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Medical significance of Caenorhabditis elegans A Aziz Aboobaker and Mark L Blaxter

Cuenovhubditis eleguns is now the model organism of choice for a growing number of researchers. A combination of its apparent simplicity, exquisite genetics, the existence of a full molecular toolkit and a complete genome sequence makes it ideal for rapid and effective study of gene function. A survey of the C. eleguns genome indicates that this ‘simple’ worm contains many genes with a high degree of similarity to human disease genes. For many human disease genes it has proven, and will continue to prove, difficult to elucidate their function by direct study. In such cases simpler model organisms may prove to be a more productive starting point. The basic function of a human disease gene may be studied in the background of C. eleguns, in which the most important interactions are likely to be conserved, providing an insight into disease process in humans. Here we consider the significance of this modality for human disease processes and discuss how C. eleguns may, in some cases, be ideal in the study of the function of human disease genes and act as a model for groups of parasitic nematodes that have a severe impact on world health.

Key words: Caenorhabditis elegans; evolution; functional genomics; gene function; genetics; human disease; model organism; parasitism; phylogeny.

Ann Med 2000; 32: 23-30.

Introduction

More than 35 years ago the free-living nematode Caenorhabditis elegans was selected by Sydney Brenner as a simple model metazoan (1). We probably now know more about the biology of C. elegans than of any other animal (2). It grows from embryo to the 1-millimetre adult in 3 days, and the transparency of the body at all stages of its life cycle allows every cell division, migration, differentiation, fusion and death to be followed in the live animal (1-3). Its anatomy is deceptively simple with the 959 somatic cells including neuronal (302 cells representing 118 different subtypes (4)), epidermal (213 cells), muscle (111 cells) and intestinal (34 cells) types. We also know the complete wiring pattern of its nervous system (5). Both forward and reverse genetics are now routine and, combined

From the Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, UK.

Correspondance: Mark Blaxter, PhD, ICAPB, King’s Buildings, University of Edinburgh, Edinburgh EH9 3JT, UK. E-mail: [email protected]. Fax: +44 131 6505450.

with powerful molecular techniques, allow rapid investigation of gene function. Over the last 35 years the tractability of the C. elegans model has made an outstanding contribution to our understanding of molecular, physiological and developmental mechanisms, many common to more complex animals, including humans. This understanding has undoubtedly had knock-on effects for medical science, often providing the foundations for investigating the molecular basis of disease processes. However, at the end of 1998 the worm acquired of a new weapon in its armoury. It is the first multicellular organism to have its complete genome sequenced (6, 7).

This ‘simple’ worm has a genome of 97 Mbp encoding more than 19 000 confirmed (ie known to be transcribed) and predicted open reading frames (6 , 7). The implications of this achievement are profound. Firstly, further study of C. elegans will be facilitated and enhanced by the complete genome sequence and will thus continue to give us insight into fundamental processes common to more complex organisms (see (8, 9) for examples). Secondly, C. elegans contains genes that bear a significant similarity to genes implicated in human disease. It is thus possible to

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24 ABOOBAKER BLAXTER

study the basic molecular functions of these important genes in a very tractable experimental system. Finally, C. eleguns is a nematode: a member of a diverse and successful group of animals (10). Included in this phylum are a large number of human parasites, which have a significant impact on world health (11). If C. eleguns is a model organism for studying our own biology, it should be an even better model for studying the important parasitic groups in the phylum Nematoda. Of relevance to all three of these issues is the evolutionary relationship between nematodes and other animal groups, particularly other model organisms and humans. Several questions arise (12). Will this affect how universal we expect future discoveries in C. eleguns to be? Also, what is the relationship between C. eleguns and the parasitic nematodes of medical importance? Which of these nematodes is C. eleguns a good model for?.

In this review we discuss the ways in which C. eleguns has been and will continue to be of importance to medical science and illustrate the importance of the C. eleguns genome project to this contribution.

Relationship between C. e/egans and other species of importance in medical research

With the completion of the C. eleguns genome project and the completion or anticipated completion of other model organism genomes, such as Escherichiu coli, yeast and the fruit fly Drosophilu (3-6), important

pathogen genomes such as malaria (18, 19) and the human genome (17), it is now more important than ever to clarify the evolutionary relationships between species. Such a framework is required to allow the interpretation and efficient application of genomic data to fuel cost-effective medical and basic research. Which model organisms provide the best test bed for which genes and processes of interest? Which genes are likely to have conserved functions as well as sequence?

