Diverse Caenorhabditis elegans genes that are upregulated in dauer larvae also show elevated...

doi:10.1006/jmbi.2000.3880 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 433±448

Diverse Caenorhabditis elegans Genes that areUpregulated in Dauer Larvae also Show ElevatedTranscript Levels in Long-lived, Aged, orStarved Adults

Vera Cherkasova, Srinivas Ayyadevara, Nejat Egilmezand Robert Shmookler Reis*

Departments of Geriatrics,Medicine, and Biochemistry &Molecular Biology, Universityof Arkansas for MedicalSciences, and Central ArkansasVeterans Health Care System -Research 151, 4300 West 7thStreet Little Rock, AR72205, USA

Present addresses: V. CherkasovaSchool of Medicine, Dept. of BiologMolecular Pharmacology, 240 LongBoston, MA 02115, USA; N. EgilmeMolecular Immunology, Roswell PaElm & Carlton Streets, Buffalo NY

Abbreviations used: cM, centiMobinding protein.

E-mail address of the [email protected]

0022-2836/00/030433±16 $35.00/0

Under adverse conditions, the nematode Caenorhabditis elegans undergoesreversible developmental arrest as dauer larvae, an alternative third lar-val stage adapted for dispersal and long-term survival. Following sucharrest, which may exceed three times their usual life-span, worms resumedevelopment to form reproductive adults of normal subsequent longev-ity. Mutations of genes in the dauer-formation (daf) pathway can extendlife-span two- to fourfold, even in adults that mature without diapause.To identify transcript-level changes that might contribute to extendedsurvival, we prepared a subtractive cDNA library of messages moreabundant in dauer than in non-dauer (L3) larvae. Six genes were con-®rmed as three- to ninefold upregulated in dauer larvae, after correctionfor mRNA load: genes encoding poly(A)-binding protein (PABP), heat-shock proteins hsp70 and hsp90, and three novel genes of uncertain func-tion. The novel genes encode a partial homologue of human activatingsignal cointegrator 1 (ASC-1), a GTP-binding homologue of a ribosomalprotein, and an SH3-domain protein. Transcript levels for all except hsp70increased during aging in two C. elegans strains, whereas the three novelgenes (and possibly PABP) were also induced to varying degrees by star-vation of adults. All six genes are expressed at higher levels in youngadults of long-lived daf mutant strains than in normal-longevity controls,suggesting that increased expression of these genes may play a protectivefunction, thus favoring survival in diverse contexts.

# 2000 Academic Press

Keywords: nematode; aging; senescence; development; diapause
*Corresponding author
Introduction

Development of Caenorhabditis elegans proceedsthrough four larval stages (L1-L4), punctuated bymolts, to the reproductively mature adult. Larvaeat the late L1 stage can respond to adverseenvironmental stimuli (food restriction and/or

, Harvard Universityical Chemistry &wood Avenue,z, Department ofrk Cancer Institute,

14263, USA.rgan; PABP, poly(A)-

ing author:

crowding) by pursuing an alternative developmen-tal pathway that arrests at a modi®ed third larvalstage, the dauer or ``enduring'' larva. Dauer larvaeare specialized for stress-resistance, extended survi-val, and dispersal (Wadsworth & Riddle, 1989);when favorable conditions return, however, theycan resume development to the adult. Dauer larvaecease feeding entirely, are intermittently motile,and have reduced metabolic activity (Klass &Hirsh, 1976; Riddle, 1988; Wadsworth & Riddle,1988; Anderson, 1978).

Several physiological and metabolic changeshave been demonstrated to be associated withentry into and exit from the dauer stage, includingsubstantial changes in intracellular pH and energymetabolism (Wadsworth & Riddle, 1988, 1989).After the L1 molt, metabolic pathways divergebetween predauer (L2d) and non-dauer (L2)

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434 Genes Upregulated in C. elegans Dauer Larvae

worms. Whereas L2 larvae committed to growthswitch from the glyoxylate pathway to the citricacid cycle, and show marked increases in isocitratedehydrogenase activity and in levels of ATP andother high-energy phosphates, this transition doesnot occur in L2d larvae destined to form dauers(Wadsworth & Riddle, 1989).

Genetic studies of dauer formation have focusedprimarily on the identi®cation of genes that affectthe switch in L1, initially directing development tothe dauer pathway, and genes in¯uencing morpho-genesis of dauer larvae. Two classes of recessivedaf (dauer formation) mutants have been analyzed:dauer-defective (daf-d), which fail to enter thedauer pathway despite induction by pheromone,and dauer-constitutive (daf-c), which commit todauer-formation at restrictive temperatures even inthe absence of induction (Riddle, 1988). Abranched dauer-formation pathway has beeninferred from epistatic interactions among mutants,and comprises alternating stimulatory and inhibi-tory effectors (Vowels & Thomas, 1992; Thomaset al., 1993; Gottlieb & Ruvkun, 1994; Larsen et al.,1995). Certain daf gene products have been shownto participate in signal transduction (Georgi et al.,1990; Estevez et al., 1993), while temporal aspectsof dauer formation are regulated by heterochronicgenes (Liu & Ambros, 1989).

Dauer arrest, over periods of 0-60 days, does notreduce adult life-span following resumption ofdevelopment (Klass & Hirsh, 1976). Since star-vation of larvae at other stages can also arrestdevelopment for more limited periods (Johnsonet al., 1984), such experiments have long been usedas evidence that aging commences only after com-pletion of development. More recently, however,mutations in the genetic program that initiatesdauer formation have been shown to greatlyextend the longevity of adult worms withoutdevelopmental arrest. Several dauer-constitutivemutants (e.g. daf-2, age1 (previously known as daf-23)), which at 25.5 �C form dauer larvae even with-out pheromone induction, develop at a permissivetemperature (15-20 �C) into adults that survive fortwice the normal life-span (Kenyon et al., 1993;Larsen et al., 1995; Morris et al., 1996). Moreover,certain double mutants have life-spans extendedby at least threefold (Larsen et al., 1995; Lakowski& Hekimi 1996). Thus, the same molecular mech-anisms that exempt the dauer larvae from agingappear to govern adult longevity. The age-1mutation, which confers stress resistance andincreased life-span, encodes a PI-3 kinase (Morriset al., 1996). This functions just downstream of daf-2, identi®ed as a nematode homologue of humangenes encoding receptors for insulin and insulin-like growth factors (Kimura et al., 1997), andupstream of daf-16, which encodes a Fork headtranscription factor (Lin et al., 1997; Ogg et al.,1997). These observations suggest that metabolicattenuation in daf-2 and age-1 mutants (both asdauer larvae and as unarrested adults) results frominterruption of a signal transduction pathway:

ligand-activated daf-2 receptor acting via the age-1PI-3 kinase to generate PIP3, which then engagesmetabolic upregulation through multiple mechan-isms including a kinase cascade and transcriptionalactivation (Kimura et al., 1997; Lin et al., 1997; Ogget al., 1997).

To identify gene-expression changes in dauer lar-vae, which may underlie both their long-term sur-vival and the extended longevity of daf-mutantadults, we prepared a subtractive library of cDNAsexpressed at much higher levels in the dauer stagethan in L3. Six genes were found to be markedlyupregulated in dauer larvae, in terms of steady-state mRNA levels, and their expression was alsomonitored during aging and starvation of adults.These dur (dauer-upregulated) cDNA clones com-prise genes encoding poly(A)-binding protein,heat-shock proteins hsp70 and hsp90 and threenovel genes. The novel genes encode a homologueof human activating signal cointegrator 1 (ASC-1;Kim et al., 1999), a GTP-binding protein homolo-gous with a ribosomal protein, and an SH3-domainprotein. Five of the dur genes showed elevatedtranscript levels during adult aging, and four werealso upregulated to some degree following star-vation. Two long-lived strains bearing mutationsin the dauer formation pathway, daf-2 and daf-2;daf-12, show greater adult-stage expression of allsix dur genes than is seen in wild-type or daf-16;daf-2 worms of normal longevity. This suggeststhat products of the dur genes may play protectiveroles, contributing to survival of the adult as wellas the dauer larvae.

