 34.5 ±0.9 (2, 100) 28.2 ± 0.9 (3, 136) 23.1 ± 0.7 (3,143) ND MQ266? clk-l(qm30) clk-2(qm37) 30.3 ±0.9 (1,50) 28.3 ±0.7 (1,50) 25.4 ± 0.7 (1,50) ND MQl24 c1k-3(qm38),' c1k-l(e2519) 36.7 ± 1.4 (2, 69) 22.7 ±0.6 (3, 99) 17.8 ± 0.5 (3, 138) 17.4 ± 1.4 (1, 10) MQ224 c1k-3(qm38),' c1k-l(qm30) 41.4± 1.7(2,79) 43.5 ± 1.5 (1, 50) 27.5 ± 1.2 (2, 105) ND MQ225 clk-3(qm38); clk-l(qm37) 33.8 ±0.9 (2, 100) 22.3 ±0.8 (3, 150) 20.6 ±0.6 (3, 144) 12.7 ±0.5 (1, 50) MQ524 gro-l(e2400) c1k-2(qm37) 21.4 ±0.9 (1,37) 12.3 ±0.6 (l, 50) ND ND MQ223 c1k-3(qm38),' gro-l(e2400) 24.4 ±0.6 (1,50) 15.9 ±0.8 (2, 82) 14.6 ± 0.4 (3, 145) ND 1J401 age-} (hx546) fer- 15(b26) 30.9 ± 1.2 (2, 100) 26.3 ± 1.2 (2, 94) 26.6 ± 0.8 (3, 149) 22.0 ± 1.5 (1, 50) MQ415 age-1(lrx546) fer- 15(b16)" ND 34.1 ± 1.7 (l,50) ND 30.6 ±0.9 (1,50) gro-l(e2400)

ND = not detennined. ·Strains carrying gro-I, clk-2, orfer-15 mutations are not fertile at 25°C, so in this experiment, wonns were allowed to hatch at 20 0 e and then wcre transferred to 25°C. • • 44

Fig. 2.1. AIl three clk-l alleles lengthen life span. Graphs show the percentage ofworms • alive on a given day after eggs were laid (day 0) at 18°C from two pooled experiments.

N2 + (0), CB4876 clk-l(e2519) (e), MQ130 c/k-l (qmJO) (-) and MQ438 clk-l(qm5I)

(..). Sample size 96,92,40 and 78 respectively. AIl three alIeles ofclk-llengthen life

span.

100

(1) 80 .-> -ca 60 ~c: CD (.) 40 ~ Q) a. 20

0 0 1 0 20 30 40 50 day

• 45

temperatures (Table 2.2). For example, at 18°C, ail Clock mutants have a mean life span • at least 3 days longer than the wild type (Table 2.2 and Fig. 2.1). Clock mutations lengthen life span by about 20-30% with sorne variation at different temperatures (Table

2.2). In the case ofc/k-l. mutations ail three alleles have a very similar effeet on life span

(Fig.2.I). c/k-l mutations display a clear temperature sensitivity for life span lengthening

life span more at low temperature than at high temperatures (Table 2.2).

The life span ofCloek double mutants: To examine how these genes internet

genetically, l made ail ofthe possible double mutant combinations containing the

reference aUele ofeach gene (see Chapter 4). Although there is a range ofseverity, aU

double mutants take longer to develop than mutants in the individual constituent genes

(see Chapter 4, Table 4.2). Sorne double mutants develop only slightly slower than the

individual c/k mutations, while others display a profound lengthening ofdeveloprnent,

lengthening cell cycles, embryonic development and post-embryonic development to as

much as four fold that ofthe wild type (Chapter 4). Since aH Clock mutations lengthen

life span, we wondered whether double mutants would display even greater life spans

than individual clock mutations. 1examined the life span ofaU double mutants strains,

except clk-I gro-I, at 15, 18, 20°C, and in selected cases at 25°C as weil. c/k-l gro-I

double mutants were not tested because they are not viable in the second homozygous

generation. Most double mutants have much longer Mean and maximum life spans than

the individual Clock mutants, especially at low temperatures (Table 2.2). For example,

the mean life-span ofc/k-I(e2519) clk-2(qm37) mutants is over 50% greater than that of

either clk-l(e2519) orclk-2(qm37) alone and almost 90% greater than that ofthe wild • type at 18°C (Fig. 2.2A and Table 2.2). Again there is a range ofseverity with sorne 46

Fig. 2.2. Cloek mutations interaet to determine life-span. Graphs show the pereentage of • worms alive on a given day afterhatehing (day 0) at 1SoC (A) N2 (0), clk-l(e2519) (e), clk-2 (qm37) (Â) and c/k-l(e2519) c/k-2(qm37) (.). Sample size (n) is ISO exeept for

clk-l c/k-2 (n=136). clk-l clk-2 double mutants live even longer than clk-l and c/k-2

single mutants. (8) N2 (0), c/k-3 (e), gro-l{Â) andclk-3; gro-l(.). n=100 exeept for

clk-3; gro-l (n=82). clk-3; gro-l double mutant have a mean life-span similar to the wild

type even though each mutation individually increases life-span.

• 100 • A 80 Q) > ---a:s 60 ...c: Q) 40 0 ~ Q) 0- 20 0 0 10 20 30 40 50 60 days

100 B

Q) 80 > ---as 60 ...c: Q) 40 0 '"- a..Q) 20 0 0 10 20 30 40 50 60 days • 48

double mutants living marginal1y longer than the single mutants, while the strongest lived • up to 3 times as long as the wild type (Table 2.2). There appears to be a correlation between the life span ofthe double mutants and the severity oftheir effect on

developmental timing, and especially that ofpost-embryonic development (see Table 4.2

for post-embryonic development data).

To determine the specificity ofthe interactions among the Clock genes 1made two

sets ofclk-l clk-2 and clk-3:clk-l doubles, one containing clk-l(e2519) and the other clk­

1(qm30). The clk-l allele qm30 is much stronger than e25/9 for aImost an phenotypes

examioed except for life span for which the two aIleles do oot appear to be different

(Wong 1994, Wong et al. 1995 and Table 2.2). clk-3(qm38);c/k-/(qm30) double mutants

have a very long Mean life span at ail temperatures, up to three fold that ofthe wild type,

and much longer than that ofclk-3(qm38):clk-1(e2519) double mutants (Tables 2.2). This

is the same pattern that was seen with the effect ofthese double mutants on development

(Chapter 4, Table 4.2). In contrast to clk-3;clk-l doubles, clk-l c/k-2 doubles \vith either

clk-/(qm30) orclk-l(e2519) develop at the same rate and have equally extended life­

spans (Table 2.2 and Table 4.2).

The interactions ofgro-l with clk-2 and clk-3 are distinct from those between

other pairs ofClock mutants. Although clk-2, clk-3, and gro-/ single mutants aIl live

longer than the wild type (Table 2.2), both gro-l clk-2 and clk-3;gro-l double mutants

have a Mean life-span similar to that ofthe wild type (Fig. 2.2B and Table 2.2). Since

clk-3(qm38); clk-2(qm37) double mutants live longer than either clk-2 or clk-3, this

implies that gro-l May suppress the long life ofclk-2 and clk-3. However, gro-l does not • suppress the increased longevity ofage-l(hx546), another mutation which confers long 49

life (Friedman and Johnson 1988). In fact the mean life-span ofan age-lfer-15; gro-l • strain is greater than that ofgro-l and ofage-lfer-15 (Table 2.2). These results suggest that the suppression ofextended life-span ofclk-2 and clk-3 by gro-l may involve a

specifie interaction between these genes and that gro-l and the clk genes may function in

a common pathway.

The interaction ofClock mutants with daf-16 and daf-2: Several other C.

elegans genes are known to affect life-span (reviewed in Chapter 1). The best studied of

these are involved in the control ofdauer fonnation (reviewed in Riddle 1988).

Mutations in the dauer-constitutive (Daf-c) genes daf-2, daf-28 and age-l can lead to an

extended life-span without entry into the dauer stage (Friedman and Johnson 1988 9

Kenyon et al. 1993, Malone et al. 1996). Mutations in the dauer defective (Daf-d) gene

daf-16 suppress extended life-span (as weIl as the Daf-c phenotype) in daf-16;daf-2 and

daf-16; age-l doubles (Kenyon et al. 1993, Larsen et al. 1995, Donnan et al. 1995,

Gottlieb and Ruvkun 1994, Ogg et al. 1997, Lin et al. 1997).

The Clock genes' effect on life-span, however, appears to involve a DAF-16­

independent mechanism. daf-16(m26) does not suppress the life-span extension seen in

clk-l, clk-3 orgro-l strains (Fig. 2.3A and Table 2.3). From the pooled results ofthree

independent trials we see that daf-16(m26) mutants live slightly shorter than the wild type

(Figure 2.3A), a result we have seen repeatedly and has also been noted by others

(Kenyan et al. 1993, Larsen et al. 1995, Malone et al. 1996). daf-16 has a similar effect

on clk-l and daf-16(m26); clk-l(e25l9) double mutants live slightly shorter than clk-

1(e2519). However, daf-16(m26); c/k-l(e2519) animals still live significantly longer than • the wild type. In these experiments, daf-16(m26),' clk-l(e2519) double mutants live 20% 50

Figure 2.3. The interaction ofclk-l with daf-16 and daf-2. A) The percentage ofwonns • alive on a given day after eggs being laid (day 0) for three pooled experiments at 18°C: N2(D), daf-16(m26)(.), c/k-l(e2519) (0) and daf-16(m26); clk-J(e2519) ce). (D)

Percent ofworms alive on a given day after fust day ofadulthood Cday 0) at 25°C: N2

(0), clk-l(e2519) (-), daf-2(eJ370) ce) and daf-2(e1370) c/k-l(e2519) (Â). n=50 for all

genotypes. Worms were raised at 20°C UDtil the first day aIl worms were adults, to

prevent daf-2(e1370) mutants from becoming dauers, and then were transferred to 25°C.

N2 and daf-2 were transferred after 3 days, c/k-l after 4 days and daf-2 clk-l after 6 days.

The adult life-span ofdaf-2 clk-l double mutants is considerably longer than that ofdaf-2

alone and almost 6 times that ofthe wild type.

• • 100-"'-.4"

Q) 80 .-> -ca 60 ...... c:: ~ 40 '- Q) CL 20

o+---~-...... --~----~" o 10 20 30 40 Days

100

CD 80 -.~ ca 60 ~c: CD 0 '- 40 a.CD 20

0 0 20 40 60 80 • days 52

Table 2.3. The mean life ± standard error for daf/6:c1k-/, daf/6;c1k-2, eJaf-/6:clk-3 and daf/6;gro-J double mutants. The pooled results ofthree aging experiments are shown for daf/6;c1k-l, daf16;c1k-2 and dafJ6;clk-3 white the results ofa single experiment are given for daf16,'gro- J. The number in parenthesis is the total sample size.

Genotype clk-}(e25 19) clk-2(qm37) clk-3(qm38) gro-l(e2400) daf/6(+),' c/k(+) 20.8 ± 0.4 (145) 21.9 ± 0.4 (145) 20.1 0.5 (141) 16.4 0.4 (47) daf/6(m26),' clk(+) 19.5 ± 0.4 (145) 19.8 ± 0.3 (144) 19.7 0.3 (148) 15.0 0.3 (46) daf/6(+); c1k(-) 25.0 ± 0.5 (141) 25.3 ± 0.6 (137) 24.9 0.5 (146) 18.7 ±0.4 (49) daf16(m26),' c/k(-) 23.5 ± 0.4 (148)* 19.0 ± 0.4 (131) 23.9 0.4 (135) 19.1 ± 0.3 (44)

* The strain MQ413 daf/6(m26); clk-J(e2519) was used in these experiments. In two ofthe three trials, cohorts ofSM510 daf

16(",26),' clk-/(e25/9) worms (kindly provided by Shin Murakami) were also tested. The life span of SM510 and MQ413 worms were not statistically different in either trial (p>0.15 for both trials using student t test).

• • 53

longer than daf-16(m26). and c/k-l(e2519) mutants live 20% longer than the wild type • (N2). This demonstrates that the shorter life span ofdaf-16(m26); c/k-l(e2519) as compared to clk-l(e2519) is not due to partial suppression ofc/k-l(e2519) long life by

daf-16(m26). Rathery this suggests that the daf-16(m26) mutationy or a mutation strongly

linked to ity bas a weak deleterious effect on life span. The results ofthree trials for daf­

16(m26); clk-3(qm38) were very similar to that ofdaf-16(m26); clk-l(e2519) (Table 2.3).

daf-16(m26); gro-l(e2400) was aIso tested once and displayed no evidence of

suppression ofgro-l by daf-16 (Table 2.3). As daf-16 does not suppress any other aspect

ofthe clk-l(e2519)y clk-3(qm38) andgro-l(e2400) phenotypesy this is consistent with clk­

ly clk-3y and gro-l aeting in a different pathway from daf-16. Our observations are

clearly different from those made with daf-16; age-l and daf-16; daf-2 double mutantsy

which live no longer than daf-16 alone (Kenyony et al. 1993, Donnan et al. 1995. Larsen

et al. 1995); clearly indieating that daf-16 strongly suppresses the long life ofage-l and

daf-2.

Surprisingly daf-16 superficially appears to completely suppress the long life of

clk-2 mutants (Table 2.3). However it is not clear whether tbis represents a specifie

suppression ofclk-2 by daf-16. or a non-specifie deleterious effect ofcombining these

two mutations. In contrast ta the effeet ofdaf-16(m26) on age-l or daf-2. daf-16(m26)

does not suppress any other aspect ofthe c/k-2 phenotype. Specifically daf-16(m26) does

not suppress the slow growth al 20°C or the embryonie lethality ofclk-2 at 25°C. AIso in

sorne trials daf-16(m26); clk-2(qm37) double mutants actually live sborter than daf­ • 16(m26). consistent with these genes baving a non-specifie deleterious interaction. 54

Thus genetic evidence suggests that clk-l, clk-3 andgro-llengthen life span by a • different mechanism than the dauer genes. Because the dauer genes and the Clock genes appear to affect life-span by different mechanisms, we examined the effect on wonn life

span ofcarrying mutations in both a Oaf-c and a Clock gene. One might predict that

strains earrying mutations in both a clk gene and a long lived dauer gene eould live very

long. This is indeed what we see, a daf-2(eI370) clk-l(e2519) strain lives longer than its

component strains at both 18 and 25°C (Table 2.4 and Fig. 2.3B). This effeet was also

seen with daf-2(e1370) clk-l(qm30) double mutants at both 18 and 25°C (Table 2.4).

Furthermore, in one trial the mean life-span and Mean adult life-span ofdaf-2(eI370) clk­

l{e2519) double mutants was nearly five and six times that ofthe wild type respeetively

(Fig. 3.38). This is the greatest multiplicative increase in mean life-span over the species

average seen in any organism by any rneans (Kenyon et al. 1993, Larsen et al. 1995).

The results for daf-2 clk-/ double mutants contrast with the result ofcombining age-land

daf-2. which, by genetie and moleeular criteria, appear to function in the same pathway.

In this case, the double mutants age-l(/L'C546); daf-2(e1370) do not live longer than daf­

2(e1370) (Donnan et al. 1996). Taken together these results confirm that the dauer genes

age-J and daf-2lengthen life span by a different mechanism than cfk-l.

Discussion

Ali Clock mutations lengtben life span: In this Chapter 1 show that aH Clock • mutations lengthen life span by between 20 and 30%. As aIl Clock mutations also display 55

Table 2.4. The interaction ofclk-l \vith daf-2. The results oftwo experiments at 18°C • and 25°C were pooled. Mean life span ± standard error ofthe mean are given with the sample size in parenthesis.

strain 18°C 25°C*

N2 N2 17.6 ± 0.5 (92) 9.6 ± 0.2 (95)

CB4876 clk-l(e2519) 20.9 ± 0.6 (83) 11.9 ± 0.6 (78)

CB1370 daf-2(e1370) 32.1 ± 1.2 (87) 24.1 ± 1.8 (40)

MQ513 daf-2(e1370) clk-l(e2519) 44.0 ± 2.3 (82) 42.9 ± 1.2 (98)

In two trials MQ130 clk-l(qm30) and MQ514 daf-2(e1370) clk-l(qm30) were also tested.

The lire span ofN2, clk-l(qm30), daf-2(e1370),anddaf-2(e1370) clk-l(qm30) were 21.0 ±

0.4 (49),27.1 ± 1.0 (40), 34.5 ± 1.4 (45) and 40.0 ± 3.4 (24) respectively at 18°C and 10.2

± 0.3 (50), 11.5 ± 0.6 (50), 22.9 ± 2.2 (17) and 28.8 ± 1.2 (50) respectively at 25°C.

*This experiment was done under the conditions described in Figure 2.3B: only adult life­

span is given.

• S6

very similar effects on developmental and behavioral timing, this implies that mutations • in these four genes may all affect the same underlying process. This data suggests that this process is fundamentally involved in setting the rates ofMany timed events and is

fundamentally involved in setting the rate ofaging.

In the case ofclk-l we know that all three alleles display a similar effect on life

span (Fig. 2.2). These results, along with the fact that the long life ofclk-l mutants can

be Urescued" by a transgene containing the wild type c/k-l sequence (Ewbank et al.

1997), confinn our earlier work (Wong 1994, Wong et al. 1995) and show that the clk-l

helps detennine life span. Our results directly contradict those ofMurakami and Johnson,

who based on the results ofa single trial, have reported that clk-l(e2519) is long lived

while clk-l(qm30) is not (Murakami and Johnson 1996). This discrepancy in results may

be explained by the fact that clk-l mutants, and clk-l(qm30) mutants in particular, display

a much higher degree ofvariability than the wild type for most phenotypes examined

including aging (Wong et al. 1995, Wong 1994, unpublished data). In Il independent

trials of40-50 wonns at 4 different temperatures, cohorts ofclk-l(qm30) wonns live from

1% shorter to 64% longer than their wild type control cohort. Ho\vever, when multiple

trials at any given temperature are pooled, we find that clk-l(qm30) lives significantly

longer than the wild type and that this effect is more pronounced at low temperature

(Table 2.2).