Varying hypotheses for animal phylogeny all have different implications for the relevance of C. eleguns (see Fig 1) (an expanded version of the phylogenetic trees in Fig 1, giving the names of all phyla, is available from URL: http://www.ed.ac.uW-mbdscienceFigure1 . html). Both morphological (Fig la , b) (20, 21) and molecular (Fig l c ) (22) phylogenetic analyses have been used to try to resolve the relationships between the animal phyla (see (10, 23) for discussions of these and the position of C. eleguns).

Some phylogenies (Fig l a ) place nematode ancestors basal to the arthropod-vertebrate split (24). This would usefully imply that genes conserved between Drosophilu and C. eleguns would also be expected to appear in the human genome (unless they had been uniquely lost). However, recent molecular phylogenetic analysis has placed nematodes and arthropods as sister taxa in a clade of moulting animals, the Ecdysozoa (22). Evidence supporting this phylogeny comes from a comparison of sets of slowly evolving orthologous proteins between yeast, flies and worms (25) and the developmental homeobox gene (Hox) family involved

a , Choanoflagellata b , Choanoflagellata

Cnidaria Cnldada

Nematoda

I P *In

Platyhelminthes P 5 ;

Arthropoda 2 p Annellda Mollusca

n

u)

I

B 8

Enhimodermata 5 Enhimodermata

5 Vertebrata 6

Vertebrata

C 1 Choanoflagellata

‘f Veriebrata

Figure 1. Relationships of the animal phyla. Three hypotheses of these relationships are represented, each having different implications for the expected similarity of the Caenorhabditis elqans genome to other species (10). a) A phylogeny based on traditional morphological criteria. b) The phylogeny proposed by Nielsen (21), wherein nematodes are considered protostomes and are grouped with other phyla having an anterior introvert organ. c) A molecular phylogeny based on that proposed by Aguinaldo and co-workers (22), joining arthropods and nematodes in a clade of moulting animals, supported by further evidence from Hox gene sequence analysis (26) and a consideration of slowly evolving orthologous proteins (25). (Adapted from (1 O).)


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MEDICAL SIGNIFICANCE OF C. E L E G A N S 25

in patterning metazoan body plans (26). If this phylogeny is correct, the two major invertebrate model organisms (Drosophilu and C. eleguns) are more closely related to each other than either of them is to humans. Thus, although many genetic networks, such as signalling systems can be conserved in self- contained cassettes (27) and recruited wholesale to perform new functions in humans, we may find that one caveat of using model organisms is that conservation between them will not always stretch to humans.

C. elegans: a stepping stone to understanding human genetic disorders

One major application of C. eleguns research has been to seed an understanding of fundamental processes which are frequently conserved to some extent in humans. This includes the basic molecular genetic components of Rus oncogene signalling (8), programmed cell death (apoptosis) (9), fibroblast growth factor signalling (28), neuronal patterning and guidance, synaptic transmission (29), sex determination (30), olfaction (3 1) and, more recently, possible clues to the processes involved in ageing and life span (32). This will continue as the novel genes identified by the genome project are studied.

C. eleguns has been used to study directly the functions of human disease genes, many of which have homologues in the C. eleguns genome (see Table 1). This allows the basic molecular functions and genetic interactions of these genes to be studied in the C. eleguns system (reviewed in (35)).

Using postsequencing functional genomics to study gene function in C. elegans

The full genome sequence of C. eleguns speeds up the process of investigating gene function by mutation analysis. Mutations identified by forward genetic screens can be mapped precisely by using classical genetics, and the corresponding part of the sequence can lead directly to the gene. Active transposons (mobile genetic elements) have been characterised in C. eleguns and can be used as molecular tags in mutagenesis experiments (36, 37). The sequence flanking a transposon insertion that is responsible for a mutant phenotype can easily be isolated and the mutated gene identified by comparison to the genomic sequence (38).

Two reverse genetic approaches have been developed: isolation of gene knock-outs by transposon insertion (39, 40) or chemical deletion (41), and the production of ‘pseudo’ knock-outs by injection of double-stranded RNA, a phenomenon called RNA interference (RNAi) (42). The mechanism of RNAi is

not yet fully understood, although the isolation of C. eleguns mutants that are resistant to RNAi has begun to elucidate the molecular events involved (43, 44). The effect is not stable, but a few days after injection it is possible to have an idea of the probable null mutant phenotype in progeny of the injected nematode (45). RNAi may also allow the knock-out of gene families by using double-stranded RNA complemen- tary to highly conserved motifs.