Results

Preparation of a subtracted cDNA library fordauer-upregulated mRNA clones

A dauer cDNA library was enriched for mRNAspecies that are much more abundant in dauer lar-vae than in L3 larvae, by two cycles of subtractivehybridization (Sive & St. John, 1988) with excessL3 mRNA. This procedure effectively depletessequences from the dauer library that are presentat comparable or greater transcript levels in L3 lar-vae. Of 240 clones analyzed on duplicate dot-blotarrays, 25 produced markedly and reproduciblygreater hybridization to dauer [32P]cDNA than toL3 probe, suggesting greater mRNA abundance inthe dauer state. These 25 clones were tested forcross-hybridization to each other in dot-blot arrays,and representative clones from each distinct (non-cross-hybridizing) set were selected for hybridiz-ation to Northern-blot ®lters.

Most subtracted-library clones correspond tomRNAs that are substantially more abundantin dauer larvae than in L3 or adult worms

Steady-state levels of transcripts putatively upre-gulated in dauer larvae were examined by hybri-dizing membrane-immobilized mRNAs, isolated

Genes Upregulated in C. elegans Dauer Larvae 435

from dauer larvae and non-dauer worms of vary-ing age, to cloned cDNA probes (excised inserts,labeled with 32P). Of 25 clones initially identi®ed asdauer-upregulated, 13 were con®rmed by quanti®-cation of multiple Northern-blot hybridizations,indicating dauer RNA levels at least threefoldhigher than in L3 larvae (e.g. see Figure 1). Basedon cross-hybridization, partial sequencing data,and sizes of hybridizing mRNA bands, these 13clones were assigned to six groups representingdistinct genes or gene families (Table 1A). Theremaining 12 cDNA clones, corresponding to 11

Figure 1. Northern-blot hybridizations of 32P-labeledcDNA clones against poly(A)� mRNA from C. eleganslarvae or adults at various ages. Autoradiographs showrepresentative hybridizations of Bristol-N2 mRNAs to aseries of labeled probes, by sequential stripping andreprobing of the same Northern-blot ®lters. Lanesmarked 4-day/st. contain mRNA from four day-oldadults that had been starved for 24 hours prior to RNApreparation. Ages are indicated as days following trans-fer of L1 larvae to feeding medium at 0-24 hours afterhatching. Bristol-N2 worms reach sexual maturity onday 4, and median mortality on day 20, at 20 �C (datanot shown, and Ebert et al., 1993).

distinct (non-cross-hybridizing) mRNA classes,produced dauer/L3 mRNA ratios of <3; theirsequences have not been determined.

32P-labeled cDNA inserts, representing each ofthe six dauer-upregulated gene families, were usedto probe multiple Northern-blot ®lters containingpoly(A)� mRNA preparations, isolated from fourindependent cohorts of worms at each of severaldevelopmental stages and adult ages. Thesehybridizations were quanti®ed by direct imagingof b-emissions, and normalized by hybridization oftotal poly(A) tracts on the same ®lters to a [32P]oli-go(dT)26 probe. In control experiments, the hybrid-ization signal was shown to vary linearly withmRNA load on the ®lters (see Materials andMethods). Poly(A)�-normalized transcript levelswere three to nine times greater in RNA prep-arations from dauer larvae than in those from L3larvae (Table 1C). Normalization of data to a con-stitutively expressed gene, cytoplasmic b-actin,implied gene upregulation by ninefold to 27-fold(Table 1B). These ratios are actually minimal esti-mates, since hybridizations to L3 samples wereweak to undetectable (Figure 1), resulting in bandquanti®cations that may consist largely of back-ground noise. The analyses were replicated formultiple RNA preparations and hybridizations,allowing the statistical signi®cance of the upregula-tion to be evaluated (see con®dence intervals (95 %CI) in Table 1).

Quanti®cations for the tested genes are summar-ized in the histograms of Figure 2. All except b-actin have been normalized in two ways: (1)adjusted for load, assessed by oligo(dT) hybridiz-ation to poly(A) tracts in mRNA, as describedabove; and (2) normalized to b-actin hybridizationresults. The relative abundances of mRNA mol-ecules per animal or per cell are best representedby steady-state transcript levels normalized to acontrol ``housekeeping'' gene such as b-actin,rather than to a constant load of mRNA (see Dis-cussion). Nevertheless, load-normalized data aregiven below (unless otherwise stated) since thisproduces a smaller apparent change with dauer-formation or aging.

The dauer-upregulated clones hybridized tosimilar extents with mRNA from dauer larvae of adaf-7 dauer-constitutive mutant (CB1372) inducedby growth at 25.5 �C (data not shown), as withmRNA preparations from wild-type (Bristol-N2)dauer larvae induced at high population density.This implies that high expression of these clones isa characteristic of the dauer state rather than aresponse to a particular means of dauer induction.

Identification and characterization of cDNAclones from the dauer-enriched library

DNA sequences, determined for at least onemember of each distinct group of clones from thedauer-enriched cDNA library, were used to screenGenBank and EMBL databases by FASTAand BLAST searches, and to screen additional

Figure 2 (legend opposite)


Table 1. Clones tested for enhanced dauer expression

Reference genes

hsp70 hsp90 PABP dur123 dur135 dur191 b-act.a cit. syn.a EF-1aa

A. Characterization of clonesNo. clones: 4 4 1 2 1 1 - - -mRNA size (kb) 2.3 2.5 2.6 1.65 2.4, 1.8,1.5 2.8 1.5 2.3 2.1B. Dauer/L3 (hybridizations normalized to b-actin mRNA)Dau-1/L3 5.0 7.2 3.1 3.9 6.5 6.0 1 0.9 3.6Dau-2/L3 11.0 24.0 10.0 14.0 13.0 33.0 1 1.7 9.5Dau-3/L3 8.5 26 11.0 19.0 15.0 25.0 1 3.3 13.0Dau-2,3/L3 mean (n) 9.3 (3) 26 (4) 11.0 (4) 17.0 (3) 15.0 (3) 27.0 (4) 1 2.5 (3) 11.0 (2)Dau-2,3/L3, 95 % CI 6.6-12 24-31 8.8-16 12-29 13-16 23-41 ± 1.7-3.6 8.9-14C. Dauer/L3 (normalized to poly(A)� mRNA)Dau-1/L3 3.1 5.1 3.0 3.1 9.4 9.0 0.79 0.6 2.8Dau-2/L3 4.2 9.1 4.0 5.2 5.0 12.6 0.38 0.6 3.6Dau-3/L3 2.3 8.8 3.1 5.6 4.2 7.3 0.33 1.1 3.7Dau-2,3/L3 mean (n) 2.9 (3) 8.8 (4) 3.3 (4) 5.5 (3) 4.5 (3) 8.7 (4) 0.34 (4) 0.90 (3) 3.6 (2)Dau-2,3/L3 95 % CI 1.8-4.2 7.7-11.2 2.8-4.8 3.8-7.3 3.9-5.0 6.8-12.9 0.30-0.39 0.60-1.3 3.6-3.7

Dauer/L3 ratios were determined from paired transcript values for the two stages, measured in the same experiment. The 95 %con®dence intervals (CI) were calculated assuming a log-normal distribution, as expected for the ratio of two normally distributedvariables, for pooled Dauer-2/L3 and Dauer-3/L3 data. Dauer-1/L3 values were outside these intervals for ®ve of the nine genestested, when normalized to oligo(dT) hybridization, and for all genes tested when data are normalized to b-actin. The number ofindependent cohorts of worms grown and harvested for RNA, and then analyzed on separate Northern blots, is given as n.

a Clones from C. elegans cDNA library (Waterston et al., 1992): b-act., cytoplasmic b-actin gene (clone cm13d3); cit. syn., citratesynthase gene (clone cm10f11); EF-1a, elongation factor-1a gene (clone cm12b2).


completed and un®nished C. elegans genomicsequences by BLAST high-stringency searching.Three genes were identi®ed by sequence similarityto previously determined DNA sequences, whereasan additional three genes could not be identi®edunambiguously by similarity to known DNAsequences (Table 1A). Each cDNA sequence wasthen analyzed for long open reading frames, andtranslated into inferred protein sequences, whichwere compared to the SwissProt and PIR proteindatabases, and checked for known sequencemotifs, using Genetics Computer Group (GCG,University of Wisconsin) programs.