The phenotypes ofClock double mutants suggest Clock genes affect a

common process: Not ooly do ail Clock mutations lengthen life span, most double

mutants live even longer than the individual Clock mutations. Most double mutants • display a strong correlation between the length oflife span and developmental rate (see 57

figure 2.4) consistent with the same defect underlying all phenotypes ofClock double • mutants. These results are also consistent with the four genes affecting the same process. This interpretation is strengthened by the fact that the Clock genes display sorne allele

specifie interactions. Based on the phenotypic and molecular characterization ofclk-1 we

know that e2519 is a weak partialloss offunction allele while qm30 is a strong putative

null allele ofclk-J (Wong 1994, Wong et al. 1995, Ewbank et al. 1997). clk-3(qm38);

clk-l(qm30) double mutants develop much more slowly and live significantly longer than

clk-3(qm38); clk-l(e2519) double mutants (Tables 2.2, 4.2). These results are consistent

with these two genes affecting the same process. The simplest interpretation ofthis result

is that c/k-J and clk-3 May act in parallel, partially redundant pathways. In contrast clk-J;

clk-l double mutants containing eitherclk-l(e1519) orclk-J(qm30) have identical

phenotypes, developing at the same extremely slow rate and have a very similar long life

span (Tables 2.2, 4.2). This suggests that the activity ofclk-2 may be required for the

residual activity ofclk-J(e2519).

gro-J appears to specifically repress the long life ofclk-2 and clk-3. This may imply a

specifie interaction ofthese genes. Howevergro-l clk-2 and clk-3; gro-l double mutants

develop substantially more slowly than clk-2. c/k-3 orgro-l. So it is possible that the

effeet ofgro-l on the life span ofc/k-2 and c/k-3 is a non-specifie a deleterious effeet of

combining these mutations. 1could not study the effeet ofeombining gro-J and c/k-I on

life span as these double mutants mostly die as dead eggs in the tirst homozygous

(matemally rescued) generation. The synthetie lethality ofgro-l clk-l double mutants is

consistent with eithergro-l having a specifie interaction with clk genes or with gro-l • having a non-specifie deleterious effect on strains carrying clk mutations. 58

Clock mutants and dauer mutants lengthen liCe span by different • mechanisms: The phenotypes ofCloek mutants and the Daf-c mutants age-/, daf-2 and daf-28 are radieally different, suggesting that these two classes ofgenes lengthen life span

by different means. Firstly, the Cloek mutants are not dauer defeetive, even at 27°C

(unpublished observations). Seeondly, the life span ofDaf-c mutants is temperature

sensitive with the mutants living even longer eompared to wild type at high temperatures

(Tables 2.2, 4.2). The relative life span ofthe Cloek mutants and Cloek double mutants is

either not temperature sensitive, or even eold sensitive (i.e. clk-/, see Table 2.2). clk­

2(qm37) and gro-/(e2400) single mutants, a10ng with many ofthe double mutant strains,

are not even viable at high temperatures sueh as 25°C. Thirdly, the Oaf-e mutations seem

to lengthen adult life span speeifieally while Cloek mutations simultaneously lengthen

many timed phenomena.

Genetie evidenee aIso suggests that Cloek mutations and the Daf-e mutations

lengthen life span by different meehanisms. We had sho\\tn previously that mutations in

daf-/6 do not suppress the long life 0 f the Cloek mutants clk-/, c/k-3 and gro-l

(Lakowski and Hekimi 1996). However, the result for clk-/ has been disputed

(Murakami and Johnson 1996). After repeating trials, we have eonfirmed our original

analysis and shown that daf-16(m26) or a elosely linked mutation has a weak deleterious

effeet on lire span. Perhaps the deleterious effeet ofdaf-16 on life span may explain the

different experimental results.

daf-16(m26) appears to suppress c/k-2(qm37). However in the light ofthe

observation that daf-16(m26) reduees life span, it is possible that this effeet may be non­ • specifie. Towards the end oftheir life oid worms often beeome very pale, 100se turgor 59

and become almost immobile. In most strains this is a indication that death is imminent • (usually within a day)~ but clk-2 mutants can survive for a surprisingly long time in this state (more than one week). Ifdaf-16 has a deleterious effect on wonns in this state. this

could explain its effect on clk-2. This is consistent with our observation that although

daf-16(m26) does not affect the time offirst death~ it accelerates the death rate once the

population starts to die (see Fig. 2.3A)~ suggesting that daf-16 May limit life span through

a non-specifie deleterious effect on decrepit wonns. Our interpretation that the effect of

daf-I6 on clk-2 longevity May not be specifie is further supported by our observations

that daf-16(m26) does not suppress the slow development~nor the 25°C lethality ofclk­

2(qm37).

It has been suggested that in the worm there is a single mechanism oflife span

prolongation which requires the activity ofdaf-16 (Kenyon~ et al. 1993, Dorman et al.

1995~ Larsen et al. 1995~ Murakami and Johnson 1996). That daf-16 activity is required

for the long life ofthe dauer constitutive (Daf-c) mutants age-I and daf-2 (Kenyon. et al.

1993, Dorman et al. 1995, Larsen et al. 1995) is generally accepted~ particularlyas daf­

16 is also required for ail other aspects ofthe phenotypes ofthese mutants (Gottlieb and

Ruvkun 1994, Ogg et al. 1997, Lin et al. 1997). However my results show that the wild

type daf-16 activity is not required for the long life ofclk-I. clk-3 and gro-l and that at

least two, and possibly multiple pathways exist that determine the life span ofthe worm.

This interpretation was confinned by our observation that daf-2 clk-l double

mutants live even longer than daf-2 or clk-l. Both daf-2(e1370) clk-l(e2519) and daf­

2(e1370) clk-l(qm30) double mutants livesignificantly longerthanclk-l ordaf-2 atboth • 18°C and 25°C. In one trial al 25°C daf-2(eI370) clk-l(e2519) even lived five times 60

longer than the wild type. This is in contrast to age-l daf-2 double mutants which live 00 • longer than daf-2. How do age-l and daF-21engtben lire span?: So why do Clock mutants and

dauer mutants live long? As reviewed in Chapter l, mutations in the dauer genes age-l

and daf-2 increase resistance to many stresses including UV, temperature and free radical

generators (Larsen 1993, Vanfleteren 1993, Mwakami and Johnson 1993). This suggests

that age-l and daf-2 mutants may live long because they have increased resistance to

Reactive Oxygen Species (ROS) which May he the cause ofaging. The sole function of

age-l and daf-2 appears to be to reguIate the activity ofthe forkhead transcription factor

DAF-16. Thus, age-l and daf-2 May live long because they rail ta repress daf-16 which

directly, or indirectly, may regulate resistance ta oxidative stress. In retrospective the

finding that daf-2 and age-l mutants May live long because they have increased stress

resistance may not be so surprising, given that dauer larvae are aIso resistant to many

stresses (reviewed in Riddle 1988).

Murakami and Johnson have reported that one allele ofcfk-l. e2519. lengthens life

span and increases UV resistance, while another allele, qm30, does not live long and is

not resistant ta UV stress (Murakami and Johnson 1996). However, we have shown that

aIl alleles ofcfk-Ilengthen lire span (Fig. 2.1, Wong et al. 1995, Ewbank et al. 1997).

Therefore, since Murakami and Johnson found that clk-l(e2519) is stress resistant while

clk-l(qm30) is not, stress resistance is oot correlated with Mean life span in c/k-l mutants.

Dow do the Clock gene lengtben lire span? So how do Clock geoes lengtheo • life span? Sorne cues come from the phenotypes ofClock mutants and double mutants. 61

Firstly, Clock mutants display a lengthened development. However, the lengthened life • span ofClock mutants is not due solely to lengthened development. Clock mutants and most doubles have significantly longer Mean adult life spans than the wild type. This

implies that Cloek mutations are not like mutations in rad-8 which only lengthen

development (Ishi et al. 1992). Secondly, among invertebrates it has been shown that life

span is often inversely related to temperature (see Fig. 1.1). This has lead to the idea that

life span is partially determined by the rate at which an organism lives (pearl 1928).

Presumably differences in the Urate ofliving" refleet differences in metabolie rates. In C.

elegans both adult life span and post-embryonic development are linearly related to

temperature and thus correlated to each other. As temperature is lowered, both mean

adult life span and the length ofpost-embryonic development increase (Klass 1977). This

is consistent with both ofthese variables being dependent on the "rate ofliving" in C.

elegans. So l plotted the length ofdevelopment against mean adult life-span for aIl

experiments done at a constant temperature (Fig. 2.4). l find, like Klass, that lowering

temperature lengthens both development and mean adult life-span in the wild type in an

apparently linear fashion (Fig. 2.4). The Clock single mutants and aH the cfk double

mutants, ctisplay a similar relation between these two life history traits. However, at any

given temperature, these strains have both a longer development and longer mean adult

life span than the wild type. It appears, therefore, that mutations in the Clock genes have

similar effeets on development and life-span as has lowering the temperature. This

relationship is not trivial, as two groups ofstrains have a very distinct relation between

adult life-span and the lengtb ofdevelopment: at any given temperature, gro-l cfk-2 and • clk-3;gro-l double mutants have a much shorter adult life-span than the wild type, while 62

Fig. 2.4. Scatterplot ofthe length ofdevelopment versus Mean aduIt Iife span. Data is •• shawn for all experiments in which strains were maintained at a constant temperature. Mean adult life-span was calculated (mean life-span - length ofdevelopment) using the

data in Tables 2.2 and 4.2. (+) N2, (e) daf-2 and age-l containing strains, (Â) gro-l

clk-2 and clk-3; gro-l double mutants, (CI) ail other strains, including Clock single

mutants and ail Clock double mutants which do not contain gro-l(e2400). A-linear

regression line is shown for the last group ofstrains. AIso included in this plot are points

for N2 (life-span = 10.7 ± 0.3 (1,200), development = 1.6 ± 0.4) and clk-l(e2519) Oife-

span = Il.7 ± 0.3 (1, 200), development = 2.6 ± 0.4) continuously cultured at 25°C. Due

to the difficulty in detennining the length ofdevelopment ofc/k-3(qm38); clk-l(qm30)

worms at 20°C (see Chapter4 Table 4.2 notes), these results were excluded.

~ 12 c CI) >. c ctS 10 • "'C c "--' • C +"" 8 C c: C C Q) 6 c e E • c c CL 0 4 • • ,. -Q) • • Q)> 2 • -C 0 0 10 20 30 40 mean adult lifespan (days) • 63

aIl strains containing either daf-2 or age-l, including daf-2 clk-l and age-l fer-15;gro-1 • strains, have a much longer Mean adult life-span, relative to development, than the wild type. However, in daf-2 clk-l and age-l fer-15:gro-1 strains, the Clock gene mutation

still lengthens both the duration ofdevelopment and adult life-span. Clock gene

mutations appear, therefore, to alter the scaling ofthese life history parameters without

affecting their linear relation. We conclude that mutations in Clock genes reduce the "rate

ofliving" rnuch like reducing temperature does. So the Clock phenotypes are consistent

with these genes controlling the rate ofliving presumably through control ofmetabolic

rates.

The identity ofclk-l strengthens tbis hypothesis. As described in Chapter 4, c/k-l

encodes a small protein ofunknown biochemical. 115 yeast homologue is localized to the

inner membrane ofthe mitochondria where it regulates respiration (Jonassen et al. 1998).

We propose that mutations in clk-l affect mitochondrial function and reduce respiration.

Reduced respiration could lead to reduced production ofROS. This could lead to damage

caused by ROS accumulating more slowly, delaying agjng and extending life span. Thus

Clock genes may lengthen life span by reducing the rate ofproduction ofROS while daf­

2 and age-l May elevate the levels ofdefenses against ROS.

• 64 • Chapter 3

The Geneties ofCalorie Restriction in

Caenorhabditis elegans

• 65

Abstract

• Low caloric intake (caloric restriction) can lengtben the life span ofa wide range ofanimais and possibly even that ofhumus (Sohal and Weindruch 1996,

Finch 1990, Rose 1991). To understand better how caloric restriction lengthens Ure

span, genetic methods and criteria were used to investigate its mechanism ofaetioD

in the nematode Caenorhabditis elegans. Mutations in many genes (etlt genes) result

in partial starvation ofthe worm by disrupting tbe function ofthe pharynx, the

feeding organ (Avery 1993). Most eat mutations tested, including multiple aUeles of

eat-I,2, 6, 18 and une-26 significantly lengtben Iife span. The strongest effects were

seen in eat-l mutant strains which can live 50% longer tban tbe wild type. In C.

elegans, two other genetically distinct mechanism oflife span extension are known: a

mechanism involving genes (daf-2, age-l, daf-16 and daf-28) that regglate dauer

formation (Kenyon et aL 1993, Larsen et al. 1995, Dorman et aL 1995 and Malone et

al. 1996), and a mecbanism involving genes (elk-l, elk-2, elk-3 andgro-l) that affect

the rate ofdevelopment and bebavior (Lakowski and Hekimi 1996, Wong et al. 1995

and Hekimi et aL 1998). 1 find that the long life ofeat-2 mutants does Dot require the

activity ofDAF-16 and that ellt-l; daf-2 double mutants live even longer than

extremely long lived daf-2 mutants. Tbis demonstrates that calorie restriction

lengtbens life span by a mecbanism distinct from tbat ofdauer formation mutants.

[n contras~ 1 find that caloric restriction does Dot further increase tbe liCe span of

long-Iived elk-l mutants, suggesting that elk-l and caloric restriction affect similar • processes. 66

• Introduction

Rodents rnaintained in laboratories are nonnally provided with excess food and

permitted to feed at will, a procedure known as ad libitum feeding. However, it bas long

been known that reducing the caloric intake from ad libitum amounts, while maintaining

adequate vitamins and nutrients (a process known as Calorie or Dietary Restriction), cao

significantly increase rodent life span (Yu 1996, Sobal and Weindruch 1996). This result

was tirst demonstrated in the 1930s by McCay and colleagues (McCay 1942) and bas

subsequently been reproduced by Many groups (Yu 1996, SohaI and Weindruch 1996).

Too drastic caloric restriction leads to acute starvation and death, bowever under

laboratory conditions, calorie intake can be reduced by up to 50% without detrimental

effects on life span (SohaI and Weindruch 1996). By such experimentaI treatments, Mean

and maximum life span bave been increased by as much as 50% (Sobai and Weindruch

1996). More recent work suggests that caloric restriction lengthens the life span ofa wide

range oforganisms including: fish, spiders and Daphnia (Sohal and Weindruch 1996).

Trials have begun with higher primates, and based on the preliminary evidence,

calorically restricted rhesus monkeys show sunilar signs ofdelayed aging to those seen in

the calorically restricted rodents (Verdery et al. 1997). These results suggest tbat caloric

restriction should extend the life span ofhigher primates and presumably even that of

humans.

Calorie restriction has been studied best in rodents and it is known that radents

undergoing calorie restriction display Many physiological changes, ineluding redueed 67

body weight, temperature7 blood glucose and insulin levels (Yu 19967 Sohal and • Weindruch 1996). However, it is unclear which ofthese changes are required for an

extended life span7 and Many theories have been proposed for how and why calorie

restriction extends longevity (Yu 19967 Sohal and Weindruch 1996, Rose 1991). Calorie

restriction, however7 remains the only experimental treatment that has been shown

repeatedly to significantly prolong the life ofvertebrates (Yu 19967 Sohal and Weindruch

1996, Finch 19907 Rose 1991).

It is in C. elegans, however, that genetic mechanisms that cao extend life span

have been best cbaraeterized7 and the worm has become the foremost animal model

system for the molecular analysis oflife span (Lithgow7 1996; Jazwinski 1996, Hekimi et

al., 1998). In C. elegans. mutations in severa! genes have been shown to prolong life

(reviewed in Lithgow 1996, Jazwinski 1996). It has also been shawn that, the mechanism

ofcalorie restriction exists in wonns7 as lowering food intake lengthens their life span

(Klass 1977, Hosono et al. 1989). Furthennore, a large group ofC. elegans mutants (eat

mutants) already exist which are calorically restricted based on their appearance and the

function oftheirpharynx, the feeding organ (Avery 1993). We could therefore use C.

elegans to study calorie restriction genetically, and to try to relate the mechanism of

calorie restriction ta the genetic factors known to extend the life span in worms.

The pharynx is a neuromuscular organ that pumps in7 concentrates and grinds up

bacteria before passing this food to the intestine (Albertson and Thomas 1976). When

wonns are placed on plentiful food the pharynx pumps over 200 times a minute (Raizen

et al. 1995). The pharynx contains about 80 nuclei and is composed mostly ofmuscle

ceUs and neurons (Albertson and Thomas 1976). The pharynx is almost completely 68

surrounded by a basement membrane that separates it from the rest ofthe worm • (Albertson and Thomas 1976). Only two neurons form connections between the pharyngeal nervous system and the rest ofthe worm's nervous system and these neurons

are not needed for normal feeding (reviewed in Avery 1993, Avery and Thomas 1997).

Consequently, the control ofpharyngeal pumping is largely autonomous ofthe extra­

pharyngeal nervous system.

Avery and collogues have studied the genetic control ofpharyngeal pumping. In a

screen for mutants with defects in pharyngeal pumping, 35 genes that affect the function

ofthe pharynx were identified (Avery 1993). The largest group ofthese genes, the eat

genes, lead to abnormal pharyngeal muscle movements and a starved appearance, without

obvious morphological abnonnalities (Avery 1993). The eat mutations fall into many

different classes affecting pharyngeal pumping in difIerent ways. For example, mutations

in sorne eat genes, such as eat-4, 5, 6 and 12, affect the strength, or the proper sequence,

ofcontractions and relaxations ofthe various muscles ofthe pharynx, leading to

inefficient feeding (Avery 1993). Mutations in other genes, such as eat-2, 8, 9 and 18,

however, do not affect the coordination ofthe muscle contraction but only drastically

reduce the rate ofpharyngeal pumping (Avery 1993, Raizen et al. 1995).

eat mutants are believed to disrupt normal pharyngeal pumping by affecting the

properties ofpharyngeal muscle and/or nervous system. Most eat mutations cause visible

defects in feeding behavior which resemble those caused by experimental alterations in

the pharyngeal muscles or nervous system (Avery 1993). For sorne eat mutants there is

even phannacological, electrophysiological or genetic evidence that they affect nervous • system or muscle (Avery 1993, Raizen et al. 1995, Davis et al. 1995). Furthennore, 69

severa! eat gene are now cloned and the identity ofthese genes strongly implies a • function in muscle or nervons system. eat-S affects cell-to ceLI coupling in the pharynx and encodes a protein that thought to he a component ofinvertebrate gap j unctions

(Starich et al. 1996). eat-6 encodes an a-subunit ofa sodium-potassium ATPase that

probablyacts in pharyngeal muscle and is necessary to maintain membrane resting

potentials (Davis et al.1995). eat-12 encodes an alphal subunit ofa voltage activated

calcium channel (Lee et al. 1997).