Clones with sequence identity to known genes

The two genes most highly represented in thedur subtracted library, at four clones each, aremembers of the heat-shock protein (hsp) family.One group of four clones showed extensivesequence identity, e.g. 98 % over 149 bp, to a C. ele-gans cDNA homologous to the hsp70A gene ofother species (GenBank locus CELHSP70), whileanother set of four clones comprises overlappingpartial cDNA sequences with 97-99 % identity with

Figure 2. Effects of dauer-formation, starvation, and agingsubtracted-library clones and three reference gene clones. Daation to [32P]oligo(dT) probe, and (excepting b-actin itself)Bristol-N2 dauer larvae to L3 larvae; ST(4-d)/L3, ratio of fouvest, to L3 larvae; 4-day/L3, 15-day/L3, 20-day/L3, ratios ofvival is 20 days. TJ1061, 4-day, 10-day/4-day, 12-day/4-daystrain TJ1061, in adult worms at four days (set to 1), ten daybars indicate �S.E.M. ranges for independent worms cultufrom each culture as a datum point. Since these are not exintervals indicated in Tables 1 and 2 provide better indices o

members of the C. elegans hsp86/90 gene family(McCombie et al., 1992), such as locus CELXT0056.The dur141 insert sequence is similar to that of thehuman gene for polyadenylate binding protein(PABP) (68 % sequence identity over 197 bp), andnearly identical with a previously isolated C. ele-gans cDNA (GenBank, CELXT00272) with markedhomology to PABP genes from several species.

Novel cDNA clones

Three cloned inserts from the dauer-speci®c sub-traction library could not be identi®ed unambigu-ously with known genes by nucleotide or aminoacid sequence similarities in the GenBank, EMBL,PIR, or SwissProt databases, despite determinationof 993 nt of dur123, 732 nt of dur135, and 781 nt ofdur191. The complete genomic sequences bestmatching these cDNA clones were translated andused for further cognate-gene and motif searches.

Clone dur123 matches multiple loci in the geno-mic sequence, including sequences tandemly dupli-cated both on chromosome II (Y53F4B; BLASTN-2.0MP p < 10ÿ77) and on chromosome I (F46A8,BLASTN p < 10ÿ65). Additional strong matches

on C. elegans transcript levels. Summary of data for sixta are normalized both relative to poly(A)-tract hybridiz-relative to b-actin transcript levels. Dauer/L3, ratio ofr day (adult) N2 worms, starved 24 hours prior to har-adult N2 worms at three ages, to L3 larvae; median sur-

: relative transcript abundances for the ts-double mutants (median survival), and 12 days (�20 % survival). Errorres and RNA preparations, treating the indicated ratiopected to be normally distributed, the 95 % con®dence

f reproducibility.


appear elsewhere on chromosome II (F54D10,BLASTN p < 10ÿ33) and chromosome I (F41D3,BLASTN p < 10ÿ32), indicating a small gene family.Sequencing of the Y53F4B cosmid (accession no.AL 132949), which contains the strongest hom-ology to dur123, has been completed recently, andthe corresponding ORF was predicted using theGeneFinder program. This predicted ORF encodesa protein of 432 amino acid residues with signi®-cant homology to the human ASC-1 protein, acti-vating signal cointegrator 1 (28 % identity, 44 %similarity over 428 amino acid residues; Kim et al.,1999). The highest degree of homology corre-sponds to a transactivating domain in ASC-1, con-taining a putative zinc ®nger motif, which includesbinding sites for several transcription factors, tran-scription integrators, and nuclear receptors (67 %identity, 76 % similarity over 70 amino acid resi-dues). More distant homologues of dur123 havebeen found in the yeasts Schizosaccharomyces pombeand Saccharomyces cerevisiae.

Dur135 hybridized to multiple transcripts(Figure 1). A major transcript at �1.5 kb was upre-gulated at least 4.5-fold in dauer larvae (Table 1, Band C), while 1.8 kb and 2.4 kb mRNAs appearedto be entirely speci®c to dauer larvae. A weakband at � 2.1 kb was visible in all samples exceptL3. The dur135 clone sequence matches a singleORF in the genomic sequence (accession no.Z69360, coding sequence 5 of clone F25H8;BLASTN p < 10ÿ50), which maps at �3.28 cM (nearstP44) on chromosome IV. This large ORF, com-prising 24 exons, would encode a protein chain of814 amino acid residues. Although the predictedprotein shows no consistent homology to anyknown protein sequence, it has several notable fea-tures. It contains unusually high percentages ofalanine (17 %) and glycine (13 %), primarily in a C-terminal domain that has weak homology (35-40 %identity over �200 amino acid residues) to severallate-embryonic plant proteins. The N terminus ofDur135, in contrast, has a very high proline content(39 % of 111 consecutive residues), including sev-eral runs of three and four proline residues, andthus shows homology to other proline-rich proteinssuch as myosin heavy chain and clathrin assemblyprotein.

Clone dur191 maps to chromosome IV betweennP52 and daf-1. Its sequence, matching uniquelywithin cosmid T07A9 (BLASTN p < 10ÿ154),includes a T-rich region (59/89, or 69 % T) withinthe cDNA. The single ORF encodes a protein of404 amino acid residues, which contains a domaincharacteristic of the S24E family of ribosomal pro-teins (similarity p < 10ÿ36). It also contains a seg-ment resembling a number of GTP-bindingproteins, and contains a P-loop consensussequence. These glycine-rich regions, linking a b-sheet and an a-helix, can interact with a nucleotidephosphate group (Saraste et al., 1990) and are themost conserved ATP/GTP-binding motifs. The clo-sest homolog to Dur191 is a GTP-binding proteinfrom S. cerevisiae (Lpg15p, GenBank entry

U432281; 55 % identity, 76 % similarity), which issimilar in sequence to a putative ribosomal proteinof Halobacterium cutirubrum, thought to function inprotein export (Shimmin & Dennis 1989). A humanhomolog, gi/3153873, shows 39 % identity and52 % similarity to the C. elegans protein.

Expression of dauer-upregulated genes isstable during extended dauer arrest (3-30 days)

To evaluate the effect of prolonged times in thedauer state, transcript levels for ®ve dauer-upregu-lated genes were monitored over a 30-day period(Figure 3). For each gene, expression was highestimmediately after entry into dauer, declined by10-45 % at day 3, and then remained relativelyconstant through day 30.

Messages most abundant in the dauer stageare also upregulated during aging of adults

Five of the six genes identi®ed from the sub-tracted library, for which mRNA levels appearedmost strongly upregulated in dauer larvae, alsoincreased their transcript levels during aging ofadult Bristol-N2 worms. The sixth, hsp70, increasedonly with respect to b-actin mRNA (Figure 2). Foreach of these genes, the effect of aging was some-what greater at 20 days (50 % mortality) than at 15days (20 % mortality), but was signi®cant (p < 0.05)at both ages. Bristol-N2 worms were aged as large-scale synchronous populations; beginning in lateL4, they were grown in the presence of FUdR toprevent reproduction (Mitchell et al., 1979; Ebertet al., 1993). We also examined strain TJ1061, whichharbors two temperature-sensitive sterilitymutations (Fabian & Johnson, 1995), allowing syn-chronously-aging cultures to be maintained at25.5 �C without FUdR. Under these conditions,relative transcript levels were elevated with age forall six genes (Figure 4; see Figure 2 and Table 2 forquanti®cations). The increase appeared slightlygreater at ten days (50 % mortality for TJ1061 at25.5 �C) than at 12 days (80 % mortality),suggesting a possible effect of selection for survi-val. These results, taken together, indicate thatincreased mRNA levels resulted from aging per serather than FUdR treatment, and was not speci®cto the Bristol-N2 strain or to growth at a singletemperature.

Does starvation induce dauer-upregulatedmRNA species in adult worms?

Because dauer larvae cannot feed, it is possiblethat dauer-speci®c gene expression could be aresponse to the starved state. In order to minimizethe representation of starvation-response genes inthe subtracted library, L3 larvae were deprived offood for about three hours just before the RNApreparation. Since this precaution would notentirely prevent isolation of such genes, Northern-blot membranes included mRNA samples from

Figure 3. Expression of dur clones during varyingintervals in the dauer state. Histograms show relativemRNA expression (ratio of dur clone hybridization tooligo(dT) hybridization) for six dauer-upregulatedclones, probing poly(A)� mRNAs from Bristol-N2 dauerlarvae at 0.2-30 days from the time at which >95 % ofworms were SDS-resistant, indicating dauer formation.Error bars indicate �S.E.M. for two probings each, withtwo hsp90 clones.

Figure 4. Northern-blot hybridizations of 32P-labeledcDNA clones against poly(A)� mRNA from C. elegansTJ1061 adults at various ages. Autoradiographs showrepresentative hybridizations of poly(A)� mRNAs to aseries of labeled probes (the six subtractive-libraryclones and b-actin), by sequential stripping and reprob-ing of the same Northern-blot ®lters. Ages are indicatedas days from transfer to feeding medium, 0-24 hoursafter hatching. TJ1061 worms grown at 25.5 �C are atroughly their median longevity on day 10 (data notshown). Quanti®cations of these hybridizations are pre-sented in Table 2 and Figure 2.


adult worms that had been deprived of food for 24hours prior to RNA preparation, for comparison tosamples from unstarved adults of the same ageand strain. Only one of the tested genes, dur123,was as strongly induced by starvation (four- to®vefold) as by aging of adults. Transcripts corre-sponding to two other clones, dur135 and dur191,were moderately elevated (1.7 to 1.9-fold) by star-vation, while PABP transcripts increased only 1.3-fold (each p < 0.05; see Table 2). No mortality was

associated with this interval of food removal;indeed, adult life-span is increased by continuousgrowth in medium containing 10 % of the usualbacterial concentration (Klass, 1977), or in axenicmedium (Mitchell et al., 1979).

Effects of dauer formation, aging andstarvation, on other C. elegans genes

Several known C. elegans cDNA clones (kindlyprovided by Ratna Shownkeen, MRC, Cambridge,UK; see Waterston et al., 1992) were also 32P-labeled and hybridized to the panels of poly(A)�

mRNAs, for comparison to clones in the subtractedlibrary. Hybridizations to a cytoplasmic b-actingene clone indicated only one-third as much b-actin mRNA in dauer preparations as in L3 larvaeor young adults (Figure 1), consistent with priorobservations of reduced b-actin transcription indauer larvae relative to adults (Dalley & Golomb,1992). Transcripts of the b-actin gene also declined

Table 2. Effects of starvation and senescence

A. Ratios of transcript levels, normalized to b-actin mRNAhsp70 hsp90 PABP dur123 dur135 dur191 b-actin cit.syn. EF-1a

(1) Starved/unstarved ratio: 4-day Bristol-N2 adults at 20�CMean (n) 0.85 (2) 1.0 (4) 1.5 (3) 4.8 (4) 1.9 (2) 2.2 ((3) 1 0.52 (2) 1.2 ((1)95% CI 0.78-0.92 0.98-1.1 1.5-1.5 4.7-5.0 1.5-2.3 1.7-2.5 ± 0.49-0.54(2) Effects of aging in Bristol-N2 adults at 20�C: 15-day/4-day ratioMean (n) 1.5 (2) 2.8 (3) 2.4 (3) 5.1 (3) 4.4 (2) 3.2 (3) 1 1.5 (2) 1.7 (1)95% CI 1.2-1.9 2.0-3.7 1.8-2.9 3.9-7.2 3.6-5.3 2.3-4.1 ± 1.4-1.6(3) Effects of aging in Bristol-N2 adults at 20�C: 20-day/4-day ratioMean (n) 1.8 (2) 3.6 (3) 3.1 (3) 6.5 (2) 7.8 (2) 4.0 (3) 1 1.2 (2) 2.4 (1)95% CI 1.2-2.6 2.4-4.9 1.9-4.4 3.6-10 4.3-12 2.6-5.4 ± 1.0-1.4(4) Effects of aging in TJ1061 at 25.5�C: 10-day/4-day and 12-day/4-day ratios10/4-day 2.6 2.3 1.5 4.2 2.4 2.5 112/4-day 4.1 4.1 2.7 8.8 4.4 4.2 1

B. Ratios of transcript levels, normalized to poly(A)� mRNAhsp70 hsp90 PABP dur123 dur135 dur191 b-actin cit.syn. EF-1-a

(5) Starved/unstarved ratio: 4-day-old Bristol-N2 adults at 20 �CMean (n) 0.70 (2) 0.93 (4) 1.3 (3) 4.3 (4) 1.7 (2) 1.9 (3) 0.90 (4) 0.65 (2) 1.0 (1)95% CI 0.55-0.86 0.85-1.1 1.2-1.5 3.9-4.4 1.6-1.9 1.7-2.0 0.80-0.93 0.40-0.95(6) Effects of aging in Bristol-N2 adults at 20 �C: 15-day/4-day ratioMean (n) 0.69 (2) 1.6 (3) 1.4 (3) 3.0 (3) 2.0 (2) 1.9 (3) 0.62 (3) 0.80 (2) 0.82 (1)95% CI 0.48-0.93 1.4-1.8 1.1-1.6 2.6-3.5 1.5-2.6 1.1-2.8 0.36-0.89 0.57-1.4(7) Effects of aging in Bristol-N2 adults at 20 �C: 20-day/4-day ratioMean (n) 0.73 (2) 1.9 (3) 1.5 (3) 3.4 (2) 3.1 (2) 2.0 (3) 0.60 (3) 0.6 (2) 0.87 (1)95% CI 0.60-0.88 1.8-2.0 1.4-1.6 3.1-3.6 2.1-4.2 1.5-2.6 0.38-0.85 0.35-3.1(8) Effects of aging in TJ1061 at 25.5 �C: 10-day/4-day and 12-day/4-day ratios10-day/4-day 2.3 2.1 1.3 3.7 2.1 2.2 0.8812-day/4-day 1.7 1.7 1.1 3.6 1.8 1.7 0.41

Ages refer to days after transfer to feeding medium, 0-24 h post-hatch. Median longevities in these experiments were �20 days forBristol-N2 at 20 �C, and �10 days for TJ1061 at 25.5 �C. Ratios were determined for paired transcript values within the same ®lter(e.g. 15-day/4-day), after normalization of each value for ®lter rehybridization to either b-actin (A) or oligo[dT] (B). A (1-3) and B (5-7): The 95% con®dence intervals (95% CI) were determined using log-transformed values, of data derived from four independentRNA preparations and hybridizations for each age or developmental stage. Not all samples were present or quanti®able on every ®l-ter, resulting in variable n values as indicated. A(4) and B(8): TJ1061 data represent single RNA preparations at each of three ages.


by 38-40 % during aging of Bristol-N2 worms, butwere unaffected by starvation (Table 2). In strainTJ1061, b-actin transcripts decreased by only 12 %at the median life-span (about ten days at 25.5 �C),but fell 59 % in those worms remaining alive at 12days (20 % of the survival population; see Table 2and Figure 2).

Citrate synthase was examined as an indicator ofmetabolic-pathway activity, since it participates inboth the glyoxylate cycle (utilized by embryos, L1,L2d, and dauer larvae), and the citric acid cycle,which replaces it in L2-L4 larvae and adults(Wadsworth & Riddle, 1989). Citrate synthase tran-script levels were not signi®cantly altered by dauerformation or aging of adults, but fell �35 % instarved young adults (Tables 1 and 2).