Many other genes are known that affect the development or fonction ofmuscle

and nervous system and mutate ta give an YD&oordinated (or Unc) phenotype. There are

over 130 une genes in C. elegans and most une strains can be easily cultured in the

laboratory because C. elegans does not need ta move ta mate or feed. Sorne ofthese

genes are among the best studied genes in C. elegans and Many have been cloned. There

are multiple causes for uncoordination, and different une mutations are known ta affect

different aspects ofthe development and function ofnervous system and muscle. Most

une mutations have little or no effect on pharyngeal pumping (Avery 1993). By testing

the life span ofune mutants, we can test whether general defects in muscle and nervous

system cao affect life span. Thus testing the life span ofune mutations is an excellent

control for effect ofeat mutations on life span.

In this chapter, 1 present work 1 have done to study caloric restriction genetically

and ta try to relate caloric restriction to other know genetic means ofextending C. elegans

life span. Based on the starved appearance ofeat mutants one would predicted that they

should be calorically restricted and live long. 1 examined the life span ofa number ofeat ~. mutants to detennine ifgenetic caloric restriction could extend life span. As a control the 70

life span ofseveral une mutants was also examined. Finally, as described in Chapter 2,

there is strong evidence that at least 2 different genetic mechanisms exist that are capable

ofextending the life span ofC. eleganst. Therefore, the interaction ofeat-2 mutations

with other genes known to affect life span was examined to determine ifthe mechanisms

that extend the life span in Clock and dauer mutants have any similarities to how caloric

restriction lengthens life span.

Materials and Methods

General methods and straiDs used: C. elegans strains were cultured as

described by Brenner(1974). Standard genetic methods for C. elegans were used

(Brenner 1974; Sulston and Hodgkin 1988). Animais were cultured at 20°C unless other

wise stated. The wild type strain was the N2 Bristol strain, derived from stock obtained

directly from the l\1RC in Cambridge England. Mutations and rearrangements used were

as follows:

LGI: eat-5(ad464), eat-/5(ad602), unc-29(eJ072), daf-J6(m26), eat-/8(ad820sd, adJ/JO)

LGII: eat-3(ad426), Ilnc-4(eJ20), eat-2(ad453, ad465, adJJJ3, adJ/ J6)

LGIII: eat-8(ad599), dpy-J(eJ), daf-2(eJ370), unc-79(qmJ2. qmJ4. e/030, e/068). elk­

J(e25J9. qm30), unc-32(eJ89). une-47(e367). unc-49(e382), eat-4(ad572, ad8J9,

/cy5), une-25(e/56. e265, e59J, e89/) • 71

LGIV: eat-IO(ad606), eat-7(ad450), une-24(eI38, e448, el172, eDj27), unc-30(eI9l,

e3lB, e596), eat-l(ad427, e2343), une-26(e205, e345, e1196, m2)

LGV: eat-6(ad467, ad601, ad792, ad997). une-46(e177). lIne-80(qm2, qm3, qm9. el069,

el272)

LGX: une-7(e5, wd7), une-9(eIOI, ec27, hs6, nr450). eat-13(ad522), une-l(e538, e7l9),

eat-17(ad707),

Aging Experiments: Aging experiments were perfonned as previously described

(Chapter 2, Lakowski and Hekimi 1996) except that experiments were begun by allowing

adult hermaphrodites to layeggs for a lirnited time (4-6 hours). Animais were cultured at

20°C unless otherwise stated. Experiments were started with 50 experimental worms per

genotype and the wild type (N2) was always included as a control. A plate of

approximately 30 spare worms was started at the same time as the experimental worms

and was treated identically except that deaths on this plate were not counted. WOrInS that

died from matricidal hatching (Bagging) were replaced by spare worms when possible, or

were discarded from the analysis when this was not possible. Statistics were calculated

using the Microsoft Excel 97 analysis ToolPak TM or Sytat5. Mean life spans were

compared using the students t-test assuming unequal variances.

Out-crossing strains: After it was found in initial experiments that many eat and

une strains live long, 1wanted to detennine ifthe effect on life span was due to the

mutation ofinterest or due to background effects. To test this all eat and Many une

mutations were back-erossed twiee to the wild type, and life spans were re-tested. In

most cases, mutations were re-isolated from the second (F2) generation by pieking single • worms with the relevant phenotype (Eat or Une) to individual plates (singling). The 72

original eat-l(e2343) strain contained a closely linked une-3I mutation. In the first out­ • cross, Eat-non-Unc recombinants were picked and allowed to selffertilize. By singling Eat-non-Unc progeny ofthese recombinants, a eat-l(e2343) strain was isolated. This

strain was out-crossed once more. eat-3(ad427) is matemally rescued and the original

strain, DA631, contained an unlinked him-8(eI489) mutation. To re-isolate the eat-3

mutation after out-cross, 20 wild type F2 progeny were singled and the phenotype ofF3

broods was scored. In the first round ofout-cross a plate was selected that was eat-3 but

not Him to remove him-8. This strain was then out-crossed once more. eat-7(ad450sd) is

semi-dominant. To outcross this strain, Eat F2 progeny were singled and the F3 broods

were scored for the absence ofany wild type progeny.

Making double mutants: No significant differences in the life-span ofeat

mutations before and after back-cross were note~ so the strain DA465 eat-2(ad465) was

used in experiments 100king at the interaction ofeat-2 with daf-l6, daf-2 and efk-l. eat­

2; elk-l double mutants were made by crossing elk-l males to eat-2 hennaphrodites, and

picking wild type (WT) Ft cross progeny. WT F2 progeny were singled and slow

growing F3 broods were examined for the presence ofEat progeny. From these broods,

several Eat progeny were singled and from one ofthese a eat-2; clk-l strain was derived.

eat-2(ad465); daf-2(e1370) double mutants were made by mating daf-21+ males to eat-2

hermaphrodites and singling FI cross progeny, which were then placed at 25°C. From F2

broods that segregated ~ dauers, Eat wonns were singled and transferred to 20°C. F3

adult Eat progeny were transferred to 25°C and allowed to lay progeny to identify

homozygous eat-2; daf-2 double mutants. F4 plates with ooly dauers were transferred to • 15°C to recover from dauer arrest, and from these plates a eat-2(ad465); daf-2(el370) 73

strain was isolated. To isolate a daf-16(m26); eat-2(ad465) strain~ eat-2(ad465) males • were mated ta daf-16(m26); dpy-l(el) daf-2(eI370) unc-32(eI89) hermaphrodites and WT FI cross progeny were placed at 25°C. WT F2 progeny were singled and F3 broods

were screened for ~ Eat and ~ Dpy Unc progeny with no dauers. Several Eat F3

progeny were single~ and from a F4 brood which segregated no DpyUne, Une or Dpy

progeny a daf-16(m26); eat-2(ad465) strain was derived.

Results

The original experiments with eat mutants: Known eat mutants have clear

defects in pharyngeal function leading to a insufficient food uptake and a starved

appearanee. Based on this phenotype we predicted that eat mutants should live long due

to calorie restriction. Ta test this hypothesis l tirst looked at life span ofsix eat strains

(Table 3.1). Ofthe six strains initially tested only three, DA531 eat-l(ad427)~ DA465

eat-2(ad465) and DA467 eat-6(ad467) reproducibly lived longer than the wild type (

Table 3.1). The DA464 eat-5(ad464) strain lived long in one oftwo trials, DA631 eat­

3(ad426); him-8(e2489) has a mean life span indistinguishable from the wild type

although its maximum life span is greater than N2 and DA521 eat-7(ad450) lived shorter

than the wild type in both trials.

These results are consistent with eat-l, 2 and 6 extending life span by calorie • restriction. However, as ail 6 strains tested have feeding defects, but only sorne lived 74

Table 3.1. The pooled results oftwo experiments in which the life span ofeat strains • received from the Caenorhabditis Genetics Center were tested. The combined sample size (n), and Mean ± standard error are given. sig trial = The number oftrials in which the

mean was significantly different from N2 (Student's t-test with unequal variances). %

diff: = the percent difference ofthe Mean from that ofthe wild type. t-test = the

significance ofthe difIerence ofthe Mean to that ofthe wild type (student t-test with

unequal variances). Straïns that lived significantly longer than the wild type (p

shown in bold, while short lived strains are underlined.

Strain Straïn n mean sig % t-test

trial diff

N2 + 97 16.4 ± 0.4

DA531 eat-l(ad427) 100 20.5 ± 0.4 2/2 +25 <0.0005

DA465 eat-2(ad464) 100 23.9 ± 0.4 2/2 1 +45 <0.0005

DA631 eat-3(ad426); him-8(e1489) 79 17.0 ± O.S 0/2 +4 0.35

DA464 eat-5(ad464) 99 18.2 ± 0.4 1/2 +11 0.001

DA467 eat-6(ad467) 46 19.3 ± 0.4 2/2 +17 <0.0005

DA521 eal-Zfad45Q) 100 14.1 ± 0.3 2/22 -14 <0.0005

1) DA465 was been tested in 6 different trials and in each and every trial it bas lived significantly longer than N2

2) In both trials DA521 lived significantly shorter than the wild type • 75

long~ 1suspected that background mutations could be affecting the life span ofsorne of • these strains. In an attempt to minimize background effects, 1out-crossed all the tested mutations twice, as weil as more alleles ofthe genes which appear to lengthen life span

and a number ofother eat mutations (table 3.2). By out-crossing these mutations twice

3/4 ofall unlinked background mutations should be removed.

Most twice-backcrossed eat mutant strains live long: 1 then re-tested the life

span ofall eat mutations. Ifgenetic caloric restriction causes long life, then mutations in

Many eat genes should lengthen life span. As expected, mutations in Many eat genes

(eat-1, 2, 3, 6, 13 and 18) significantlyextend life span. Ifmutations in the 6 eat genes

increase life span by caloric restriction, then different alleles ofa single gene should show

similar effects. Indeed, we find that all four tested alleles ofeat-2 and eat-6, as weil as

both alleles ofeat-1 and eat-18, significantly increase life span (Fig. 3.1, Table 3.2).

OfaIl ofthe eat genes tested the strongest effect was seen with eat-2 mutants,

which can live over 50% longer than the wild type (Fig. 3.1, Table3.2). The life span

extension ofeat-2 is comparable ta other previously characterized long-lived mutants,

such as clk-1(e2519) and daf-2(e1370) (Lakowski and Hekimi 1996), and is ofa similar

magnitude to the effect ofcaloric restriction on the life span ofmammals (Sobal and

Weindruch 1996). The effect ofeat-2 on life span is also robuste The life span ofeat­

2(ad465) has been determined 7 times and in each and every trial it lives substantially

longer than the wild type, with an average lengthening oflife span of47% over the wild

type (Table 3.1, 3,2. and unpublished data).

Three ofthe mutations tested did not lengthen life span. The mutations eat­ • 5(ad464) and eat-lO(ad606) do not affect life span (Table 3.2). I! is Dot clear why these 76

Table 3.2: Mean life span ofa number ofeat mutant strains. Sample size (n), mean lire • span ± the standard error ofthe mean and percent difference from the wild type are given. The final column displays the probability that the Mean lire span is the same as the wild

type (Student's t-test). Straïns that lived significantly longer than the wild type (p

are shown in bold, while short lived strains are underlined. Three separate experiments

were performed at different rimes each with the wild type (N2) as a control.

strain genotype n mean %diff t-test

ExPeriment # 1 N2 + 50 21.6 ±0.6 MQS81 eat-l(ad427) 50 28.8 ± 1.0 +33 <0.0005 MQS82* eat-l(e2343) 50 23.9±0.9 +11 0.03 MQ573 eat-5(ad464) 50 20.2 ±0.5 -6 0.07 MQS74 eat-7(ad45Q) SO 14.0 ± O.S -35 <0.0005

Experiment # 2 N2 + 50 19.5 ± 0.5 MQ643 eat-2(ad453) 50 26.5 ± 0.7 +36 <0.0005 MQS94 eat-2(ad465) 50 25.1 ± 0.7 +29 <0.0005 MQ644 eat-2(adll13) 50 28.4 ± 0.9 +46 <0.0005 MQ631 eat-2(adll16) 36 30.6 ± 1.4 +57 <0.0005 MQ646 eat-18(ad820sd) 50 26.9 ± 1.0 +38 <0.0005 MQ649 eat-18(ad1110) 38 22.4 ± 0.9 +15 0.008 • 77

Table 3.2 (coot.): • strain genotype n Mean %diff t-test

Experimeut # 3 N2 + 50 19.9 ± 0.5 MQ701 eat-3(ad426) 50 22.0 ± 0.9 +11 0.04 MQ584 etlt-6(ad792) 50 27.1 ± 0.6 +36 <0.0005 MQ583 eat-6(ad467) 50 27.2 ± 0.7 +37 <0.0005 MQ591 eat-6(ad601) 50 22.8 ± 0.7 +15 0.001 MQ645 eat-6(ad997) 39 23.6 ± 0.7 +19 <0.0005 MQ700 eat-lO(ad606) 31 21.5 ± 0.8 +8 0.12 MQ596 eat-13(ad522) 50 26.0 ± 0.6 +31 <0.0005

* A high proportion ofeat-l(e2343) wonns died from a prolapsed gonade Ifthese worms

are subtracted from the analysis the Mean life span increases significantly. [also tested

the life span ofeat-8(ad599), eat-15(ad602) and eat-/7(ad707) however the sample sizes

for these strains were too low give reliable results due to high levels ofmatricidal

hatching. eat-4(ad572), eat-4(ad819) and eat-4(ky5) were a1so tested but MOst ofthe

worms crawled offthe plates and died from desiccatioo.

• 78

Figure 3.1. Four alleles ofeot-2 lengthen life span. The percentage ofworms alive on a

• given day after eggs being laid for a single experiment: N2 (Â)~ eat-2(ad465) (0), eat-

2(ad453) (.), eat-2(adll13) (~), and eat-2(adll16) (e). Mean life spans are given in

Table 3.2. The death ofthe last surviving eat-2(adll16) worm is not shown. This wonn

died on day 80.

100 ,....--11111!!1!~~------,

Q) 80 > ---ca ....., 60 c: Q) o 40 '- Q) a.. 20 o+---.--..-.op--~~--"",,:~~-=:~~ a 1 0 20 30 40 50 Day

• 79

mutations do not extend life span, but it is possible that these mutations might lead to a • too weak feeding defect to noticeably affect life span. Altematively, these mutations might produce deleterious pleiotropic effects which mask any positive effects ofcaloric

restriction on life span. Surprisingly, the only known allele ofeat-l, ad450, actually

shortens life span (Table 3.2). The significance ofthis result is unclear as eat-7(ad450)

has a very unusual phenotype (Avery 1993). ad450 is semi-dominant mutation and the

nature ofits pumping defect is unclear. When eat-7 mutants are disturbed they appear to

pump nonnally, however when theyare left alone they cease pumping, consequently it is

difficult to study the phenotype because viewing the worms usually disturbs them.

The severity ofthe feeding defect correlates witb lile span in eat-2 and eat-6

mutants: Thus, 14 of 17 eat mutations tested significantly lengilien life span, strongly

suggesting that most eat mutants live long due to calorie restriction. This bypothesis is

strengthened by the observation that in both eat-2 and eat-6 mutants, the severity ofthe

eating defect appears to correlate with life span. Mutations in eat-2 appear to ooly affect

the rate ofpharyngeal pumpiog (Raizen et a/. 1995). As K1ass bas shown that food intake

is linearly related to pumping rate (Klass 1997), pumping rate is a reliable measure of

nutritional status. In the one trial in which four alleles were tested (Table 3.2, Fig. 3.1), 1

find that the two eat-2 alleles with the slowest pumping rates, eat-2(adl113) and eat­

2(adI116) (Raizen et al. 1995), live longer than a clearly weaker allele, eat-2(ad453). In

this trial a strong allele ad465 had a shorter average life span than ad453, increasing life

span 29 and 36% respectively. However the life span ofeat-2(ad465) has been tested 7 • times and in this trial it had one ofits weakest effects, increasing life span much less than 80

its 47% average. Although the feeding defect in eat-6 mutants is more complex, the case • is even dearer for eat-6 mutants. Because mutations in eat-6 cause feeble contractions and slow delayed relaxations ofpharyngeal muscles (Davis et al. 1995), pumping rate is

not a good measure ofthe feeding defect and indirect measures are needed. In eat-6

mutants, progeny production rate is a good measure ofthe feeding defect, with the more

severe aIleles producing progeny more slowly than less severe alleles. Based on life span

data (Table 3.2) we come up with the same allelic series (ad60l

as Davis et al. for other phenotypes such as progeny production rate (Davis et al 1995).

Most une mutations do Dot lengthen Iile span: The fact that long-lived eat

mutants display a range ofdefects with different underlying molecular or anatomical

causes, strongly suggests that these mutants live long because ofthe only phenotype they

share in common: restricted food intake. It remains possible, however, that the eat

mutants live long not because ofcaloric restriction, but rather as a consequence ofsorne

pleiotropic effects on the nervous system and/or muscles. To test ifthis is the case, we

examined the life span ofa number ofuncoordinated (une) mutants, most ofwhich also

clearlyaffect nervous system or muscle function, leading to movement defects (reviewed

in Chalfie and White 1988). By testing the life span ofune mutants we test whether

general defects in muscle and nervous system can affect life span. Testing une genes also

tests whether reduced movement can affect life span.