A C. elegans clone corresponding to elongationfactor 1 alpha (EF-1a) was also tested, since thecorresponding mRNA levels have been reported toincrease during aging of C. elegans (Fabian &Johnson, 1995), but to decrease or remain constantin aging Drosophila (Shikama et al., 1994; Shikama& Brack 1996). We found 3.6-fold higherexpression of EF-1a in C. elegans dauer larvae thanin L3 larvae; transcript levels declined slightly (13-18 %) during aging of adults, but were not per-turbed by starvation (Tables 1 and 2).

Dauer-upregulated genes are also induced inlong-lived dauer-pathway mutants

A number of temperature-sensitive mutations inthe dauer-formation pathway also confer greatlyextended life-span to adult worms that hadmatured at a permissive temperature (Kenyon et al.,1993; Larsen et al., 1995; Tissenbaum & Ruvkun,1998). We tested Bristol-N2 (wild-type) and thedauer-pathway mutants daf-2(e1391) (mean survi-val 3.8� wild-type at 25 �C; Tissenbaum &Ruvkun, 1998), daf-2(e1370);daf-12 (mean survival3.5� wild-type at 25 �C; Larsen et al., 1995), anddaf-16;daf-2 (mean survival the same as wild-typeat 25 �C; Larsen et al., 1995), to determine whetherincreased expression of dur genes, characteristic ofthe dauer stage, is found in young adults (day 5)of long-lived mutant strains. For all dauer-upregu-lated genes tested, hsp70, hsp90, PABP, dur123,dur135, and dur191, we observed markedlyincreased transcript levels in the long-livedmutants relative to the normal-longevity controlstrains (Figure 5). Expression of dur123 and dur135was not detectable in young worms of normallongevity (Bristol N2 and daf-16;daf-2) but appearedin long-lived mutants daf-2 and daf-2;daf-12. Theother four dur clones appeared upregulated bytwo- to fourfold in the long-lived mutants, relative

Figure 5. Study of expression of the dur genes in thedauer pathway mutants. Expression of six dur genesand b-actin was assayed as described for Figure 4,except that total RNA was used in this gel (8 mg/lane),rather than poly(A)� mRNA. All worms were ®ve day-old adults, grown at 20 �C.


to the two control strains, when normalized tocytoplasmic b-actin as a reference gene. Similarresults were obtained for two independent exper-iments using separate expansions of each strain.These data suggest that enhanced dur geneexpression may be functionally related to extendedlongevity.

Discussion

Normalization of transcript levels

The evaluation of mRNA steady-state levels byNorthern-blot analysis usually involves normaliza-tion of hybridization signals with respect to a con-trol RNA species. Ideally, results would becorrected to re¯ect mRNA molecules per cell, butthis is an elusive goal in practice. Rehybridizationof ®lters to cloned cDNA probes representing``housekeeping'' genes such as b-actin is a common

procedure (Sambrook et al., 1989) to normalizeloads of total RNA or of partially puri®ed poly(A)�

mRNA. Hybridization to [32P]oligo(dT) permitsadjustment for uneven loading of poly(A)�

mRNAs (Harley, 1987; Dalley & Golomb, 1992) butmay be misleading in developmental or agingcomparisons, since constant mRNA loads can rep-resent very different numbers of nematodes orcells. Cell number increases little in C. elegansbetween L3 and late adulthood, whereas growth issubstantial and continuous over this period; it isachieved primarily through increased cell volume,accompanied by a parallel increase in RNA con-tent. A ®xed load of poly(A)� mRNA would thuscontain more worm-equivalents of mRNA fromyoung than aged adults, leading to under-esti-mation of age-dependent upregulation per wormor per cell. In the histograms of Figure 2, mRNAhybridization results for each clone have been nor-malized to the corresponding levels of [32P]oli-go(dT) hybridization, and also relative to transcriptlevels of cytoplasmic b-actin. The choice of b-actinis arbitrary, since none of the reference genes main-tained constant transcript levels in all comparisons(Figure 2). This correction, which may reduce sys-tematic bias during development and subsequentgrowth (see above), also confers lower coef®cientsof variation (SD/mean) than hybridization tooligo(dT) probe (e.g. see Tables 1 and 2), whichintroduces far more background noise due to thediffuse size distribution of poly(A)� mRNA. Theeffect of normalization mode on our results may besummarized as follows. When data are adjusted bypoly(A)� mRNA content, the dauer/L3 ratios forthe six dur clones and EF-1a range from 3 to 9, athird of the actin-normalized ratios (9 to 27); incontrast, the consequences of starvation are essen-tially unaffected by the mode of normalization.Age-dependent changes are attenuated to an inter-mediate degree when normalized to poly(A)�

mRNA (see Table 2). Under either treatment of thedata, however, the same ®ve of the six subtracted-library clones showed the largest transcriptelevations, for each C. elegans strain tested at twoadvanced ages. In Bristol-N2, hsp70 was the leastenhanced (or instead decreased) with senescence,while in strain TJ1061, PABP showed the smallestincrease.

Implications of enhanced heat-shock geneexpression in dauer larvae

In most eucaryotes, including nematodes, heatshock and other environmental stresses inducecoordinate expression of stress-response genesincluding hsp16, hsp70 and hsp86/90. Even withoutheat shock or other stress exposure, dauer larvaeand aged adults express elevated levels of hsp86/90mRNA (Figure 1 and Dalley & Golomb, 1992),although the other components of the generalstress response are not coordinately induced.Expression of hsp70 is three times greater in dauerlarvae than in L3, but declines slightly with aging


or starvation of N2 adults; it was elevated abouttwofold in aging TJ1061 adults at 25.5 �C (Tables 1and 2). hsp16 expression does not occur at detect-able levels in dauer larvae (Dalley & Golomb,1992), and has not been evaluated in aging adults.

Hsp90 proteins are believed to serve as chaper-ones for many proteins, including a variety of pro-tein kinases (Craig et al., 1993; Hendrick & Hartl,1993). Moreover, Hsp90 interacts with steroid hor-mone receptors to facilitate receptor activation bysteroids, and to prevent transcriptional activationin the absence of steroids (Craig et al., 1993;Hendrick & Hartl, 1993). The elevation of hsp86/90transcript levels, in both dauer larvae and olderworms, may re¯ect ``conservational'' functions ofHsp90 proteins required in both states, e.g. stabil-ization of proteins to delay their degradation,and/or attenuation of transcriptional activation.Although such functional roles are speculative atthis point, it is of interest that two heat-shock pro-teins (Hsp26 and Hsp84) are induced in sporula-tion of yeast and in oogenesis of Drosophila (Kurtzet al., 1986), both of which mark transitions tolong-term cell survival in a temporarily non-divid-ing state.

Hsp90 expression is known to be regulatedthrough mRNA stabilization (Dalley & Golomb,1992); this may be mediated in part by poly(A)-binding protein (PABP), for which transcripts aremore abundant in the dauer stage than at L3.PABPs regulate message stability and translationalef®ciency (Sachs & Deardorff, 1992; Stambuk &Moon, 1992), and thus could serve to reduce meta-bolic demands due to rapid mRNA turnover andinactive polysomal ribosomes.

In evaluating the elevated transcript levels ofheat-shock and other genes in dauer larvae, weconsidered the possibility that they may have beenstress-induced by the treatment with detergent(1 % (w/v) SDS) used to isolate pure dauer popu-lations. Increasing the recovery time between SDSexposure and RNA preparation to three days(Dauer-2), from about three hours allowed in otherpreparations (Dauer-3), did not reduce the abun-dance of any dur-homologous mRNA (Table 1),although expression of heat-shock mRNAs hasbeen shown to peak about two hours after induc-tion and returns to baseline levels within six hours(Dalley & Golomb, 1992). In another group ofdauer larvae (Dauer-1), prepared without any SDStreatment, dauer upregulation of several dur geneswas moderately reduced relative to Dauer-2 andDauer-3 (Table 1), as would be expected for a less-pure dauer population; it remained in the three- toninefold range, however, for all six genes. Thus,the results for both control preparations (Dauer-1and Dauer-2) argue against a stress response as theprincipal mechanism for dur gene upregulation indauer larvae. This conclusion is consistent withprevious observations of increased hsp90 and hsp70transcript levels in dauer larvae prepared by ®l-tration, relative to L3 larvae (Dalley & Golomb,1992).

Features of the novel dauer-upregulated genes

The gene represented by cDNA clone dur123was the most profoundly affected by starvation.Several related genes or pseudogenes are found onchromosomes I and II, suggesting a multigenefamily. The predicted ORF of the best-matchinglocus encodes a homologue of human activatingsignal cointegrator 1 (ASC-1; Kim et al., 1999).ASC-1 is a transcription coactivator of nuclearreceptors, which associates with speci®c com-ponents of the RNA polymerase II complex, withnuclear receptors, and with transcription integra-tors SRC-1 (Onate et al., 1995; Yao et al., 1996) andCBP-p300 (Goldman et al., 1997) via a putative zinc®nger motif. ASC-1 is thought to play an importantrole in establishing distinct coactivator complexesunder different cellular conditions (serum star-vation, for example). The putative zinc ®nger motifin ASC-1 is suf®cient for activation of transcription,and this motif shows the strongest homology tothe predicted dur123 ORF. This suggests thatDur123 may participate in activation of transcrip-tional complexes speci®c to the response to star-vation, including dauer larvae (which do not feed),and aging worms, which may undergo chronicpartial starvation due to slowed pharyngeal pump-ing. Since transcriptional coactivators are thoughtto form distinct complexes, thus activating andintegrating various signaling pathways, we cannotexclude the possibility that Dur123 may have bothoverlapping and distinct functions during star-vation, aging and dauer formation. It would thusbe informative to identify other dauer-formationpathway proteins with which Dur123 interacts.

Dur135 detected multiple bands on Northernblots, two of which were not seen in any non-dauer RNA preparations. Such multiple transcriptsmay represent products of alternative RNA spli-cing, given that the cloned probe lies near the 30-end of the dur135 gene (data not shown). Thedur135 sequence matches well to only one locus inthe C. elegans genomic sequence, thus precludingthe possibility of a multigene family. The encodedDur135 protein, although lacking substantial iden-tity with previously reported protein sequences,shows weak but extended homology with someplant embryonic proteins near its C terminus,where alanine and glycine-rich domains are situ-ated. Glycine confers high ¯exibility in proteinfolding and thus allow kinks in polypeptide chains(Schulz & Schirmer, 1979), and may also play arole in protein-protein interactions (Bandziulis et al.,1989). The N terminus of Dur135 contains manyproline residues, some of which ®t the pattern of aminimum consensus sequence for SH3 (Src hom-ology 3) binding domains (Ren et al., 1993). Unfor-tunately, these features provide insuf®cient cluesas to the function of Dur135, although the presenceof dauer-speci®c transcripts suggests some rolespeci®c to the dauer stage.

The dur191-encoded protein sequence containsan unusual domain typical of ribosomal proteins,


and a P-loop motif suggesting binding of ATP orGTP (Saraste et al., 1990). Moreover, its closesthomologs are a yeast GTP-binding protein and anarchaebacterial protein thought to be ribosomaland to function in protein export (Shimmin &Dennis, 1989). Ribosomal activity and ``docking''may well be regulated in accord with the verydifferent energetic priorities of dauer versus repro-ductive stages, possibly via ATP or GTP binding;such a role for Dur191 remains speculative at pre-sent, but would be consistent with modulation ofdur191 expression by food intake. Increasedexpression of several genes encoding GTP-bindingproteins, including several of unknown function,has also been reported in muscle cells from senes-cent mice (Lee et al., 1999).

Aging C. elegans adults upregulate the samegenes most elevated in dauer larvae

Surprisingly, ®ve of the six subtractive-librarygenes for which transcripts were most elevated inthe dauer stage also showed increased expression,by factors of 1.5 to 3.7, during aging of N2 andTJ1061 adults (Table 2). Nevertheless, aging adultsdid not achieve transcript levels as high as those ofdauer larvae. We also determined transcript levelsfor three reference genes, encoding EF-1a, b-actin,and citrate synthase, relative to poly(A)� mRNAload, and observed moderate declines with age(Table 2). Since an earlier study found age-depen-dent mRNA declines for nine of 11 clones from asenescent-worm cDNA library (Fabian & Johnson1995), these combined results suggest that senes-cent down-regulation of genes may be the norm inC. elegans. In screening clones that were not upre-gulated in dauer larvae, we noted that the greatmajority declined in transcript level, or remainedconstant, with senescence (data not shown). Thus,the observation of increased expression duringsenescence, for ®ve of six highly dauer-upregulatedclones, seems unlikely to be fortuitous.

Role of starvation in dauer-specificgene expression

Because dauer larvae do not feed, it is possiblethat dauer-enhanced gene expression may be aresponse to starvation. This is particularly ger-mane, since both development and senescenceappear to be transiently arrested by starvation oflarvae for up to ten days (Johnson et al., 1984), andbecause aged worms also show a markedreduction in pharyngeal pumping (Hosono et al.,1980; Johnson & Simpson, 1985).

The effect of starvation per se, on transcript levelsof dauer-upregulated and control genes, wasassessed by comparing mRNA levels in four day-old adults that had either been starved for 24hours or had been fed continuously since the L1stage. Of the cDNA clones tested, dur123 was byfar the most responsive to starvation, whichinduced a greater than fourfold increase in its tran-

scripts. dur135 and dur191 transcripts increased 1.7and 1.9-fold after starvation, whereas PABPshowed a smaller but still signi®cant response(Table 2). To the extent that these genes are induci-ble by starvation, we may have attenuated theobserved effects of dauer formation and aging byrinsing all worms free of bacteria, and then main-taining them in buffered medium for three hoursjust prior to mRNA preparation. This bacteria-freeinterval was included so as to allow digestion ofenteric Escherichia coli (which would otherwise con-tribute substantially to total RNA), and to reduceenvironmental and physiologic variation amongpreparations.

Remarkably similar results were obtained for alldauer-upregulated genes tested during extendedperiods of developmental arrest: transcript levelswere highest initially, fell moderately by day 3,and then remained essentially constant throughday 30. This pattern suggests that induction ofthese genes may form part of a dauer-speci®c pro-gram. If it were instead a secondary response tostarvation, with only the absence of feeding pro-grammed, such induction would not be expectedto decline (or even to hold steady), but rather toincrease during a period of prolonged starvationfollowing dauer formation.

Inter-relation of dauer and senescent states

Of six genes identi®ed by their high transcriptlevels in dauer larvae, relative to the L3 andyoung-adult stages, ®ve also show increasedexpression during aging. This suggests some simi-larity between the dauer stage and senescence, per-haps re¯ecting common responses to adverseconditions, environmental in the case of dauer lar-vae, but internal (physiological) conditions insenescence. For those genes also induced by star-vation, the common feature could be adaptation tofood deprivation, since dauer formation is favoredby starvation and characterized by a cessation offeeding, whereas aged worms show a markedreduction in the rate of pharyngeal pumping(Hosono et al., 1980; Johnson & Simpson, 1985). Itis not unlikely, then, that genes induced during thedauer stage, senescence and starvation, wouldinclude those involved in adaptation to caloricrestriction, a process that can extend life-spanin many species (Feuers et al., 1993) includingC. elegans (Klass, 1977), possibly by limiting thegeneration of oxidative metabolites (Feuers et al.,1993; Honda et al., 1993). A different mechanismmust account for increased hsp70 and hsp90 tran-script levels in dauer larvae, since these genes werenot induced by starvation (Table 2).