In the initial analysis ofune strains, mostly received from the Caenorhabditis

Genetics Center, 1 found that mutations in une-l. 4, 6, 7 and 29 clearly do not lengthen

1 ad792 is a cold sensitive allele. The allelic series is correct as sbown at 25° and 20°C, however at 15°C • ad792 may he the strongest eat-6 allele (Davis et al. 1995). 81

Table 3.3: The life span oforiginal une strains. The total number oftrials is • given. Ifa single trial was done the Mean for this trial is shown, however ifmultiple trials were done the average ofthe means the standard errorofthe means is given. Finally the

percent difference ofthe Mean from that ofthe wild type is given. Strains that repeatedly

lived longer than the wild type are shown in boldo

strain Genotype trials mean sig trial %diff N2 + 5 16.1 ± 0.8 CB538 unc-l(e538) 2 15.3 ± 1.4 0/2 -5 CB719 unc-l(e719) 2 16.6 ± 1.8 112' +3 Ca120 unc-4(e120) 1 17.4 0/1 2 +8 CB68 unc-6(e68) 1 16.4 0/1 +2 CB5 unc-7(e5) 1 16.3 0/1 +1 NCI06 unc-7(wd7) 1 14.9 0/1 -7 FH85 unc-9(ec27) 1 15.3 0/1 -5 NS450 unc-9(nr450) 1 15.0 0/1 -7 HH29 unc-9(hs6) 1 14.3 111 1 -11 CBIOI unc-9(elO/) 2 20.4±4.0 112 +27 CB138 une-l4(e138) 4 20.2±0.4 4/43 +25 CB448 unc-24(e448) 1 16.5 0/1 +2 CB927 une-l4(e927) 2 19.4 ± 0.8 2/2 +20 CBl172 unc-24(el/72) 2 16.9 ± 1.2 0/22 +5 CB156 une-25(el56) 3 22.1 ± 0.5 3/3 +37 CB205 une-26(elOS) 3 22.6 ± 2.3 3/3 +40 CBI072 unc-29(el072) 3 14.7 ± 0.2 1/3 1 -9 CBlû30 unc-79(el030) 3 17.6±1.2 113 +9 CB1272 unc-80(e1272) 3 20.7 ± 1.2 3/3 +29 1) Significantly shorter lived than N2 2) Tested once more. Result not significantly different from N2 • Tested 4 other times and 3/4 trials significantly longer Iived than N2. 82

life span (Table 3.3). Surprisingly 1 found that a large proportion ofthe initial set of • strains tested lived longer than the wild type including strains containing alleles oflllle-9. une-24, une-25 une-26, une-80 and possibly une-79 (Table 3.3). The result for une-9 was

clearly Dot due to this gene as three other aileles have no effect on life span (Table 3.3).

The results for une-79 and une-24 \vere sorne what ambiguous: the only tested allele of

une-79lived long in one out of3 trials, while two out offour une-24 aIleles lived long in

ail trials. The only tested alleles ofune-25. 26 and 80 alilived reproducibly longer than

the wild type. To see ifthese effects were due to the une mutation, or due ta background

mutations, 1 out-crossed the une-24, 25, 26. 79 and 80 mutations twice along with more

alleles ofthese genes. When 1 examined the life span (Ifthese out-crossed strains, it

became clear that the long life on most ofthe une strains mentioned above appears to be

due to background mutations. AlI four une-24 alleles have a mean life span

indistinguishable from the wild type after out-cross (Table 3.4) indicating that une-24

does not affect life span and that the strains ca 138 unc-24{e138) and CB927 unc­

24(e927) contain life-extending mutations that are unlinked or only weakly linked to UllC­

24. After out-cross, most unc-25. 79 and 80 alleles have a life span indistinguishable

from the wild type (Table 3.4) indicating that these genes do not affect life span.

However, the strains MQ587 une-25(e156). MQ638 une-80{e1272) and possibly MQ603

une-79(el068) do live longer t&'l.an the wild type indicating that they contain life­

extending background mutations closely linked to the respective une mutations. The fact

that une-25 does not lengthen life span was confinned by the analysis ofthe life span of

une-30, 46. 47 and 49 straÏns. unc-25 encodes Glutamic Acid Decarboxylase (GAD) an • enzyme essential for the biosynthesis ofthe neurotransmitter GABA (Y Jin and B. 83

Table 3.4: Mean life span ofa number ofune mutant strains. Columns are as described • for Table 3.2. Strain Genotype n Mean % diff t-test

Experiment # 1 * N2 + 50 19.0 ± 0.5 MQ545 unc-24(eI38) 50 19.2 ± 0.6 +1 0.79 MQ546 unc-24(e448) 50 19.1 ± 0.4 +1 0.85 MQ547 unc-24(e927) 50 19.0 ± 0.5 0 0.95 MQ548 unc-24(eI172) 50 19.7 ± 0.4 +4 0.25

Experiment # 2 N2 + 50 21.7 ± 0.6 MQ613 unc-25(e265) 50 22.8 ± 1.1 +5 0.42 MQ612 unc-25(e59I) 50 22.1 ± 0.9 +2 0.71 MQ611 unc-25(e89I) 50 22.5 ± 1.0 +4 0.49 MQ633 unc-49(e382) 50 23.1 ± 0.6 +7 0.11 MQ603 une-79(elO68) 50 24.1 ± 0.9 +11 0.03 MQ604 une-80(eI069) 50 21.6 ± 0.6 0 0.95

Experiment # 3 N2 + 50 19.9 ± 0.5 MQS87 une-25(e156) 50 25.1 ± 0.7 +26 <0.0005 MQS92 une-26(e205) 50 28.7 ± 0.6 +44 <0.0005 MQ647 une-26(e345) 34 29.2± 0.8 +47 <0.0005 MQS74 une-26(eI196) 50 27.3 ± 0.7 +37 <0.0005 MQ648 une-26(m2} 39 26.8 ± 0.7 +35 <0.0005 • MQ632 unc-3Q(e596) 50 18.2 ± 0.5 -9 0.02 84 • Table 3.4 (cont.): Strain Genotype n Mean % cliff t-test

Experimeot # 4 N2 + 50 21.6 ± 0.6 MQ575 unc-30(e191) 50 20.0 ± 0.6 -7 0.06 MQ588 unc-3Qfe3/8J 50 16.9 ± 0.5 -22 <0.0005 MQ578 unc-46(e177) 50 21.5 ± 1.0 0 0.96 MQ579 unc-47(e367) 50 20.5 ± 0.5 -5 0.16

Experiment # 5 N2 + 50 19.9 ± 0.7 MQ636 unc-79(elO30) 38 19.1 ± 0.7 -4 0.43 MQ635 unc-79(qm12) 50 19.7 ± 0.5 -1 0.89 MQ634 unc-79(qm14) 50 21.9 ± O.S +10 0.05 MQ638 unc..80(e1272) 50 22.0 ± 0.7 +11 0.03 MQ60S unc-80(qm2) 50 21.2 ± 0.7 +7 0.18 MQ609 unc-80(qm3) 50 20.1 ± 0.6 +1 0.83 MQ637 unc-80(qm9) 50 19.5 ± 0.6 -2 0.67 *unc-24 aIleles have been tested 4 times with similar results.

• 85

Horvitz personal communication). The genes une-3D. 46, 47 and 49 are aIso known to • drastically reduce GABA signaling (Rand and Nonet 1997) and mutations in these genes also do not lengthen liCe span (Table 3.4). So l have shown that 14 unc genes (une-J. 4, 6,

7,9,24,25,29,30,46, 47, 49, 79, and 80) do not lengthen life span. Although the life

span ofune mutants has not been systematically studied, sorne previous work has shown

that at least 5 other une genes (une-2, une-15, une-20. une-54 and une-78) do not lengthen

life span (Johnson 1984).

Mutations in une-l6 lengthen lüe span: The only une gene examined that

clearly lengthens life span is lIne-26. After back-crossing four alleles ofune-26 to the

wild type twïce, l determined that ail four alleles significantly lengthen life span (Table

3.4). However, as unc-26 mutations are known to have a starved appearance and a

feeding defect (Avery 1993), the long life ofthese mutants is probably also due to caloric

restriction. So 14/14 tested une genes that do not affect pharyngeal pumping do not

lengthen liCe span while 7/10 genes that disrupt normal pharyngeal pumping do lengthen

life span. This, along with the other evidence presented, indicates that eat genes indeed

lengthen life span by calorie restriction.

Tbe two otber genetic mecbanisms oflife extension in C. elegans: As life

span extending mechanisms can be analyzed genetically in C. elegans (Kenyon et al.

1993, Larsen et al. 1995, Donnan et al. 1995 and Lakowski and Hekimi 1996), we

wondered whether any ofthe known genetic factors extending life span involve the same

mechanism as calorie restriction. As discussed in Chapter 2 at least two different genetic

mechanisms for extending life span have been identified in C. elegans. One mechanism • involves the partial activation ofthe dauer pathway by mutations in the genes age-J, daf-2 86

and daf-28 (reviewed in Kenyon 1996, Lithgow 1996), while the other involves the • reduction ofphysiological rates by loss-of-function mutations in the Clock genes clk-l, clk-2, clk-3 and gro-l (Lakowski and Hekimi 1996, Wong et al. 1995). Evidence for the

independence ofthese mechanisms was presented in detail in Chapter 2, however l will

briefly review it here. age-I and daf-2 are involved in an insulin-Iike signaling cascade

that regulates the activity ofthe forkhead-like transcription factor DAF-16 (Kimura et al.

1997, Morris et al. 1996, Gottlieb and Ruvkun 1994, Lin et al. 1997, Ogg et al. 1997).

Loss-of-function mutations in daf-I6 strongly suppress the extreme long life ofthe dauer

mutants age-I and daf-2 (as weU as ail other phenotypes ofdaf-2 and age-Il (Gottlieb and

Ruvkun 1994, Larsen et al. 1995, Kenyon et al. 1993, Dorman et al. 1995, Lin et al.

1997, Ogg et al. 1997). However mutations in daf-16 do not suppress the long life ofthe

Clock mutants c/k-l, clk-3 and gro-l (Lakowski and Hekimi 1996, and Chapter 2)

suggesting that Clock mutants lengthen life span by a mechanism distinct from that of

age-l and daf-2. Consistent with this interpretation, daf-2 clk-l double mutants live

significantly longer than either daf-2 or clk-l. This is in contrast to the effect of

combining age-l(hx546) and daf-2(eI370), which based on genetic and molecular data

are thought ta function in the same pathway. age-l(hx546); daf-2(eI370) double mutants

do not live longer than daf-2(eI370) (Dorman et al. 1995).

Calorie restriction lengthens life span by a mechanism distinct from that of

dauer mutants: To test whether calorie restriction extends life span by the same

mechanism as the dauer genes, l tested whether daf-16 could suppress the long life ofeat­

2(ad465). The results ofthese experiments are very sunilar to those for daf-16(m26); clk­ • 1(e25I9) and daf-16(m26); clk-3(qm38) double mutants (see Chapter 2). 87

Figure 3.2. The long life ofeat-2(ad465) is not suppressed by daf-/6(m26). The

• percentage ofwonns alive on a given day after eggs being laid (day 0) for two pooled

experiments: N2 (0), daf-16(m26) (.), eat-2(ad465) (0) and daf-16(m26),· eat-2(ad465)

(e). Mean life spans are 19.7 ± 0.5 (100). 17.4 ± 0.3 (l00), 26.3 ± 0.6 (100) and 23.6 ±

0.6 (57) respectively.

100

al ao > ---ca ....., 60 C al Co) 40 1.- 0) CL 20 0 0 1 0 20 30 40 day

• 88

Although daf-I6(m26); eat-2(ad465) double mutants live slightly shorter than eat­ • 2(ad465). daf-I6(m26); eat-2(ad465) double mutants still live longer than the wild type (Fig. 3.2). Aga~ daf-16(m26) mutants live slightly shorter than the wild type (N2) (Fig.

3.2, Larsen et al. 1995 and Chapter 2), suggesting that the daf-/6(m26) mutation or a

mutation closely linked to it, has a weak deleterious effect on life span. This implies that

the shorter liie span ofdaf-I6(m26); eat-2(ad465) as compared ta eat-2(ad465) is not due

to partial suppression and in fact, eat-2(ad465) mutants live 34% longer than the wild

type, while daf-16(m26); eat-2(ad465) double mutants live 36% longer than daf-I6(m26).

The fact that daf-16 does not suppress the long life ofeat-2 suggests that the dauer

mutations and eat mutations lengthen life span by different mechanisms. Based on this

observation one would predict that eat-2(ad465); daf-2(e1370) double mutants could live

longer than daf-2(eI370) and this is indeed what we round (figure 3.3A). This result is

very similar to the effect seen when combining the effects ofdaf-2(e1370) and c/k-

1(e2519) (Lakowski and Hekimi 1996 and Chapter 2), and contrast \vith the effect of

combining age-I(hx546) and daf-2(e1370).

Calorie restriction may lengthen lire span by the same mechanism as clk-l

mutations: To examine whether calorie restriction and Clock gene mutations lengthen

lire span by a similar mechanism 1 examined the life span ofeat-2(ad465); clk-l(e2519)

double mutants. l find that eat-2 clk-l double mutants do not live longer than either of

the single mutants (Figure 3.38). One interpretation ofthese results is that eat-2 and clk­

1 mutations May affect a common process. As both eat-2 and clk-l mutations lengthen • life span by a mechanism distinct from that ofthe dauer genes, this interpretation is 89

Figure 3.3. The interaction ofeat-2 with da.f.2 and clk-l. A) The percentage ofwonns • alive on a given day after eggs being laid (day 0) at 20oe: N2(D), eat-2(ad465)(.), daf­ 2(eI370) (.6.) and MQ413 eat-2(ad465); daf-2(e1370) (e). Mean Iife span ± standard

error ofthe mean, with sample size in parenthesis, are 21.9 ± 0.8 (50),26.3 ± 0.9 (50),

34.1 ± 1.6 (66) and 41.6 ± 2.1 (60) respectively. eat-2(ad465); daf-2(eI370) worms live

significant1y longer than either eat-2(ad465) or daf-2(eI370) (p<0.0005 and p=0.005

respectively). B) The percentage ofworms alive on a given day after eggs being laid (day

0) for two pooled experiments at 20o e: N2 (0), eat-2(ad465) (.), c/k-l(e2519) (e) and

eat-2(ad465); clk-l(e2519) (0). Mean life spans are 19.7 ± 0.5 (l00), 26.3 ± 0.6 (100),

25.1 ± 0.9 (100) and 27.5 ± 0.8 (100) respectively. eat-2(ad465); clk-l(e2519)lives ooly

marginally longer than c/k-l(e2519) (p=O.05) and no longer than eat-2(ad465) (p=O.23).

In one trial c/k-l(qm30) and eat-2(ad465); c/k-l(qm30) were scored. The results for this

experiment were: N2, 21.9 ± 0.8 (50); eat-2, 26.3 ± 0.9 (50); clk-l, 24.1 ± 1.4 (50) and

eat-2; clk-l, 26.5 ± 1.5 (50).

• • 100--- ....------, A Q) 80 --> -as 60 +Jc: ~ 40 ~ Q) CL 20

O-l----~-~DII-...... ---.--=~-.....IIIW o 20 40 60 80 day

100 B

Q) 80 --> -ca 60 +Jc: Q) 0 40 ~ Q) a. 20 0 0 20 40 60 80 • day 91

consistent with other genetic data. In particular, one interpretation consistent \vith these • results is that caloric restriction may work in part by lowering the activity ofelk-l.

Discussion

Many etlt genes can lengthen liCe span: These results have severa! implications

for our understanding ofcaloric restriction and aging. At least seven genes were

previously known to lengtben the life span ofC. e/egans (Lithgow 1996~ Jazwinski 1996,

Hekimi et al. 1998). l have now shown that at least seven eat genes can also extend life

span by calorie restriction. Over twenty five genes known to cause a starved phenotype

and it is estimated that there are at least 60 in the C. e/egans genome (Avery 1993).

Mutations in 7Il0 tested eat genes clearly increase life span, suggesting that there may be

forty or more '4aging" genes ofthis type. l have also identified at least 5 background

mutations in une strains which lengthen life span. These strains did not appear to develop

slowly and were not dauer constitutive, even at 27°C (unpublished observations). It is

difficult to see subtle phenotypes on the background ofstrong une mutations, so these

mutations may have a weak Eat phenotype. Ifnot then, the background mutations in

these strains identify other novel genes involved in the regulation ofaging. These results • indicate that a great many genes regulate life span. 92

Why some ellt mutations might not lengtheD liCe span: As all eat mutants • appear starved, one might expect that an eat mutations should increase life span. Indeed most eat mutations do increase life span, but not ail. There could be severa! explanations

for why not all eat mutants live long. It is possible that sorne eat mutants are not

calorically restricted enough to noticeably lengthen life span. Conversely, sorne mutations

may lead to tao drastic caloric restriction and actually shorten life span. It has been noted

in many animais, including C. elegans. that severe starvation limits life span (see Fig. 1.2,

Klass 1977, Johnson et al. 1984). It is also possible that sorne eat mutations have

deleterious pleiotropic effects which counteract the effect ofcaloric restriction on life

span.

Reducing pbysical movement does not lengthen liCe span: Before l began this

study, it was not clear how many genes could lengthen life span in C. elegans. The

effects ofonly a small number genes on life span have been properly documented in C.

elegans. One class ofgenes that had not been systematically examined was une genes.

Mutations in une genes cause uncoordinated movement or even paralysis. Mutations in

most une genes, like eat genes, are thought to affect either muscle or nervous system

function or development. However most une mutations do not affect the function ofthe

pharynx (Avery 1993). Thus une mutations also make excellent controls for the effects of

eat genes on life span. 1examined the effect ofmutations in 15 une genes upon life span.

These une genes were selected because they have a range ofcauses for their movement

defects. For example, mutations in UIIC-4 and une-30 encode homeodomain proteins that

are necessary for certain neurons ta express their proper fate (reviewed in Ruvkun 1997), • unc-6 is required for the proper migration ofneuronal growth cones (reviewed in Antebi 93

et al. 1997), and une-25. une-46. une-47. and une-49 are required for GABA mediated • neurotransmission (reviewed in Rand and Nonet 1997). 1 find that 14 ofthe 15 une genes tested do not increase life span. Mutations in une-29 and une-30 May actuaIly reduce life

span (Tables 3.3 and 3.4). This indicates that reducing physical movement has little or no

effect on C. elegans life span. The only une gene that clearly lengthens life span in une­

26. However, as une-26 mutants have a known feeding defect (Avery 1993), it is

parsimonious to suggest that it is the feeding defects, and not the movement defects, in

these wonns that lengthen life span.

Background problems in the analysis ofaging: Although most mutant stains

in C. elegans have been generated from the standard reference strain (N2), background

effects cao still be a problem. In the process ofstudying the life span ofune mutants, 1

discovered that a large proportion ofune strains contain background mutations that extend

life span. 1even found that, although at least two strains containing different une-24

alleles lived long, the effect was not due to mutations in une-24. It is not clear why 50

Many ofthe une strains tested harbor such background mutations, but it may just be a

coincidence. It is aIso possible that because une mutations usuaIly cause severe

phenotypes, mild background mutations can not easily be seen in these backgrounds.

However, Many une strains available frOID the Caenorhabditis Genetics Center have only

been backcrossed once to N2 after the mutagenesis, 50 they are likely to hacbor many

background mutations. Background mutations have also been a problem in the analysis

ofthe life span ofspe-26 mutants (Gems and Riddle 1996). Based on these results care

must be taken when trying to link a quantitative phenotype, such as life span, to genotype. • This suggests, that to show that mutations in a gene lengthen life span, one needs to show 94

tbat more tban one allele lengthens life span and that long life is tightly linked to the • mutations. By these criterion, mutations in age-l, daf-2, c/k-l, eat-I, eat-2, eat-6, eat-I8 and unc-26 clearly affect life span. Fonnally, as ooly a single allele ofdaf-28. c/k-2, c/k­

3, gro-I, eat-3 and eat-I3 have been shown to lengthen life span, it is possible that the

reported effects ofany one ofthese genes could be due to background effects. However,

in mast cases, long life is known to be genetically linked to the gene ofinterest, and as

genes with similar phenotypes clearly lengthen life span, it is likely that the effect on life

span is indeed due to these genes.