Recent observations in other laboratories indicatethat genes in the dauer pathway initiate alterationsthat can greatly extend the duration of both theadult and dauer stages. Several temperature-sensi-tive dauer-constitutive mutants, when allowed tomature at a non-permissive temperature, havegreatly extended adult life-spans over a range of


temperatures (Kenyon et al., 1993; Larsen et al.,1995; Lakowski & Hekimi, 1996). daf-2 was ident-i®ed as a member of the insulin receptor genefamily (Kimura et al., 1997), while age-1 (daf-23) is aPI-3 kinase (Morris et al., 1996), which in mamma-lian cells is an indirect substrate (via phosphory-lated IRS1 or IRS2) of the insulin receptor. Theallele-speci®city of interactions between daf-12 anddaf-2, such that some double mutants extend adultlife-span by 3.5-fold, while others have abbreviatedlongevity (Larsen et al., 1995), suggests that daf-12may encode the corresponding ligand, or a proxi-mal substrate of the receptor tyrosine kinase. daf-16appears to function downstream of daf-2 and age-1,since daf-16;daf-2 and daf-16;age-1 double mutantsshow neither a temperature-sensitive, dauer-consti-tutive phenotype nor extended longevity of adults(Kenyon et al., 1993; Larsen et al., 1995; Lin et al.,1997; Ogg et al., 1997). Identi®cation of the daf-16gene product as the Fork head transcription factor(Lin et al., 1997; Ogg et al., 1997) thus implies thatboth dauer-formation and life-span extension areeffected through transcriptional regulation. Wehave assessed the nitrogen metabolism of C. ele-gans adults by measuring their ammonia pro-duction. We found that daf-2, daf-2;daf-12, and daf-2clk-1 mutants excrete much less ammonia nitrogenthan wild-type worms, in inverse proportion totheir extended life-spans (2.0, 2.4, and 2.7� wild-type, respectively), whereas daf-16;daf-2 wormshave both normal longevity and normal ammoniaproduction (J. Thaden et al., unpublished results).This is consistent with a metabolism-regulatoryrole of daf-2 in nematodes, at least partially analo-gous to the function of insulin receptor in mamma-lian cells (Kimura et al., 1997).

Expression of all six dur genes was elevated inyoung adults of the long-lived mutants daf-2 anddaf-2;daf-12, relative to the wild-type control strain.The daf-16;daf-2 double mutant, which displaysnormal longevity, also has wild-type levels of durgene transcripts. These data suggest a causalrelationship between enhanced expression of thesegenes and increased longevity. It is possible thatthey form part of a genetic program elicited by avariety of stresses (including starvation, crowding,and senescence), to enhance survival. Furtherstudy of dauer-enhanced and dauer-speci®c tran-scripts, of which only a few, encoding cuticle pro-teins, had been reported previously (Cox et al.,1989; Sebastiano et al., 1991), should aid in theanalysis of molecular changes shared by long-liveddaf-c mutant adults and wild-type dauer larvae.The common induction of four dauer-enhancedtranscripts during starvation and normal agingsuggests that they may play similar roles in theseprocesses.

Related observations in other aginganimal systems

Intriguing parallels exist between our obser-vations on dauer-upregulated genes in C. elegans,

and molecular responses to aging in evolutionarilydistant species such as yeast, fruit-¯y and mouse.A SIR4 gain of function mutation, discovered byselection for resistance to starvation, also confersresistance to other stresses and increases yeast life-span (Kennedy et al., 1995). A number of stressresponse genes, including those encoding heat-shock proteins, have been found to be upregulatedduring aging of S. cerevisiae mother cells (D. Sin-clair & L. Guarente, personal communication), andincrease two- to threefold in muscle from senescentmice relative to young adults (Lee et al., 1999).Heat-shock genes are also induced constitutivelyduring aging of Drosophila (Fleming et al., 1988),apparently in response to accumulation of modi-®ed, partially denatured proteins (Niedzwiecki &Fleming, 1990). Thus, age-dependent upregulationof stress-response genes in diverse species mayimplicate a common protective mechanism againstdeleterious effects of aging.

Materials and Methods

Accession numbers of new sequence data

The sequences referred to herein as dur123, dur135,and dur191 have been assigned GenBank accession num-bers U52070, U52071, and U52072, respectively.

Strains utilized

Caenorhabditis elegans wild-type strain (var. Bristol-N2,wild-type), and the dauer-constitutive ts-mutant strainCB1372 daf-7 (e1372) were obtained from the Caenorhab-ditis Genetics Center (St. Paul, MN). Strain TJ1061 wasprovided by Thomas E. Johnson (University of Colorado,Boulder, CO). Strains JT5488 daf-2(e1370); daf-12(m20)and JT7098 daf-16(m26); daf-2(e1370) were provided byJames Thomas (University of Washington, Seattle), andstrain CB1391 daf-2(e1391) was provided by Gary Ruv-kun (Harvard Medical School, Boston, MA). Adults ofthe latter three strains were synchronized and harvestedat day 5 for RNA preparation, without use of FUdR (seebelow).

Growth of C. elegans and isolation of specific stages

All strains were routinely propagated at 20 �C asdescribed (Brenner, 1974; Sulston & Hodgkin, 1988;Ebert et al., 1993). Eggs isolated from gravid adults byalkaline hypochlorite treatment (Emmons et al., 1979)hatched over 24 hours without food, accumulating asgrowth-arrested L1 larvae. Unhatched eggs wereremoved by repeated settling. On addition of E. coli at109/ml, larvae resumed development with entry into theL2 stage; the bacterial concentration was kept at thislevel unless otherwise indicated. L3 larvae were isolatedafter a further 24 hours at 20 �C, whereas synchronizedadult cultures were established following addition of100-mM FUdR beginning late in L4, to inhibit productionof progeny while maintaining the normal life-span of�20 days at 20 �C (Mitchell et al., 1979; Ebert et al., 1993).The ts-sterile double mutant TJ1061 (spe-9(hc88) emb-27(g48)) (Fabian & Johnson, 1995), obtained from T.E.Johnson (University of Colorado, Boulder), allowedpreparation of synchronously aging populations without


exposure to FUdR. These worms were propagated at15 �C, and transferred as L1 larvae to 25.5 �C (non-per-missive temperature) to establish non-proliferating cul-tures with a median life-span of 10-11 days, somewhatshorter than the reported mean of 13.9 days at 25.5 �C(Fabian & Johnson, 1995). All worms were starved forabout three hours just prior to RNA isolation, in order toapproximate the metabolic state of non-feeding dauerlarvae. This interval also allows complete digestion ofpreviously ingested E. coli; even 30 minutes of food with-drawal is suf®cient to eliminate most bacterial RNAfrom these preparations (data not shown). Worms wereconcentrated by centrifugation through 30 % (w/v)sucrose (Ebert et al., 1993) before RNA preparation. Mostaged worm cultures were also centrifuged in 15 % Per-coll/sucrose step-gradients (Fabian & Johnson 1995) toeliminate dead worms; however, omission of this stephad no noticeable effect on Northern hybridizationresults (data not shown), suggesting that RNA contribu-ted by dead worms is largely degraded.

Dauer-constitutive CB1372 worms, bearing a tem-perature-sensitive daf-7 mutation, were cultured for sev-eral days in liquid culture medium at 25.5 �C (Sulston &Hodgkin, 1988). Dauer larvae arising constitutively werethen selected by resistance to SDS as described below,and harvested for RNA extraction three hours later.Dauer larvae were induced in the Bristol-N2 (wild-type)strain by growth at high population density (�60,000worms/ml) without subsequent refeeding, and were sep-arated from other stages by either of two procedures: (a)dauer larvae were sieved through a 15 mm Nitex nylon®lter, which retains larger worms, then washed repeat-edly in S buffer (Brenner, 1974) and allowed to settle bygravity to remove L1 larvae. The resulting dauer-enriched preparation was termed Dauer-1. (b) Wormswere suspended in 1 % SDS for 30 minutes, a treatmentthat is lethal to all stages except eggs and dauer larvae,and were then separated from debris by centrifugationthrough cold 30 % sucrose (Sulston & Hodgkin, 1988;Cassada & Russel, 1975). After treatment with SDS, butprior to RNA isolation, dauer larvae were maintained inS-basal medium for either three days (Dauer-2) or threehours (Dauer-3).