The relationship between life extension by calorie restriction and by the

dauer or Clock mutations: It has been suggested that because daf-2 encodes an

insulin like-receptor, and that daf-2 mutations lead ta alterations in metabolism, that there

are parallels between life extension by calorie restriction and by daf-2 mutations (Kimura

et al. 1997). However, my results strongly suggest that daf-2 mutations lengthen life span

by a mechanism distinct from that ofcalorie restriction. Life extension by caloric

restriction does not require the activity ofDAF-16 and eat-2; daf-2 double mutants live

even longer than daf-2 or eat-2 mutants, consistent with these genes lengthening life span

by different mechanisms. Rather, calorie restriction appears to lengthen life span by a

mechanism similar to that ofmutations in the gene c/k-I. eat-2; c/k-I double mutants live

no longer than eat-2 mutants and only marginally longer than c/k-I mutants. This

suggests that eat-2 and c/k-l may lengthen life span by affecting the same process.

Insofar as eat-2 and c/k-l cao he said to he involved in a genetic pathway, c/k-l is acting • dawnstream ofeat-2. eat-2 mutations are thought to primarily affect pharyngeal pumping 95

(Raizen et al. 1995), while clk-l acts at the level ofsingle ceUs and is presumably present • in all eeUs (Wong et al. 1995, Ewbank et al. 1997, reviewed in Hekimi et al. 1998). It is not clear how calorie restriction lengthens life span, but the leading

hypothesis is that calorie restriction May reduce oxidative stress eaused by free radicals

produced in the mitochondria (Yu 1996, Sobal and Weindruch 1996). clk-l bas been

cloned and its yeast homologue (CATS/COQ7) has been clearly implicated in the

regulation ofmitochondrial function (Proft et al. 1996, Marbois and Clarke 1996, Ewbank

et al. 1997, reviewed in Hekimi et al. 1998). Cat5p/Coq7p bas been loealized to the inner

membrane ofthe mitochondria where it affects Coenzyme Q synthesis (Jonassen et al.

1998). Recently, clk-l has also been localized to the mitochondria and clk-l mutations

have been shown to reduce respiration (Hekimi personal communication). Thus the long

life ofclk-l mutants might result from these animals baving lower levels ofrespiration

with concomitant slower production offree radieals. This suggests that calorie restriction

may affect life span by a similar mechanism. It is even possible that calorie restriction

eould lengthen life span in part by down-regulating the activity ofclk-l, thus reducing

mitochondrial function and oxidative stress.

• 96 • Chapter 4

The Caenorhabditis elegans Clock genes: four

Bovel genes that affect developmental and

behavioral timing•

• 97 • Abstract

[n a screen Cor maternally rescued viable mutations in Caenorhabdilis

elegans, mutations in tbree genes, clk-1, clk-2 and clk-J, were identified that affect

developmental and behavioral timiDg (Hekimi et al. 1995). These three genes along

with gro-1 (Hodgkin and Doniach 1997) derme the Clock class ofgenes. Mutations

in the best studied geDe ofthis class, clk-1, affect the function ofa large number of

pbenotypic traits that bave a temporal component (Wong et aL 1995; Wong 1994).

clk-l worms display aD increase in the lengtb ofembryonic and post..embryonic

developmen~a lengthening ofthe period ofrhytbmic adult behaviors and an

increase in meaD and maximal liCe span (Wong et aL 1995). Here [ sbow tbat clk-2,

clk-3 and gr0-1 sbare many features in common witb clk-l and tbat tbese four genes

are Dovel. Ail double mutant combinations oftbese four geDes display a more severe

phenotype than any mutation in a single Clock gene. Tbese observations, along witb

some complex genetic interactions among tbese genes suggest that ail Clock genes

May affect a common process. Tbe genetic mapping oftbe Clock genes bas allowed

clk-l andgro-l to be cloned. clk-2 is also weil positioned genetically and pbysically

and attempts to clone this gene are under way•

• 98

Introduction

• A number ofgenetic sereens in the nematode Caenorhabditis elegans have

identified genes required for a vast anay ofprocessesy including Donnai developmen~

movement and behavior (Brenner 1974, Hodgkin and Brenner 1977y Hodgkin 1980,

Ferguson and Horvitz 1985). Mutations that affect these processes can show Many

different modes ofinheritancey and screens have been carried out to search for mutations

which have a dominant-effecty a simple recessive-effect or have a maternal-effect

(Brenner 1974y Kemphues et al. 1988y Mains et al. 1990y Park and Horvitz 1986).

Special procedures must often be taken to recover matemal-effect mutationsy and these

type ofmutations are usually poody represented in more general screens (Capowski et al.

1991 ~ Isnenghi et al. 1983). However the analysis ofmaternaI-effeet mutations can be

very enlightening. For exampley by analyzing maternaI-effect mutations in the fruit fly

Drosophila melanogaster the earliest events ofdevelopment have been identified

(reviewed in Lawrence 1992). Mutations affecting these early events ofdevelopment

often have such profound consequences for later development that they lead ta embryonie

lethality. Based on these observations, screens for maternaI-effect lethal mutations have

also been carried out in C. elegans and have identified genes involved in the earliest

patterning events in the WOOD (Isnenghi et al. 1983y Priess et al. 1987, Kemphues et al.

1987, Mains et al. 1990y Scbnabel and Schnabel 1990a,b).

One class ofgenes that had not been systematically pursued, were those geoes

whose mutational disruption does not oecessarily lead to lethality, but which display a • matemaI-effect due to a requirement for ooly very low levels ofgene expression or to a 99

function particularly early in development. To identify ne\v genes involved in the • development ofthe worm, a screen for matemal-effect mutants was carried out (Hekimi, Boutis and Lakowski 1995, Boutis 1995). After screening 30, 000 genomes, 41

mutations were identified that fall ioto 24 complementation groups. Only two ofthese

complementation groups define previously identified genes. The remaining 22 genes can

be divided ioto seven phenotypic classes. Six ofthese classes resemble known classes of

zygotic genes. Howeverone ofthese classes, the Clock (Clk) class, appears to have a

novel phenotype, a pleiotropic alteration ofdevelopmental and behavioral timing (Wong

et al. 1995; Wong 1994).

The best studied gene ofthis class is the gene cfk-J, where clk stands for 'abnormal

function ofbiological gocks'. In the sereen five putative alleles ofthe gene clk-l were

recovered (Hekimi et al. 1995). Subsequent analysis bas shown that qm47 is not allelic to

clk-J and that qmJI probably represents a re-isolate ofthe original e25J9 mutation,

leaving three known mutations: e25J9, qm30, and qm5J (Ewbank et al. 1997). Anne

Wong did a detailed phenotypic analysis ofclk-J alleles (Wong et al. 1995; Wong 1994)

and showed that mutations in this gene affect the function ofa large number of

phenotypic traits that have a temporal component. The phenotype ofclk-J mutants was

discussed in Chapter l, but 1 will briefly review it here. cfk-J mutants display a

lengthening ofembryonic and post-embryonic development, as weIl a lengthening ofthe

periods ofrhythmic adult behaviors ineluding swimming, pbaryngeal pumping and

defecation cycle. Ail clk-J alleles also lengthen both Mean and maximum life span

(Wong et al. 1995, Cbapter 2). AlI clk-J phenotypes are fully matemally rescued (Wong • et al. 1995). Based on this, and other aspects ofthe clk-J mutant phenotype, it has been 100

hypothesized that a general mechanism oftiming control, or a cloc14 is present in C. • elegans, and that clk-l affects the function ofthis clock (Wong et al. 1995). In order to find other genes that May interact with clk-l and be involved in this

putative clock, we looked for other mutations that had a similar phenotype to clk-l

mutants. In particular we were interested in mutations which had the four following

properties: 1) maternaI rescue 2) slow development 3) slow defecation and 4) no

obvious deleterious effects. This suite ofphenotypes we have called the Clock phenotype

(Hekimi et al. 1995). In the same screen in which the clk-l alleles were isolated, three

other mutations were recovered, which have a very similar phenotype to clk-l mutations

but helong to distinct loci (Boutis 1995, Hekimi et al. 1995). These mutations define two

novel genes, clk-2(qm37) and clk-3(qm38, qm53) (Hekimi et al. 1995). A mutation in a

fourth gene, gro-l for 'iIl1wth rate abnonnal', was identified by Jonathan Hodgkin from a

\vild C. elegans isolate from Palm City California on the basis ofits slow growth

(Hodgkin and Doniach 1997). Subsequent reappraisal ofthe gro-l(e2400) mutant

phenotype revealed that it also reduced the defecation rate and was maternally rescued

(Wong et al. 1995). Pamela Larsen has also found it shows increased longevity and

resistance to heat shock (personal communication). Given the similarities ofgro-1 to the

clk genes it is surprising that no new gro-I alleles were recovered in the maternaI viable

screen. However, maternal-effect viable mutations were recovered at a relatively low

frequency in the screen, so it would have been unlikely ifail genes that can mutate to give

a Clock phenotype had been identified in the screen (Hekimi et al. 1995).

In this chapter, 1present the genetic mapping ofclk-l, 2, 3 and gro-l. 1 also • extend the phenotypic characterization ofclk-2. 3 and gro-l and investigate the îOl

interaction ofthese three genes with each other and with dk-l. By this process 1 establish • that these four genes are novel and define a new phenotypic class ofmutations.

Materials and Methods

General methods and strains: C. elegans strains were cultured as described by

Brenner (1974). Animais were cultured at 20°C unless otherwise stated. Wild type was

the N2 Bristol strain. Mutations and rearrangement used were as follows:

LGI bli-4(e937)

LGII sqt-l(sc3), dpy-IO(el28), rol-6(el87), rol-l(e9l). clk-3(qm38. qm53), eat-2(ad450),

unc-52(e444, e669su250ts), jDp

LGill daf-2(el368), emb-32(g58), emb-2(hc58), emb-l(hcl), unc-79(el030), emb­

7(hc66), dpy-I7(el64), gro-l(e2400), clk-J(e25J9), mel-3J(s2438), me/(jb7), lon­

I(el85), sma-4(e729), embl6(gI9), sma-3(e49l), lin-39(nl760), clk-2(qm37), lin­

I3(n387), ncl-I(el865), mab-5 (e1239), unc-36(e25l), unc-32(eI89), emb-34(g62), emb­

24(q40), vab-7(e1562),sDp3, qDp3, sDfl2l, nD.f20

LGIV unc-3l(e928), him-5(e2467ts)

LGV dpy-ll(e224)

LGX lon-2(e678)

ScoriDg Clock phenotype in genetic experiments: The slow growth

phenotype ofclk-J, 2, 3 and gro-l was used to score the presence or absence ofa • particular Clock mutation in recombinants from genetic mapping experiments. Usually 102

plates were started with gravid adult hermaphrodites and three days later the • developmental stage ofthe oldest progeny was determined. At this point the oldest N2 worms should be young adults, while all Clock mutants are al least 1 day from reaching a

similar developmeotal stage. In sorne cases it was oecessary to allow the gravid

hermaphrodites to only lay progeny for a short period (generally less than 6 hours) to

better stage the progeny. In these cases the hennaphrodites were allowed to lay progeny

for a short time and then removed.. a process we caU a Iimited laying. Scoring clk-3 on

the background ofsorne mutations, such as une-52, was often difficult and two rounds of

scoring often were needed. In the fust round, recombinants were grouped into those

which definitely did oot contain clk-3, those which did and those that were questionable.

The questionable recombinants and the clk-3 recombinants were re-scored in a limited

laYÎng to confirmldecide genotypes. To confirm. the presence ofc/k-2 in strains the ability

ofrecombinants to grow at 25°C was often determined, since c/k-2 containing strains

displaya fuUy penetrant embryonic lethal phenotype at 25°C (see results).

Linkage 8nalysis for clk-3: To detennine to which chromosome clk-3 mapped,

the strain DA438 bli-4(e937) I, rol-6(e187), daf-2(e1368ts) vab-7(e1562) III; une­

31(e928) IV, dpy-Il(e224) V, /on-2(e678) X(Avery 1993) was used. DA438 cootains at

least one morphological Marker in the central region 00 each ofthe six chromosomes.

c/k-3(qm38) males were mated to DA438 hennaphrodites. Three hundred

morphologically nonnal F2 worms were placed singly on plates (singled) and the

developmental rate ofF3 broods were scored. Slow developing broods (homozygous for

c/k-3(qm38) were scored for the segregation ofall DA438 markers except daf-2. • Unlinked markers should be present in 2/3 oraU c/k-3 broods while a linked marker 103

should be present in less than 213 ofaIl clk-3 broods. After weak linkage to rol-6 on LGII • was found~ linkage experiments were done to markers on the right and left arm ofLG II using standard procedures.

Mapping maternai viable mutations. For the most part standard genetic

methods for C. elegans were used for both strain construction and genetie mapping

(Brenner 1974~ Sulston and Hodgkin 1988). However mapping maternaI viable mutations

requires sorne adjustments to score phenotypes.

Three or mu/ti-point mapping: In experiments where a Clock mutation ( c ) was

placed in trans to two recessive morphological mutations (a and b), recombinants were

picked in the standard manner (i.e. phenotypically A-non-B and B-non-A recombinants

were selected from the progeny ofable heterozygotes). However to score whether the

recombinant chromosomes contained the Clock mutation~ between 8-24 non-AB FI

progeny from each recombinant were singled~ and the F2 broods were scored for any AB

progeny. The growth rate ofbroods which segregated no AB progeny was scored to

detennine ifthe recombinant chromosome contained the Clock mutation. In cases where

a Clock mutation was placed in cis to t\vo morphologicaI mutations and mapped relative

to a third morphological mutation (d) in trans (i.e. a c bld), the presence ofthe Clock

mutation on the recombinant chromosome could he scored from the growth rate ofthe

heterozygous FI progeny ofthe recombinant (i.e. a (c?)la c b). In cases where two Clock

mutations (cl and c2) were mapped relative to each other by placing them in trans (i.e.

clk-l(e25l9) andgro-l(e2400)~at least two morphologicaI mutations were used. A-non­

B and B-non-A recombinants ofa cl ble2 (or a cl bld c2 e) heterozygotes were picked • and the presence ofthe cl mutation could be scored from the growth rate ofthe 104

heterozygous FI progeny ofthe recombinant. The presence ofe2 could only be scored by • singling non-A Cl B FI progeny ofrecombinants and examining the growth rate ofF2 broods that segregated no A Cl B progeny to score the presence ofthe c2 mutation. In

the case ofthe cross ofdpy-17 gro-l sma-4 / llne-79 clk-1 lon-l. it was possible to easily

identify the homozygous recombinant progeny ofSma-non-Dpy recombinants because

theyaH picked up une-79. However, since homozygous Dpy-non-Sma recombinants

could not be easily identified and scoring these recombinants represented much more

work than scoring the Sma-non-Dpy recombinants, the Dpy-non-Sma class of

recombinants was not fully analyzed.

Mapping clk-2 relative to 00-13: The cross clk-2(qm37) unc-32(e189)//in­

13(n387) was done at 25c C to use the strict lethality ofclk-2 at this temperature (see

results) to screen for recombinants. In this experiment Unc progeny ofclk-2 une-32/lin­

13 hennaphrodites were pooled in groups offive or ten on plates and these plates were

sereened 2-3 days tater for progeny. Any plate with progeny must have eontained at least

one recombinant [i.e. a c/k-2(qm37) unc-32(e189)/ + unc-32(e189)]. It was assumed that

there was only one recombinant per plate, and the progeny were screened for the multi­

vulva (Muv) phenotype, whieh is indicative oflin-13(1l387) mutation. Sorne progeny

were plaeed singly on plates and their progeny were re-scored to confirm the initial

assessment.

Two point mapping: For the two point mapping ofclk-3 relative to une-52, FI

Une progeny ofelk-3(qm38) une-52(e669su250ts)/+ + were singled and the growth rate

ofthe F2 progeny were scored. Due to the difficulty ofscoring clk-3 on an une-52 • background, ail questionable cases were re-scored using a limited laying approach with 105

Many F2s to confinn the scoring. For all other two-point mapping experiments a slightly • different approach was used. To directly determine the genetic distance between a Clock mutation ( c ) and a genetically close recessive morphological Marker ( a )

phenotypically-A FI progeny of c aI+ + heterozygotes were picked and usually pooled

in groups offive, IO or 20. The growth rate ofthe fastest developing F2 progeny was

scored. Ifone or more ofthe FIs placed on the plate contained a recombinant

chromosome (i.e. c a/+ a) then the fastest F2 progeny would develop at a wild type rate

and would clearly develop faster than the fastest progeny on sibling plates that contained

only c ale a FIs. This plate was scored as containing at least one recombinant. To

determine the genetic distance between the e and a mutations the equation

(4.1)

was used where / is frequency ofplates with no recombinants, n is the pooling size (i.e. 5,

10 or 20) and d is the genetic distance.

Details ofmapping experiments: The details ofmost mapping experiments

and all other mapping done for the maternai-viable screen paper (Hekimi, Boutis and

Lakowski 1995) have been sent to the Caenorhabditis Genetics Center (CGC) and are

available through the C. elegans electronic data base AceDB. A summary ofmapping

data used to position clk-l, 2, 3 and gro-l is given in Tables 2.2, 2.4 and 2.5.

1 Ifthe recombination rate is p, then the frequency ofplates with no recombinants is f=[l/4{l-p)2D]/II4 or • f=(l-p) 2n. Ta get equation 4.1 solve for p and note tbat the distance in cM is l00p. 106

Calculation ofConfidence iutervals for genetic mappiug data: To calculate • 95% confidence intervals for genetic mapping data the fonnulae 22.26 and 22.27 from Zar were used (Zar 1990).

Complementation tests: Clock mutations have little affect on male mating

efficiency so homozygous Clock males were used ta perform complementation tests.

Out..crossing clk..2 and clk-3: Prior ta any detailed analysis clk-2(qm37) and

clk-3(qm38) were each crossed to N2 ten times ta remove most ofthe mutagenized

background. c/k-1 aileles had previously been crossed to N2 20 rimes (e2519), 10 times

(qm30) or five rimes (qm51) (Wong, et al. 1995).

Transferring extrachromosomal arrays: To transfer c/k-l rescuing arrays

between clk-l and gro-l backgrounds, dpy-17(e164) was used as a marker to confinn that

backgrounds had been altered. For example to transfer the qmEx80[ZC400. pRF4] array

from a clk-l(e2519) to agro-l(e2400) background, dpy-17(e164) males were mated to

clk-l(e2519); qmEx80[ZC400. pRF4] hennaphrodites. Many FI roUing male progeny

(dpy-17/clk-1; qmEx80) were then crossed to gro-1(e2400) hermaphrodites and L31L4 F2

rolling hermaphrodite progeny were singled. F3 broods were screened for the presence of

~ Dpyprogeny, ta remove the c/k-l(e2519) mutation from the background (i.e. to

identify dpy-17/gro-l .. qmEx80 progeny). Non-Dpy F3 progeny were singled to isolate a

gro-l(e2400); qmEx80[ZC400. pRF4].

Characterization ofClock strains: Most phenotypic traits ofclk-2. clk-3 and

gra-1 strains and Clock double mutants were quantified using the same methodology as

Wong (Wong 1994, Wong et al. 1995). However for Table 4.2, eggs were allowed to • hatch for up ta 12 hours and 25 worms were then scored for their developmental stage 107

every 12 hours until all became adults. The time noted is the length oftime between the • midpoint ofthe hatching window to the midpoint ofthe window in which the Median worm became an OOult. The stated error is the SUffi ofthe length ofthe hatching window

and the adulthood window divided by two.

Construction ofClock double mutants: AlI Clock genes map to LG mexcept

clk-3 (LG II) (see results). To constnlct Clock double mutant strains containing c/k­

3(qm38). the strain MQ209 clk-3(qm38) II; dpy-I7(eI64) III was constructed. dpy-I7was

used in trans to balance LGill Clock mutations which are ail closely linked to dpy-I7.

Homozygous males containing LG III Clock mutations (c) were crossed to MQ209.

Thirty phenotypically wild type F2 progeny ofc/k-3(qnz38)/+; dpy-17(e164) / c were

singled. F3 broods \vere scored for growth rate and the presence ofsorne Dpy progeny.

For each desired double, a strain was derived from a brood which took one day longer

than wild type to develop, and segregated ~ Dpy progeny (a putative clk-3(qm38) II: dpy­

17(e164)/c m strain). Double mutants were derived from these balanced strains by

singling morphologically wild type progeny, 1/3 ofwhich should be double mutants.

Two ofall the doubles constructed this way, clk-3(qm38) II; clk-l(e2519) ID and c/k­

3(qm38) II; clk-2(qm37) fi, were viable as homozygous strains and were maintained as

such. The other two doubles were maintained as balanced lines and double homozygotes

were obtained from the progeny ofbalanced beterozygotes.

Double mutant strains containing two closely linked LG ID Clock mutations were

constructed by using appropriate f1anking markers to pick recombinants and then

removing those flanking markers by outcrossing. In the case ofclk-l(e2519) c/k-2(qm37) • double mutants, Unc-non-Lon progeny ofunc-79(eI030) clk-l(e2519) lon-l(el85)/dpy- 108

17(e164) clk-2(qm37) mothers were singled and their progeny were scored for Dpy • progeny. One out ofnine recombinants segregated no Dpy progeny and Une FI progeny fonn this recombinant segregated extremely slow growing F2 progeny [putative unc­

79(el030) clk-l(e2519) clk-2(qm37) worms]. dpy-17(e164) males were crossed to these

Une progeny to produce a unc-79(el030) clk-l(e2519) clk-2(qm37)/dpy-17(e164) s~

from which 300 phenotypically wild type progeny were singled. By this procedure, an

unmarked clk-l clk-2 double chromosome can be recovered two ways in this cross: 1) As

a morphologically wild type wonn which segregates extremely slow growing progeny, ~

ofwhich are Une (a putative clk-l c/k-2/unc-79 clk-l clk-2 mother) or 2) as a

morphologieally wild type wonn which segregates aIl fast developing progeny (i.e. wild

type rate), ~ ofwhich are Dpy and none ofwhich are Unc (a putative dpy-17/clk-1 clk-2

mother). In this cross a type 1) wonn was recovered, from which homozygous c/k­

l(e2519) c/k-2(qm37) worms segregated. This double homozygote strain was not viable

so dpy-17(e164) males were crossed to the double nlutants to generate the balaneed strain

MQ141 clk-l(e2519) c/k-2(qm37)/dpy-17(e164). Similar procedure were used to

generate clk-l(qm30) clk-2(qm37) andgro-l(e2400) clk-2(qm37) double mutants except l

started with the strains unc-79(el030) clk-l(qm30) sma-4(e729) and unc-79(eI030) gro­

1(e2400) sma-4(e729) respectively. Again the double mutant eombinations were not

viable as homozygotes and were maintained as lines balanced over dpy-17(e164).

Agro-l(e2400) clk-l(e2519) strain was construeted in the following manner. N2

males were crossed to unc-79(el030) gro-l(e2400) lon-l(e185) hermaphrodites and cross

progenymales were mated to dpy-17(e164) clk-l(e2519) sma-4(e729) hennaphrodites. • Twenty five Sma-non-Dpy recombinants ofunc-79(eI030) gro-l (e2400) lon-l(e185)/ 109

dpy-17(e164) clk-l(e2519) sma-4(e729) hermaphrodites were picked. One ofthese • recombinants segregated 114 UncSma progeny which only produced dead eggs or extremely slow growing progeny (a putative unc-79(eI030) gro-l(e2400) clk-l(e2519)

sma-4(e729) strain). dpy-17(e164) males were mated to these UncSma progeny and Unc­

non-Sma recombinants were singled. One such recombinant segregated very slow

growing progeny and no Dpys ( a putative unc-79 gro-l c/k-l sma-4/unc-79 grO-1 c/k-l

recombinant). This recombinant was mated with dpy-17(eI64) males to generate a unc­

79(eI030) gro-1(e2400) clk-l(e2519)/dpy-17(e164) strain. Complementation tests to this

strain confinned the presence ofboth gro-l(e2400) and clk-l(e2519). Wild type progeny

ofthe unc-79 gro-l clk-l/dpy-17 strain were singled until a brood was identified which

segregated 114 Dpy progeny and no Unc (A putative dpy-17/gro-1 clk-l strain). One tOOd

ofall WT progeny frOID this strain segregate only dead eggs or a very few extremely slow

developing progeny. As gro-Iclk-1 double mutants are not viable in the second

homozygous generation., they were not studied further.

Results

The phenotype ofclk-l mutants: The phenotype ofclk-l mutants has been

studied in detail by Wong (Wong 1994, Wong et al. 1995) and is briefly described in the

introduction.

Initial mapping ofclk-l: Hekimi found that clk-l(e2519) was linked to dpy-17(eI64) • (Wong et al. 1995). Three point mapping placed clk-l between dpy-17 and unc-32., very 110

Table 4.1: Genetic mapping data used to position c1k- J, and gro- J. cross results mapper clk·} c1k-J/ dpy-17 lUlc-32 dpy-17 1 clk-I 20 tlllc-32 8. Hekimi c1k-l/dpy-/7 lUlc-32 dpy-17 2 clk-/ 39 wlc-32 B. Lakowski dpy-/7 clk-2 / tlllc-79 clk-I /0"-/ uIlc-79 8 dpy-17 0 clk-/ 1 1011-/ B. Lakowski dpy-J 7clk-I/ ++ 4 / 725 Dpys develop quickly p = 0.3 cM S. Hekimi sDp3 includes c/k-l(e2519) B. Lakowski sDf121 deletes clk-J(e25J9) J. Ewbank gro-1 dpy-J 7gra-/ tllle-32 / /0"-/ dpy-J 71 gra-/ 0 JOIl-l 12 tllle-32 J. Hodgkin l gro- J/ dpy- J7 IlIIe-32 dpy-J 7 4 gro-J SI tme-32 B. Lakowski dpy-J 7gra-II + + 01220 Dpys develop quickly p < 0.2 cM B. Lakowski gra-Iloll-JI ++ 21250 Lons develop quickly p = 0.4 cM B. Lakowski dpy-J 7gro-J sma-4 / wlc-79 clk-Iloll-I 84 Dpy non 8ma recombinants dpy- J7 1S gra- J B. Lakowski 69 sma-4 sDp3 includes gro-l (e2400) B. Lakowski sDI121 deletesgro-J(e2400) B. Lakowski

• • t t 1

Table 4.1 (cont.) cross results mapper clk-l and gro-l gro-l/dpy-/7 c/k-/ 1011-1 dpy-/77 gro-/ 0 c/k-/ 11 10"-/ B. Lakowski

dpy-J 7gro-J sma-4 1Imc-79 clk-/ /01l-} Imc-79 44 dpy- J7 S gro- J 0 clk- J 4 /011- J B. Lakowski dpy-J 7gro-l s",a-4 1WIC-79 c/k-} /011-1 Sma non Dpy recombinants B. Lakowski

dpy-} 7 22 gro-} 3 clk-} 27 /01l-} 27 8",a-4

The clk-l and gro-l region clpy- /7gro- J8",a-4 / 'mc-79 c/k- / 10ll-} 20 whole broods scored, 3184 WT, 1004 Lon, 895 B. Lakowski DpySma, 25 Sma, 18Dpy; 43/5126; p=0.84cM dpy-/710ll-// + + 12/ B. Lakowski 1011- / 8ma-41 + + B. Lakowski

1) results from Hodgkin and Doniach 1997.

The allele e2519 was used in ail clk-} mapping. • • Figure 4.1: A simplified genetic map of LGIII in the vicinity ofc/k-/ and gro- J

gro-J

clk-I L R

unc-79 ced-4 dpy-J 7 /on-/ 1 sma-4 unc-32

1 sDf/2/ daf4 1

sDp3 o O.S 1.0 1,11111,,11 cM

• • 113

close to dpy-J7 (Wong et al. 1995~ Fig. 4.1 Table 4.1). Two point mapping placed clk-l • 0.3 cm from dpy-J7 (Wong et al. 1995~ Fig. 4.1~ Table 4.1).To confirm this mapping~ l repeated the dpy-17 unc-32/clk-1 mapping and got aImost identical results to Hekimi

(Table 4.1).

Initial characterization ofgro-l: gro-l shows Many similarities to clk-l

including slow development (Hodgkin and Doniach 1997, Table 4.2), slow defecation [87

± 12 S (0=5)] and maternai rescue, however gro-l(e2400) complements ail alleles ofcik-l

(Wong, et al. 1995,). The gro-l(e2400) phenotype has not been studied in detail however

in the course ofculturing gro-l wonns it was noticed that they are not viable at 25°C,

becoming sterile after one generation. This suggests that gro-l is required for sorne

aspect ofgermline development or function.

Initial mapping ofgro-l: Based on genetic mapping by J. Hodgkin, gro-l

appeared to map very close to cllc-I(Table 4.1, Hodgkin and Doniach 1997). To

detennine just how close gro-l maps to clk-l. l extended the mapping ofgro-l. By a set

ofsimilar crosses to those done for clk-l. l detennined thatgro-l maps between dpy-17

and unc-32 very close to dpy-17 (Fig 4.1, Table 4.1). By two point mapping 1determined

that gro-l maps to 0.3 cm from /on-l and close to dpy-17 (Table 4.1). Based on this

extended mapping both clk-l and gro-l map roughly 0.2 cM to the right ofdpy-17 on

LGIII (Figure 4.1) and couId map to the same locus.

Deficiencies, duplicatioas and complementation tests for clk-l and gro-l:

To help retine the mapping results and to help detennine the genetic properties ofgro­

l(e2400) and the clk-J mutations~ these mutations were placed over deficiencies (Dt) and • duplications (Op). The large duplication sDp3 is able to complement the slow growth of 114

Table 4.2 Length ofpost-embryonic development ofClock strains at 15, 18, and 25°C. • strain Genotype Development (days) 15°C 18°C 20°C 25°C·

N2 N2 4.1 ± 0.4 2.5 ± 0.3 2.4 ± 0.3 1.5 ± 0.5

CB4876 clk-l(e2519) 5.0 ± 0.4 3.0 ± 0.3 2.9 ± 0.3 2.5 ± 0.4

MQ130 clk-l(qm30) 6.6 ± 0.4 4.0±0.3 3.9 ± 0.3 2.5 ± 0.4

MQ125 clk-2(qm37) 5.1 ± 0.4 3.0 ± 0.3 2.9 ± 0.3 4.3 ± 0.4

MQ131 clk-3(qm38) 5.0 ± 0.4 3.5 ± 0.3 2.9 ± 0.3 2.4± 0.4

MQ230 gro-l(e2400) 7.6 ± 0.4 4.1 ± 0.4 3.4 ± 0.3 2.9±0.3

MQ141 clk-l(e2519) clk-2(qm37) lù.O ± 0.4 6.0 ± 0.4 6.5 ± 0.5 ND

MQ266 clk-l(qm30) clk-2(qm37) 9.0 ± 0.4 6.5 ± 0.4 7.0 ± 0.5 ND

MQ124 clk-3(qm38); clk-l(e2519) 7.1 ± 0.4 5.8 ± 0.3 4.1 ± 0.4 4.5 ± 0.5

MQ224 clk-3(qm38); c/k-l(e2519)t 11.4 ± 0.5 10.5 ± 0.5 12.0 ± 0.4 ND

MQ225 clk-3(qm38); c/k-2(qm37) 5.5 ± 0.5 3.9 ± 0.3 3.8 ±0.3 5.5 ±0.5

MQ524 gro-l(e2400) clk-2(qm37) 8.9±0.4 4.4±0.3 ND ND

MQ223 clk-3(qm38); gro-l(e2400) 10.0 ± 0.4 8.0± 0.4 5.5 ± 0.5 ND

TJ401 age-l(hx546) fer-15(b26) 4.3 ± 0.3 3.0 ± 0.3 2.4± 0.3 1.5 ± 0.5

MQ415 age-l(hx546) fer-15(b26); ND 3.6 ± 0.4 ND 3.3 ±0.3

gro-l(e2400)

ND = not determined. • worms were raised at 20°C until they hatched and were then transferred to 25°C. : It was difficult to accurately detennine when clk-3(qm38); clk-l(qm30) wonns completed development, especially at 20°C~ because these worms • never develop a functional gennline. The number given is a maximum estimate. 115

both clk-l mutations and gro-l(e2400). while the small deficiency sDf121 deletes both • clk-l and gro-l (Fig 4.1, Table 4.1). However it was difficult to determine ifthe strains haploid for clk-l(e2519), clk-l(qm30) orgro-I(e2400) over sDf121 had a stronger

phenotype, so it could not be detennined genetically ifany ofthese mutations is nuU. The

phenotypes ofclk-l mutations and gro-l(e2400) over deficiencies and duplications is

fuUy consistent with these alleles acting as simple recessive, maternal-effect mutations.

Since it was not clear what the null phenotype ofclk-l and gro-l was,

complementation tests were perfonned to two maternai effect lethal (mel) mutations

which map in the region. However mel-31(s2438) and melûb7) complement both clk-l

and gro-lfor growth rate and lethality.

DetermiDing the genetic position ofmarkers in the clk-l and gro-l region:

The ability to interpolate genetic and physical position ofa gene from genetic mapping

data, requires that the position ofthe genetic markersused be weil established. Although

the markers unc-79, dpy-17. lon-l and sma-4 have been used extensively by the C.

elegans community, the genetic separation between these markers had not been clearly

determined and in successive genetic maps released by the CGC (i.e. 1991, 1993, and

1995), the absolute position ofthese genes varied widely. To detennine the separation

between these markers l perfonned a number ofgenetic mapping experiments. Results

from multi-point mapping indicated that the distance between unc-79 and dpy-17 was

roughly five times the distance between dpy-17 and /on-l (Table 4.1). To detennine the

genetic separation between dpy-J7, /on-l and sma-4. 1did three two point mapping

experiments (see Table 4.1). By counting .aIl progeny from 20 broods ofdpy-17 gro-l • sma-4 / unc-79 clk-l lon-l l detennined that the distance between dpy-17 and sma-4 is 116

0.84 cM. Using double mutants in cis 1detennined that dpy-17 lies 0.49 cM frOID /on-1 • and /on-l lies 0.35 cM from sma-4. Is gro-l(e2400) an aUele ofclk-l? From the similar genetic positions gro-

l (e2400) and c/k-l(e2519) and their sunilar phenotypes, we suspected that e2400 could

he an unusual allele ofc/k-1 that complemented the other three alleles. To determine if

e2400 is a complementing allele ofc/k-1 1tried to separate gro-1(e2400) from c/k-

l (e2519). First 1 tried to separate these t\vo mutations by picking Lon recombinants of

gro-l / dpy-17 clk-l /on-l hermaphrodites (dpy-17 is epistatic to /on-1 so one cao only see

one elass ofrecombinants). However after picking and scoring 19 recombinants no

events elearly separated e2400 and e2519 which indieated that these two mutations were

very elosé. Beeallse in this tirst cross we failed to convincingly separate e2519 and

e2400. 1 designed a cross by which it wOllld be possible to separate these two mutations

regardless oftheir relative positions. The initial idea was to piek Une-non-Lon and Lon-

non-Une recombinants ofdpy-17 gro-l sma-4 / unc-79 c/k-l /on-1 hermaphrodites,

however this strategy was abaodoned when it was realized that 5/6 ofall reeombination

events screened by this procedure occurred between unc-79 and dpy-17 in a region which

was not ofmterest. 1then took advantage ofthe surprising possibility to distinguish Dpy-

non-Sma and Sma-non-Dpy worms from DpySma doubles to map gro-1 roughly 0.03 cM

: Ninteen recombinants were picked and Il contained gro-/ but not c/k-J while 7 contained clk-J but not gro-/. One recombinantcontained clk-/ and when homozygous developed extremely slowly. was sterile and could not he maintained. Although we suspected that this recombinant could contain both clk-/ and gro-/. al the rime ofthis mapping we did not know what the phenotype ofany Clock double mutant was like. 50 this recombinant was excluded from the analysis because it could not he 5cored completely. However. in the Iight oflater results (see below) it is likely that this un-scored recombinant contained both • c1k-J andgro-/. We analyzed this cross as a fallure to separate clk-/(e25/9) andgro-/(e2400) 117

3 or 15 kb to the left ofclk-1 • This suggests that the two mutations are in separate genes, • however, the possibility existed that e2400 and e2519 were two mutations at two separately mutable locations within a genetically large gene.