RNA isolation

Worms (1-2 g) were frozen in liquid nitrogen andground to a ®ne powder in a porcelain mortar cooledwith liquid nitrogen. RNA was extracted using acid phe-nol (Sambrook et al., 1989) or using the Qiagen RNAextraction kit; polyadenylated RNA was puri®ed by twocycles of binding to oligo(dT)-cellulose. The concen-tration of mRNA was determined by measurement ofabsorption at 260 nm in quartz capillary tubes, using aShimadzu spectrophotometer (model UV160U).

Construction of a subtracted cDNA library

Poly(A)� mRNA was isolated from dauer and L3 lar-vae as described above. A cDNA library was constructedusing a commercial protocol and materials (Stratagene),with an initial yield of �106 plaques, of which >95 %were recombinant. After ampli®cation in E. coli PLK-F0(recAÿ, mcrAÿ, mcrBÿ) to a titer of �1010 pfu/ml, single-stranded phage was rescued from the cDNA librariesand puri®ed (Schweinfest et al., 1990). Poly(A)� RNAfrom L3 larvae was biotinylated, and employed for sub-tractive hybridization (Sive & St. John, 1988), using a ten-

fold excess of L3 poly(A)� RNA in the ®rst subtractioncycle and the same absolute amount of poly(A)� RNA inthe second cycle. Single-stranded DNA surviving bothcycles was converted to double-stranded DNA andtransfected into E. coli strain DH5.1 (Hanahan, 1983).

Initial characterization of clones by dot-blot andNorthern-blot hybridization

Plasmid DNAs were isolated from 240 clones of thedauer-enriched library (representing �10 % of this sub-tracted clone pool) by standard procedures (Sambrooket al., 1989), and were screened in duplicate dot-blotarrays for differential hybridization to [32P]cDNA fromdauer versus L3 larvae. Inserts were excised from thoseclones most differentially represented in cDNA (dauer>L3), 32P-labeled (Feinberg & Vogelstein, 1983), andtested for cross-hybridization to other inserts; representa-tive insert fragments were then selected to probe againstNorthern-blot ®lters. Aliquots of poly(A)� RNA (0.05-0.5 mg) or of total RNA (8 mg) were electrophoresedthrough formaldehyde/1 % agarose denaturing gels(Davies et al., 1986), and transferred to nitrocellulose ®l-ters. These were hybridized under standard conditions(Sambrook et al., 1989), with 32P-labeled inserts excisedfrom selected clones. When probed with [g-32P]oligo(dT)26 (see below and Harley, 1987), ®lters werehybridized for 16 hours at 25 �C and then washed in0.2 � SSC, 0.1 % SDS at 20 �C.

Hybridization signals were quanti®ed by directimaging of b-emissions with phosphor screens and alaser-scanning photodetector (Molecular Dynamics),using ImageQuantTM software. In order to monitor load-ing, retention, and availability of poly(A)� mRNAsamples, Northern-blot ®lters were then hybridized to[g-32P]oligo(dT)26. Quanti®cations of these rehybridiza-tions, and of similarly-probed mRNA slot blots, agreedwell with poly(A)-mRNA loads estimated from absorp-tion at 260 nm.

Tests for dose-response linearity of hybridizationsignal to RNA on Northern-blot filters

We tested the linearity of hybridization to 32P-labeledprobe, for varying inputs of target RNA. Poly(A)�

mRNA from C. elegans dauer larvae was loaded in atwofold dilution series from 500 to 4 ng per lane; eachload was supplemented to a total of 0.5 mg with humanpoly(A)� RNA, which alone produced no detectablehybridization. For several dauer-upregulated clones, ofvarying mRNA abundance, a linear response to poly(A)�

mRNA input occurred over a similar response range(data not shown), extending up to inputs of 0.05 mg forgenes expressed at high levels (hsp90, dur123, dur191),and to at least 0.5 mg for genes with less-abundant tran-scripts (dur141, dur135). Northern-blot ®lters used in sub-sequent experiments contained approximately constantmRNA loads of either 0.05 or 0.5 mg/lane, as appropriatefor probing of high or low-abundance species, with theexception of Figure 5 (8 mg of total RNA/lane). In anycase, the observed departure from linearity would leadto underestimation of inter-group differences.

Statistical procedures

Differences between means of small samples weretested for statistical signi®cance by the Behrens-Fishert-test, a modi®cation of Student's t-test with increased


stringency appropriate for small sample size or forpopulations of unequal variance (Kenney & Keeping,1951). Con®dence intervals were calculated by aniterative algorithm (Microsoft Excel), following logar-ithmic transformation of ratios. Although sample sizewas insuf®cient to test goodness of ®t, a ratio of nor-mally distributed parameters will have a log-normaldistribution.

Nucleotide sequence analyses of selected clones

Nucleotide sequences of inserts from selected cloneswere determined by the dideoxy-mediated chain ter-mination method (Sanger et al., 1977), using Sequenase2.0 reagents (USBC, Cleveland OH), and were com-pared to data in the GenBank and EMBL nucleic aciddatabases to identify known DNA sequences with sig-ni®cant similarity. Polypeptide translations from clonesequences (in six reading frames) were screenedagainst the PIR and SWISSPROT protein databases todetermine related protein sequences. Searchesemployed the FASTA protocol (Pearson & Lippman,1988) or BLASTIN (Altschul et al., 1997), and GeneticsComputer Group (GCG, University of Wisconsin) soft-ware.

Analyses of clones corresponding to characterizedgenes from a C. elegans cDNA library

Over 1500 clones from a C. elegans cDNA library, con-structed in lambda SHLX2 by C.H. Martin and M. Chal-®e, were previously isolated and their 50-terminalsequences determined; of �1200 genes thus character-ized, >30 % were identi®ed by similarity to known genes(Waterston et al., 1992). Clones identi®ed as cDNAsencoding EF-1a (elongation factor 1, alpha chain: clonecm13d3), citrate synthase (cm10f11), and cytoplasmic b-actin (cm12b2), were kindly provided to us by R.Shownkeen (University of Cambridge, UK). Phagestocks, grown in E. coli strain MC1061 and prepared asplate lysates, were converted to plasmid clones by lox-mediated recombination in a P1-lysogenic host. Follow-ing ampicillin selection for plasmid retention, recoveredplasmid DNA was used to transfect E. coli strain XL1-Blue for ampli®cation; plasmid DNA was again recov-ered by standard procedures (Sambrook et al., 1989).

Hybridization to filter arrays of yeast artificialchromosome (YAC) genomic clones

Novel genes were mapped by hybridization to ®lterarrays of C. elegans genomic DNA fragments, cloned inyeast arti®cial chromosomes (YAC arrays; Coulson et al.,1988). These arrays, kindly provided by A. Coulson andR. Shownkeen (MRC, Cambridge, UK), were hybridizedfor 16-20 h at 65 �C in 6 � SCP (1 � SCP � 0.1 M NaCl,30 mM sodium phosphate buffer, and 1 mM EDTA,pH 6.2), containing 0.9 % (v/v) Sarkosyl. Filters werethen washed three or four times in 0.5 � SCP, 0.5 % SDSat 50 �C, cooling to room temperature over 20 minutes.

Acknowledgments

This work was supported by grant R01-AG09413 fromthe National Institute on Aging. Several worm stockswere supplied by the Caenorhabditis Genetics Center,

funded by the NIH National Center for ResearchResources. We thank Sergey Sokol (Harvard MedicalSchool, Boston MA) for kindly providing laboratoryspace for several experiments; Thomas E. Johnson (Uni-versity of Colorado, Boulder) for providing strainTJ1061; James Thomas for strains JT7098 and JT5488;Gary Ruvkun for strain CB1391; and Ratna Shownkeenand Alan Coulson (Sanger Center, University of Cam-bridge, UK) for kindly providing us with ordered YACclone arrays spanning the C. elegans genome, and forsupplying clones cm10f11, cm12b2, and cm13d3, interalia, from the C. elegans cDNA library constructed by C.Martin and M. Chal®e.

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Edited by M. Yaniv

(Received 15 December 1999; received in revised form 21 April 2000; accepted 17 May 2000)

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