The genetic information above was used to predict the physicallocation ofclk-l

and gro-l. By injection ofcosmids spanning the predicted clk-l region, It was found that

clk-l could be phenotypically rescued by the cosmid ZC400 (Ewbank et al. 1997). 1

transferred one rescuing extrachromosomal array, qmEx80{ZC400; pRF4j, from a clk-

1(e2519) to a gro-l(e2400) background and found that qmEx80 does not rescue gro-l.

To confirm that this anay still contained the clk-l rescuing activity it was then transferred

from agro-l background to a clk-l(qm30) background and was found to fully rescue the

developmental defect ofqm30. These results indicate thatgro-l and clk-l are different

genes.

Phenotypic cbaracterization ofclk-2(qm37) m: The gene clk-2 is defined by

a single temperature sensitive allele qm37. Between 15°C or 20°C clk-2 worms develop,

defecate and move slowly. For example, at 20°C clk-2 worms have a generation rime of

approximately 2.9 days (Table 4.2) and an inter-defecation period of62 ± 15 s (n=25). At

15°C clk-2 worms appear dark and healthy. At 20°C they appear slightly pale and

segregate a significant proportion ofdead eggs, and a few percent oflarvae fail to develop

into adults. Consequently, c/k-2 worms have a very small brood size (81 ± 33 progeny

n=10) at 20°C. Embryonic development ofclk-2 wonns appears to be ooly slightly

.3 Because dpy-/ 7 is epistatic to lon-/ and 10n-l is epistatic to sma-4 1expected that dpy-/7 would be epistatic to sma-4, however the two mutations have additive effects. Both dpy-/ 7 and sma-4 sborten the length ofworms by up to ~. dpy-/7 sma-4 double mutants are extremely short approximately '4 normal body length and it is possible to distinguisb Dpy-non-Sma and Sma-non-Dpy worms from DpySma • doubles. 118

slower than wild type and highly regular (16.3 ± 0.4 br. (n=15) for clk-2 vs. 13.6 ± 0.3 • hr. (n=17) for N2). Ifclk-2 worms are transferred to 25°C from a lower temperature, they start to lay dead eggs within 5 hours after transfer. Eggs laid in the first 5 hours May

hatch but often fail to complete post-embryonic development, and those that further

develop, become pale, thin and completely sterile adults. Some clk-2 worms which arrest

as larvae at 25°C can become fertile adults ifthey are then transferred to 15°C. M.

Lupien has done a time course on the lethality of25°C clk-2 (personal communication)

confinning these initial observations. After 10 hours at 25°C, no clk-2(qm37) progeny

ever develop into adults. These results indicate that clk-2 is an essentiai gene with both

embryonic and post-embryonic functions.

Complementation tests for clk-2: Based on the sunHar map positions ofclk-2

and /in-I3 (see below and Table 4.3), and the facts that both genes are matemally rescued

and are not viable at 250 C, we suspected that qm37 could be a unusual allele oflin-I3.

However, when 1 perfonned a complementation test, we found that clk-2(qm37)

completely complements lin-13(n387). At 20°C lin-I3(n387) 1clk-2(qm37) unc-32(eI89)

heterozygotes develop and defeeate at a wild type rate at 200 C and appear dark and

healthy, with no obvious vulval defeets. At 250 C heterozygotes are viable and segregate

wild type (WT), uneoordinated (Une) and Multivulva (Muv) progeny. Both the Muv and

Une progeny are sterile and the WT progeny again segregate WT, Unc and Muv progeny.

clk-2(qm37) aIso complements two other genes whieh map to the region ncl-I(e/865) and

lin-39(n/760). Severa! temperature sensitive embryonic lethaI mutations (emb) are • known which map to the same general region ofchromosome ID as clk-2, however, ail of 119

Table 4.3: Genetic mapping data used to position clk-2. cross results mapper clk-2 clk-2 / dpy-J 7wlc-32 clpy-J 7 14 clk-2 18 Imc-32 B. Lakowski c/k-2 /1011-1 rmc-36 1011- 1 47 c/k-2 23 rmc-36 B. Lakowski dk-2 / sma-4 mab-5 rillc-36 sma-4 35 cJk-2 3 mab-5 14 'mc-36 B. Lakowski sma-3 c1k-2 IInc-36 / /in- /3 sma-3 18 c1k-2 0 lin- / 3 10 IInc-36 B. Lakowski Ii,,-J3/clk-2 rl1lc-32 cJk-2 3 lill-J3 49 rl1lc-32 M. Lupien sma-3 clk-2 rl1lc-36 / lill-39 sma-3 40 /i,,-39 0 dk-2 33 'mc-36 M. Lupien cJk-2 wlc-36 / ++ 5/630 Unes develop quiekly p=0.4 cM B. Lakowski IIDf20 does not delete clk-2 B. Lakowski qDp3 includes clk-2 B. Lakowski Complements emb-l, emb-2, emb-7, emb-J6. emb-24, emb-25, emb-32, emb-34, "cI- J, Ii,,- J3, li,,-39

• • Figure 4.2: A simplified genetic map ofLG1I1 in the vicinity ofclk-2

clk-2 1 1 L R dpy-/7 /on-/ sma-4 sma-3 lin-39 1mab-5 unc-36 unc-32

qDp3

o QS

cM

• • 121

these complement clk-2 for bath slow growth at 20°C and lethality at 25°C, therefore, it

•• appears likely that qm37 defines a novel gene.

Mapping ofclk-2: clk-2 was found to be tightly linked ta dpy-17 on LGill by P.

Boutis (Boutis 1995). In a series ofmapping experiments, l determined that c/k-2 lay

about halfway between dpy-17 and unc-32, in the interval between /on-l and unc-36

(Table 4.3, Fig 4.2). By the four factor cross sma-4(e729) mab-5(e1239) unc-36(e251) /

clk-2(qm37), clk-2 was positioned 0.05 cM to the left ofmab-5 in the Middle ofthe C.

elegans Hox gene cluster near /in-13 (Table 4.3, Fig 4.2). In a tirst attempt to map clk-2

relative to lin-13, these two genes were not separated, confirming the close proximity of

these mutations (Table 4.4). Consistent with the three point mappillg c/k-2 is contained

within the Duplication qDp3 and is not deleted by the deficiency nDflO (Table 4.3, Fig

4.2). Attempts to place clk-2 over the Deficiency nDf17, which should delete clk-2, were

unsuccessful. There are two possible explanations for tItis: 1) nDf17 is hard to maintain

and may have been lost in the mapping stain or 2) clk-2(qm37)/nDf17 heterozygotes die

as dead eggs. l did not distinguish between these possibilities, however given the

temperature sensitive lethality ofqm37 it possible that the null phenotype ofc/k-2 is

embryonic lethality and that qm37/nDf17 is lethal. Recently, M. Lupien has refined the

position ofclk-2. He found that c/k-21ies close, but to the left of/in-i3 and it was not

separated from /in-39 (Table 4.3, Fig 4.2). Formally we have shown that c/k-2 must lie

between sma-3 and /in-i3, near lin-39 (Fig. 4.2).

Charaeterization ofclk-3: clk-3 is defined by two aileles, qm38 and qm53.

The two alleles appear to be very similar, but ooly qm38 has been extensively studied. Of • the genes, gro-i, clk-2 and c/k-3, clk-3 wonns look the most like clk-i. c/k-3 worms are 122

embryonic development

25 ------.--_._-----_..__ ._..-._-.__ .,_ ... ~_. ----_.. - .------_."_._---...._------... --_. __ .- ---- ~

20

CIl m15 '&• '- .!E 10 :::1 Z

5

IIlr n o III nIL, , i i i i 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 lime (hr)

• N2 0 clk-3(qm3B)

Figure 4.3 Embryonic development ofthe wildtype (N2) and c1k-3(qm38) at 20°C. The mean length ofembryonic development ± the standard deviation is 13.7 ± 0.8 hr. (n=84) for N2 and 18.1 ± 1.5 hr. (n=88) for c/k-3.

• • 123

large dark and healthy worms, which appear slightly uncoordinated when moving • backwards. They have a slightly smaller brood size than wild type [238 ± 31 (n=10)]. clk-3 worms also develop (Table 4.2) and move slowly. clk-3(qm38) embryonic

development at 200 e is slower than wild type and displays a greater variance (Fig 4.3).

clk-3(qm38) worms also have a slow pumping rate [115 ± 33 pump/min. (n=10 worms

scored for 5 minutes)]. l have found that clk-3 worms display a slow, aperiodic

defecation cycle at 200 e [Mean 150 s, range 69- 496 s (n 30 inter-defecation periods)],

while at 25°e they defecate much faster and are much more regular [69 ± 10 s (n=16

worms for five inter-defecation periods each)]. However, when J. Lemieux scored

defecation in c/k-3(qm38) at 200 e he found it to be slower than wild type but not very

irregular (Personal communication).

Linkage analysis ofclk-3 Using the strain DA438, clk-3 was not found to be

strongly linked to the centrai region ofany ofthe chromosomes. Ooly the results for

LGII (ro/-6) differed significantly from the expected 2/3 for unlinked loci by a chi­

squared test, suggesting that c1k-3 is approximately 15 cM from ro/-6, or on one ofthe

two arms ofLGII. 1 then tested linkage to markers on both ofthe arms ofLGII and found

that elk-3 was ooly very weakly linked to sqt-2 on the left arm ofLGII and that clk-3 is

strongly linked to une-52 on the right arm ofLGII (Table 4.4, Fig. 4.4).

ComplementatioD tests for clk-3: Several genes lie near une-52 and none of

these genes has a phenotype sunilar to clk-3. In limited laying experiments lin-7, cad-l

and ace-3 mutants were found to develop at the same rate as the wild type and much

slower than clk-3(qm38). Consequently, 1 decided that complementation test to these loci • were unnecessary. 124

Table 4.4: Genetie mapping data used to position clk-3. cross results mapper clk-3/DA438 (LGI b/i-4, LGII rol-6, LGIII 69 homozygous c/k-3: LGI BIi- 41; LGII Roi 33; B. Lakowski daf2 l'ab-7, LGIV mle-31. LGV dpy-I J, LGIII Vah 50; LGIV Une 48; LGV Dpy 46; LGX LGX /011-2) Lon 42. c1k-3/sqt-3 23/114 Sqt F2 homozygous for clk-3 B. Lakowski clk-3/ulle-52(e444) 0/117 Une F2 homozygous for clk-3 B. Lakowski c/k-3 / dpy-JO Ime-52(e444) c/py-IO 100 c/k-3 2 ""e-52 B. Lakowski c1k-3 /1'0/-1 ,me-52(e444) 1'0/-/ 38 clk-3 2 ""e-52 B. Lakowski c1k-3 / ro/-l.me-52(e669st4250ts) 1'01-1 16 clk-3 l ""e-52 B. Lakowski rol-l c1k-3 une-52(e669su25Ots)/eat-2 clk-3 3 eat-2 1 une-52 B. Lakowski clk-3 'me-52/ + + 9 / 439 Une progeny develop quickly p= 1cM B. Lakowski }Df2 does not delete clk-3 B. Lakowski

Ali clk-l mapping was performed with qm38. In mapping experiments with 'mc-52 1initial1y used e444 which is severely paralyzed from the L3 stage onward. However it is difficult to score c/k-3 on a ,me-52(e444) background 50 1requested and began to use the temperature sensitive al1ele e669slI250ts which only becomes completely paralyzed after one clay ofa adulthood when raised continuously at 20°C..

• • Figure 4.4: A simplified genetic map of LOU, with a blowup ofthe vicinity ofclk-3 L R 1 '1 ..: , sqt-2 dfJJ'-JO ro/-I ""e-52 .... .' .' " .' ,." .' ...... ' " ...... " .'.' .... " ..... 1

clk-3 eal-2 /il1-7 une-52 1 jDfl

o O.S

cM

• • 126

Mapping ofclk-3: Ta confirm that elk-3 layon the right ann ofLGII it was mapped • between dpy-IO and une-52 and then again between rol-l and une-52. Both experiments placed clk-3 on the far right arm ofLGll near, but to the left, ofune-52 (fig 4.4, Table

4.4). Two point mapping places elk-3 about 1 cM from une-52 and consistent with this

position. elk-3lies outside the deficiencyjDj2 (fig 4.4, Table 4.4). By multi-point

mapping clk-3 was mapped to the left ofeat-2 making elk-3 the left-most weil mapped

gene in the gene dense region near the right end ofLGII (Fig 4.4, Table 4.4). This means

that the left boundary for where elk-3 lies is not weIl defined genetically or physically.

The genetïc interaction ofthe Clock genes: In order to investigate the genetic

interaction ofthe Clock genes, aU six possible double mutant strains containing the

reference aIleles ofthese genes [elk-l(e2519), elk-2(qm37), clk-3(qm38) andgro-l(e2400)

] were constructed. 1have also generated elk-l clk-2 and clk-l clk-3 double mutants with

qm30, the strongest elk-l allele. AlI double mutants have a more severe phenotype than

their constituent single mutations, and develop, defecate and move extremely slowly. For

example, elk-l(e2519) elk-3(qm38) worms have a Mean embryonic development time of

25.5 ± 2.8 br. (n=14) at 200 e as compared to 13.3 ± 0.4 br. (n=23) for N2. 1 attempted ta

score defecation in clk-l(e2519) elk-2(qm37) and clk-3(qm38); elk-l(e2519) double

4 mutants but it was 50 slow and irregular that it was very difficult to score • AlI double

mutant combinations are still matemally rescued and the matemal-effects ofeach gene in

a double mutant appear ta be independent. The 2S oe lethality ofelk-2 and gro-l is not

suppressed in any ofthe double combinatioDS. The various double mutant strains differ in • the severity oftheir timing defects. In Table 4.2 the approximate length ofpost 127

embryonic-development is given for ail single and double mutant combinatioDS. Other • phenotypes display a similar range ofseverity and the severity ofdifIerent phenotypes appears to be correlated. In general the mildest defects are seen in clk-2(qm37) clk­

3(qm38) double mutants which have a generation time ofapproximately 3.8 days at 20°C

as compared to 2.4 days for N2 and 2.9 days for both qm37 and qm38. The most severe

defects are seen in clk-l(qm30) clk-3(qm38) wonns which have a generation time of

approximately 12 days at 20°C. In this strain early embryonic cell cycles are lengthened

four-fold over the wild type (Hekimi personaI communication) and embryonic

development takes roughly 3 days, as opposed to 13 hours in N2 at 20°C. l aIso see some

interesting allele specific interactions. clk-3(qm38); clk-l(qm30) double mutants have a

much more severe defect than clk-3(qm38); clk-l(e2519) double mutants. This contrasts

with clk-l(e2519) clk-2(qm37) and clk-l(qm30) clk-2(qm37) double mutants which have

equally severe phenotypes.

Many double mutants display synthetic phenotypes. Most double mutants are

essentially sterile and have to be maintained as balanced strains. Only two double mutant

combinations are bomozygous viable: clk-l(e2519) clk-3(qm38) and clk-2(qm37) clk­

3(qm38). The e2519 qm38 strain is only barely viable and bas become sterile several

times during culture and bad to be recovered from starved plates or from frozen stock.

clk-l(qm30); clk-3(qm38) worms coming from matemaIly rescued mothers often arrest

development as abnonnaI larvae, while those that do not arrest, develop into small and

pale adults whicb are completely sterile and appear to lack a gennline. The tirst non-

• 4 For example one qm38: e2519 worm did notdefecate in 25 min. (wild type worms defecate every 50s). 128

matemally rescued generation ofgro-l clk-l double mutants often die as dead eggs and • were not examined in much detail. It was noticed in the course ofcu1turing the double mutants that sorne ofthe

strains appeared to live a very long time. Since it was known that mutations in c/k-l and

gro-l extend life span (Wong et al. 1995, P. Larsen personal communication) it seemed

likely that this could be a general property ofClock mutants which might be more

pronounced in double mutants. Ifthis were the case, we could use aging to study further

the genetic interaction ofthese genes. Although there are several other timing phenotypes

we couId study with double mutants, sorne ofthese would be technically very difficult to

quantitate and compare. Studying aging has the advantage that since an wonns must die,

aIl strains can be scored and compared for their longevity. Chapter 2 contains a detailed

characterization ofaging in Clock single and double mutants and was discussed in detail

there. However, as discussed in Chapter 2, life span ofclk strains is strongly correlated

with the length ofembryonic development.

Discussion

clk-l, cil-l, clk-3 and gro-l are Bovel genes: The preliminary characterization

ofmutations in cfk-2, clk-3 and gro-l suggested that these genes shared similarities with

clk-l and may also affect the function ofthe putative central biological clock (Hekimi et

al. 1995). Upon further inspection, we have found that cfk-2, c/k-3 andgro-J share Many • similarities with clk-l and it is clear that these four genes define a new phenotypic class of 129

mutations, the Clock class ofgenes. Based on this data and the detailed genetic mapping, • clk-l, clk-2, clk-3 andgro-l are novel genes. An essential elass ofgenes: Based on the temperature sensitive lethality ofclk­

2 and gro-l both ofthese genes appear to he essential genes. The synthetic lethality of

most Clock double mutants and especially clk-3(qm38); c/k-l(qm30) double mutants

suggests that clk-l and clk-3 also have very important biological funetions. The reduced

growth rate and brood size induced by ail Clock mutations means that there would be

strong selective pressure in the wild to rnaintain Clock gene funetion.

The Cloek genes interact genetieally: Clock double mutants display a mueh

more severe phenotype than any of~'te individual Clock mutants. This observation along

with allele specifie internctions suggests that these four genes May affect the same

underlying process and may even physically internet.

Positional cloning: In the process ofgenetieally mapping the Cloek genes 1

developed the expertise and the tools neeessary for genetic analysis and strain

construction. The tools developed to map the Clock genes were used to construct Clock

double mutant strains and were used repeatedly throughout the rest ofthis thesis to study

the genetics ofagjng in C. elegans. However the primary motivation behind the genetic

mapping experiments was to help positionally clone the Clock genes

The most common, and often the quickest way, to clone genes in C. elegans that

are defined by mutations, is use a positional cloning strategy. In this approach the

mutation ofinterest is mapped between the nearest cloned flanking markers, and the

genetic mapping data is used to estimate the physical position ofthe gene. With a good • estimate ofthe physical position, a small number ofclones cao be picked to attempt 130

transfonnational rescue ofthe mutant defect. With the aid ofmy genetic mapping wor~ • clk-l and gro-l have now been cloned. clk-2 is weil positioned and efforts are now underway to clone this gene as weIl. clk-3lies in a region ofthe genome in which it is

more difficult to clone genes due to a paucity ofgenetic markers and poor cosmid

coverage.

The physicallocatioD ofclk-l: The genetic mapping ofclk-l placed this gene

in a approximately SOOkb region ofChromosome m bet\veen the cloned genes ced-4 and

daf-4 (subsequently both dpy-17 and lon-l have been cloned). In this region ofLGIII

there is a Iinear relationship between physical distance and genetic distance (Bames et al.

1995). Based on my genetic mapping in this region and an estimate ofthe distance

between ced-4 and dpy-17 (0.2 cM), clk-l was predicted to be in the vicinity ofthe

cosmid ZC401 by linear interpolation. By injection ofa large number ofcosmids

spanning most ofthe region between ced-4 and daf-4, clk-l was localized to the cosmid

ZC400 (Ewbank et al. 1997) which overlaps ZC401.

The molecular identity ofclk-l: By transformational rescue using sub clones

and deletion constructs generated from ZC400, clk-l was cloned and shown to encode a

phylogenetically very weil conserved protein of 187-residues (Ewbank et al. 1997). The

mutations in the three alleles e2519, qm30 and qm51 were found. qm30 has 590 base pair

deletion that eliminates part ofthe second last and all ofthe last exon, while qm51 is a

point mutation which eliminates a splice acceptor site (Ewbank et al. 1997). Both of

these alleles have similar phenotypes and are putative Dull mutants (Wong 1994, Ewbank

et al 1997). A third weaker allele, e2519, has a point mutation causing a glutamic acid to • lysine alteration in a conserved residue (Ewbank et al. 1997). 131

The yeast gene encoding the homologue ofwonn CLK-l has been identified at • least twiee in genetic sereens (Proft et al. 1995, Marbois and Clarke 1996), and is known as CAT5/COQ7. CatSp/Coq7p is localized to the inner mitochondrial membrane

(Jonassen et al. 1998). cat5/coq7 mutants do not synthesize ubiquinone (coenzyme Q), a

lipid-soluble two-electron carrier, which is essential for respiration and consequently for

non-fermentative growth (Marbois and Clarke 1996, Jonassen et al. 1998). CAT5/COQ7

appears to indirectly affect gluconeogenesis through its reduced Coenzyme Q levels

(proft et al. 1996, Jonassen et al. 1998). Thus CAT5/COQ7 appears to be important in the

regulation ofmetabolism. The exact biochemical function ofthe CAT5/COQ7 is not

know, however, this appears to be evolutionarily conserved as both clk-l and its rat

homologue, cao rescue the phenotype ofcat5/coq7 yeast mutants and restore growth on

glycerol (Jonassen et al. 1996, Ewbank et al. 1997).

The physicallocatioD ofgro-l: The polymorphism qmPl. present in the

original gro-l strain and detected with the cosmid FllCll, is absent in a dpy-/7(e164)

gro-/(e2400) strain indicating the gro-l must lie to the right ofthis polymorphism (J.

Ewbank personal communication). Genetic mapping places gro-l to the left ofclk-l

indicating thatgro-l must lie in the region between FIICII and ZC400. By linear

interpolation using genetic mapping dat~ gro-/ lies 0.03 cM (or roughly 20kb) to the left

ofclk-l. 95% confidence intervals for this mapping place gro-l between 0.15-0.01 cM

(53-4 kb ) to the left ofclk-l. Recently gro-l has been cloned using this mapping data

and found to lie on the cosmid ZC395 (J. Lemieux personal communication). Efforts are

now underway to molecularly characterize this gene (J. Lemieux persona! • communication). clk-2 1 1

1 • 1 1 • 1 2 1 • 1 3 1 • 1 4 lin-}3 lin-39 mab-5 L 1 R -, 1 1 1 M .. 1 1 : ~ -400 -350 -300 -250 -200 -ISO -100 -SO 0 +SO +100 C07H6 kb __ RI3A5 T04A6

Figure 4.5: The 950/0 confidence intervals for c:lk-2 mapping, using the known physical position of lin-J9. lin-/J and mah-5 and assuming a linear relationship between physical and genetic distance in the region. The figure is drawn relative to /in- / J, with the scale in ki lobases (kb). Mapping experiments are: 1) .vnra-J 18 clk-] 0 lin- J3 t0 "nc-36, 2) clk-] 3 Ii,,-!349 IInc-32, 3) .r;nra-4 35 clk-2 3 mah-5 14 Il11c-36 and 4) sma-J 421in-39 0 clk-] 31 Il11c-36. Based on 95% confidence intervals,clk-2 should lie within the approximately 60 kb region indicated. The approximate extent of some relevant cosmids is shown below the figure.

• • 133

The physicallocation ofclk-2 and prospects for cloning: Genetic mappiog • experiments place elk-2 between sma-3 and /in-I3, and near lin-39, in the middie ofthe C. elegans homeobox cluster. Again~ in this region ofLGm there is a linear relationship

between genetic and physical distance (BarDes et al. 1995)~ so the position ofelk-2 cao be

better estimated by linear interpolation. l detennined the 95% confidence intervals for the

elk-2 mapping data to help define better the position ofelk-2 (see Fig.4.5). Ail ofthe

mapping data is consistent with the gene clk-2 beingjust to the right oflin-39 towards the

left end ofthe cosmid R13A5. B. McCright is now attempting to clone c/k-2 by

transfonnational rescue. The entire region where e/k-2 is likely to lie bas been sequence~

which should facilitate the identification ofe/k-2.

The physicallocation ofclk-3 and prospects for clonÎng: Two and three

point mapping places clk-3 on the right arm ofLGn~ 1 cM to the left ofune-52, to the

right ofrol-I and to the left ofeat-2 (see Fig. 4.4). This region ofLG II ooly contains

two cloned genes une-52 (Rogalski et al. 1993) and lin-l (J. Simske personal

communication.) and the metric (i.e. the rate ofrecombination per kb ) is different in the

regioos to the left and to the right of lin-7 (Bames et al. 1995) so it is very unreliable to

extrapolate to the physical position ofe/k-3 from the genetic data. A commonly used

approach to clone genes that lie in poorly characterized regions is to try to reflne the

mapping using Restriction Fragment Lengili Polymorphisms (RFLPs). To help clone /in-

7~ J. Simske identified a number ofpolymorphisms between the wild strains N2 and AB2

5 in the vicinity oflin-1 • The two polymorphisms he identified to the left of/in-l (C13D12

S DNA is digested with EcoRI and/or Hm and polymorphisms are detected by the foUowing cosmids • C13D12 (gaP?), C37G2 (gaPI4). F26Hll (gaP/7). C13B4(gaPI6) C13BIO(gaP?) And C54G9 (gaP?» 134

and C37G2 (gaPl4» could be useful in localizing cfk-3. In an attempt to localize clk-3 • further, J Lemieux has identified additional RFLPs to the left of/in-7 spanning Most of the physical distance between ro/-l and lin-7 (J. Lemieux persona! communication).

• 135 • Chapter 5

Summary and Conclusions

• 136

Many genes, and at least three mecbanisms determine ure span in C elegans • When 1 started doing work on aging in C. elegans a revolution was in progress. With the discovery that daf-2 lengthened life span, new interest was focused on aging

(Kenyon et al. 1993). This was partIy because there was a clear mechanism to explain

why daf-2 mutants lived long; presumably they activated part ofthe dauer program

(Kenyon et al. 1993). It was also shown that life span was amenable to genetic analysis

because the long life ofdaf-2 could be suppressed by mutations in daf-16 (Kenyon et al.

1993). In the last three years a series ofpapers bave radically changed how we think

about aging in C. elegans. FUst, it was shown that mutations in daf-23 couId also

lengthen life span and that again this effect was suppressed by daf-16 (Larsen et al. 1995).

Then it was shown that the long life ofage-l was also suppressed by daf-161inking most

long lived mutations to a common udevelopmental" pathway (Dorman et al. 1995).

Finally it was shown that age-l(hx546) bas a Daf-e phenotype at 27°C and that age-l was

alleHe to daf-23. thus simplifying this pathway (Malone et al. 1996). Until this point age­

l (hx546) remained as a bit ofan enigma; it was a mutation that robustly lengthened life

span but it was not clear how. During this time new results suggested that the long life of

spe-26 may be an artefact (Gems and Riddle 1996). This suggested that in C. elegans

there May he only a few aging genes and that these genes affect a single mechanism of

life span extension (Kenyon et al. 1993, Larsen et al. 1995, Donnan et al. 1995,

Murakami and Johnson 1996). In the meantime it was shown that mutations in clk-l

could also lengthen life span (Wong et al. 1995). So efforts were made to try to tie aging • in clk-l to this pathway (Murakami and Johnson 1996). However, the phenotype ofclk-l 137

mutants does not resemble that ofthe dauer mutants, suggesting that clk-l affects Iife • span by a different mechanism from that ofthe dauer genes (Wong et al. 1995). Througb the work presented in this thesis, l have shown that clk-l lengthens

life span by a mechanisms that is genetically distinct from that ofthe dauer genes.

Mutations in three other genes, c/k-2, c/k-3 and gro-I a111engtben life span and produce a

very sunilar phenotype to that seen in c/k-I mutants. Most Clock double mutants display

more severe timing defects than the individual mutants and can live extremely long. Most

Clock mutant and Clock double mutant strains display a strong cOflelation between the

rate ofdevelopment and mean adult life span. 1 also show that c/k-3 and gro-l lengthen

life span by a mechanism that is a1so genetically distinct from that ofthe dauer genes.

Based on the sÏInilarities in the phenotypes and the genetic interactions ofthese four

genes, we have proposed that clk-2, c/k-3 andgro-I lengthen Iife span by the same

mechanism as clk-l (Lakowski and Hekimi 1996).

1 have also mapped these four genes with respect to their closest flanking

markers. The results ofmy genetic mapping experiments have been used ta help

positionally clone c/k-I (Ewbank et al. 1997) and gro-I (unpublished) and may help in

the cloning ofc/k-2 and c/k-3. The identity ofc/k-l gives us sorne insight into how Clock

genes may affect Iife span (Ewbank et al. 1997) and will be discussed below.

It bas been shown in Many species that reducing calorie intake can significantly

lengthen life span (reviewed in Sobal and Weindruch 1996). 1show that mutations in

seven genes that affect pharyngeal pumping and thus reduce calorie intake, eat-I, eat-2,

eat-3, eat-6, eat-13, eat-18 and unc-26, cao significantly lengthen life span. Surprisingly, • not all eat mutations lengthen Iife span, perhaps because sorne mutations have too weak 138

an effect on feeding behavior or because they have deleterious pleiotropies that counteract • the effeet ofcaloric restriction on life span. However, mutations in a large number of control strains do not affect life span demonstrating that the effeet ofeat mutations on life

span is due to the reduction ofcaloric intake. l also show that eat-2 lengthens life span by

a mechanism that is genetically distinct from that ofthe dauer genes, but may share sorne

similarities with that ofclk-l. This suggests that there may he common mechanisms of

animal aging.

l have show for the tirst rime that mutations in the nine genes elk-2, dk-3, eat­

l, eat-2, eat-3, eat-6, eat-13, eat-IB and une-26 can lengthen lire span. In total, mutations

in 14 genes are DOW known to lengthen the life span ofC. elegans. l have also found that

severa! strains containing une mutations aIso harbor background mutations that lengthen

life span, suggesting that there May be Many other genes that cao affect life span. These

results suggest that life span is not only a polygenic trait, but also affected by Many

discrete physiological processes. l have also shown that life spao is extremely plastic. By

making various double mutant combinations the life span ofC. elegans cao be varied

extensively and can even be lengthened up to five fold.

Dow genes determine liCe spaD

The Dauer genes age-l, daf-2, daf-16 and daf-28: As discussed in Chapter 1,

the effect ofthe dauer genes age-l and daf-2 on life span appears to he solely mediated by

the gene daf-16 (Kenyon et al. 1993, Larsen et al. 1995, Donnan et al. 1995). It is not

clear how daf-28 affects life span but it May also modulate daf-16 activity (Malone et al.

1996). daf-16 encodes a forkhead-like transcription factor (Lin et al 1997, Ogg et al. • 1997) that presumably directly regulates the transcription ofsorne genes necessary for the 139

long life seen in dauer larvae and in age-l and daf-2 mutants (Hekimi et al. 1998). The • molecular targets ofdaf-16 are not kno~ but based 00 a oumberofstudies, daf-16 appears to control the response to Many stresses in C. elegans, including oxidative stress.

age-l and daf-2 mutants have elevated activity ofthe enzymes superoxide dismutase and

catalase relative to the wild type late in life (Larsen 1993, Vantleteren and de Vreese

1995). Thus the long life ofthese dauer mutants May he due to increased defenses against

reactive oxygen species (ROS), the chemicals that may cause aging (reviewed in Hekimi

et al. 1998).

The Clockgenes clk-l, clk-2, clk-3 and gro-l: Clock mutants display a

pleiotropic a1teœ.tion ofbehavior and developmental timing as weIl as lengthened adult

life spans (Wong et al. 1995, Lakowski and Hekimi 1996 and this thesis). The phenotype

ofthe Clock mutants is consistent with these mutants having a slower Urate ofliving".

The effect ofClock mutations on development and life span can be largely mimicked by

raising wild type worms at a lower temperature. It is not known what causes this slower

rate ofliving but it may result from reduced metabolic rates. The gene clk-l has been

cloned and found to encode a small protein ofunknown biochemical fonction (Ewbank et

al. 1997). Similar gene sequences have been identified in yeast and mammals and this

gene family appears to have a conserved function (Jonassen et al. 1996, Ewbank et al.

1997). The yeast homologue ofclk-l. CAT5/COQ7, is localized to the inner

mitochondrial membrane where it is known to affect the levels ofcoenzyme Q

(ubiquinone) (Jonassen et al. 1998). Recent work in C. elegans also places clk-l in

mitochondria and clk-l mutations have been shown to reduce the rate ofrespiration ofC. • elegans mitochondria in vitro (S. Hekimi personal communication). This suggests that 140

clk-l activity is required for normal mitochondrial function and that clk-l mutations may • reduce respiration in vivo. This May explain why c/k-l mutants, and by extension cfk-2. clk-3 and gro-l mutants, live long. Lower rates ofrespiration could slow aging by

slowing the production ofROS. Slower production ofROS could lead to ROS-generated

damage accumulating more slowly and thus to more graduaI aging.

The Eat genes eat-l, eat-2 , eat-3, eat-6, eat-13, eat-18 and unc-26: Mutations in

eat genes affect pharyngeal pumping and reduce the worm's caloric intake (Avery 1993).

In other systems it has been shown that caloric restriction reduces ROS-associated

damage (reviewed in Sohal and Weindruch 1996). It has been suggested that caloric

restriction also reduces metabolic rates, however the evidence for this is mixed (SohaI and

Weindruch 1996). l have found that eat-2 extends life span by a mechanism that does not

require DAF-16 but May share something in common with clk-l. This suggests that, in

the wonn at least, caloric restriction might also lengthen life span by reducing metabolic

rates. Caloric restriction could reduce metabolic rates by down regulating the Clock

genes.

AU genes that lengthen life span in C. elegans have pleiotropies

AIl known genes that cao lengtben life span in the worm have other pleiotropies

and have important functions in regulating worm development, behavior and Metabolisme

These pleiotropic phenotypes should drastically reduce evolutionary fitness. eat

mutations reduce the food intake ofworms 50 drastically that they appear starved even

under high food conditions (Avery 1993). Many eat mutants can not eat certain types of

bacteria and die ofstarvation on these food sources (Avery 1993). Sorne eat mutations • have aIso been shown to drastically reduce brood size (Davis et al. 1996). Clock 141

mutations reduee developmental rates, feeding rates and brood size (Wong et al. 1995 and • Chapter 4). Furthermore c/k-2 andgro-l mutants are not viable at high temperatures, while c/k-l(qm30) mutants have severely reduce viability at high temperatures. The dauer

mutants age-l, daf-2 and daf-28 become dauers at high temperatures even under good

conditions for wild type growth (Riddle 1988). Mutations in daf-2 and age-l have aIso

recently been shown to reduce viability and fertility (Tissenbaum and Ruvkun 1998). The

fact that mutations in all ofthese genes drastically reduce evolutionary fitness supports

the antagonistie pleiotropy theory ofaging and suggests that their affect on life span is

probably indirect.

Most ofthese mutations were actually isolated on the basis ofsorne pleiotropic

character and then subsequently shown to affect life span (Riddle and Alberts 1997,

Avery 1993, Hekimi et al. 1995). Mutations in only one gene, age-l, have been found

directly in screens for long lived mutations in C. e/egans (Klass 1983, Duhon et al. 1996).

It was once thought that the original age-l aIlele, hx546, had no phenotype except for its

effect on life span, and age-l became a model for gerontogenes (Johnson and Lithgow

1992). Gerontogenes have been defined as genes that "cause aging as a result oftheir

nonnaI function" (Johnson and Lithgow 1992). However, evolutionary theory suggests

that since aging is not adaptive, that genes specifically involved in the control oflife-span

should not exist (Rattan 1995). age-l initially stood as a challenge to this view point,

however age-l bas since been found to be allelie to the dauer-defective gene daf-23 and to

be dauer-defective itselfat 27c C. age-l(hx546) bas aIso recently been shown to reduee • brood size (Tissenbaum and Ruvkun 1998). It has also been shown that ail age-l 142

phenotypes are suppressed by daf-16. AlI this evidence now points ta age-I(hx546) being • involved in the control ofdauer fonnation. There is no evidence in C. elegans for genes that specifically control aging.

However, the pleiotropies ofgenes that affect life span can be very informative. The

genetie and phenotypic analysis ofeat, Clock and Dauer genes has shed Iight on how

these genes detetmine life span (reviewed in Hekimi et al. 1998, this thesis). Sînce ail

genes that cao lengthen life span are likely ta be pleiotropic, the pleiotropies ofnewly

identified uagingn genes should be actively examined. Only by understanding the biology

ofthese mutants cao the effect ofthe mutation on life span be properly understood.

Implications ofthis work for the study ofhumo aging

The link between aging in clk-J mutants and calorie restriction suggests that there

may be a universal mechanism ofaging and that studying invertebrates may tell us about

this general mechanism ofaging. Results from work in Many systems, including C.

elegans are consistent with free radicals produced in the process ofrespiration being

major factors in aging.

l have shown that there are Many genes that affect longevity in C. elegans. This

suggests that the genetie hasis oflongevity in mammals could he extremely complexe

Based on work in C. elegans on the dauer, eat and Clock mutants, genes that affect stress

responses, the ability ta acquire, ingest or digest food or respiration and mitochondrial

function could also play a raIe in human aging. • 143

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