There is now a variety of methods established to study the expression patterns of genes (46): whole- mount immunolocalization using specific antibodies (47); in situ mRNA hybridization with DNA or RNA probes (48); and transgenic expression studies using green fluorescent protein (GFP) and lacZ reporter constructs (49, 50). All of these techniques have been optimized to allow high throughput screening and analysis. In addition, it is now possible to study the trancriptional response of an entire genome by using DNA microarrays (51). Such arrays are now being made for C. eleguns and will provide another powerful tool for assaying subtle differences caused by varying transcriptional states (documentation is available from URL: http://cmgm.stanford.edu/-kimlab). These powerful postsequencing functional genomics techniques together with the exquisite genetics of C. eleguns, its fully characterized anatomy and full genome sequence make the study of gene function a comparatively rapid and inexpensive process. This full molecular toolkit can be used to study the genes of interest, particularly those related to human disease genes (see Table 1).

Studying human diseases within the worm

A number of human disease gene homologues in C. eleguns have already been studied to some extent giving important clues to human gene function. For example egl-1 SIFGFLRRET (52), srnu-4/DPC4 (53), cyk-I/DFNAl (54), vub-3PAX6 (55), fer-lLGMD2B (56), sel-l2/AD3/AD4 (57-59), lov-1PKD1 (62) and pkd-2PKD2 (60) have all been studied to some extent (see Table 1). Perhaps the best example thus far is that of the sel-12 gene, a homologue of human presenilin genes 1 and 2 (PSI and PS2, see Table 1). Mutations in these genes are responsible for early onset of the neurodegenerative disorder Alzheimer’s disease (61). It was found that wild-type human PSI and PS2 can substitute for C. eleguns sel-12 in vivo and that most of the mutants identified resulted in reduced activity (62). This also allowed the regions of the gene dispensable for function to be identified. sel-12, and by extension presenilins, were shown to be involved in the activity of the Notch signalling pathway (57, 58). This has suggested possible new mechanisms for the causes of the disease (63). Further work has begun to identify C. eleguns genes that regulate sel-12 level or activity, which could provide important candidates for

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Table 1. Positionally cloned human disease genes and their Caenorhabditis elegans homologues. A database of 108 positionally cloned human disease genes was obtained from http://genome,nhgri,nih.gov/clone (see also (33)), and the protein sequences of the genes were compared with the full proteome of C. elegans by using the C. elegans Wormpep protein database (http://www,sanger,ac,uk/Projects/ C-elegans/blast-server.shtml) using BLASTp (34). This Table only presents those human genes that hit a C. elegans gene with a BLASTp probability of less than 1.0e4, indicating a strong match. A full version of the Table is available at http://www.ed.ac.uk/-aaa/human/ table1 .html. Where more than one C. elegans gene provided a strong match, only the highest hit is listed, and the total number of strong hits is in parenthesis after the probability score. A total of 52 genes have strong matches in C. elegans. Both OMlM (Online Mendelian Inheritance in Man: http://www.ncbi.nlm.nih.gov/Omim/) and ACeDb (A C. elegans database; http://www.sanger.ac.uk/Projects/C_elegans/webace-front-end. shtml) were used to find descriptions of the gene products. egl- 15, abl- 7 , sel- 12, vab-3, cyk- 7 , fer- 1, npc- 7 , sma-4 and pkd-2 are all C. elegans genetic loci that correspond with the genes, some of which have been studied extensively. EGF, epidermal growth factor; FGF, fibroblast growth factor; TGF, transforming growth factor.

Disease Gene Accession C. elegans BLASTp Description, locus name symbol number gene probability

(< i .oe40)

Aarskog-Scott syndrome Achondroplasia Adrenoleukodystrophy, X-linked Agammaglobulinaemia, X-linked Alagille syndrome Alzheimer's disease (chromosome 14) Alzheimer's disease (chromosome 1) Amyotrophic lateral sclerosis Angelman syndrome Aniridia Ataxia telangiectasia Barth syndrome Basal cell nevus syndrome Bloom syndrome Chondrodysplasia punctata Congenital nephrotic syndrome Cystic fibrosis

Cystinosis Deafness Dementia, hereditary multi-infarct Denatorubral pallidoluysian atrophy Diastrophic dysplasia Duchenne muscular dystrophy Dystonia Glycerol kinase deficiency Gonadal dysgenesis Hereditary multiple exostoses, type 1 Hereditary multiple exostoses, type 2 Hereditary nonpolyposis colon cancer Hereditary nonpolyposis colon cancer H yperexplexia Hypophosphatemic rickets

FGDl FGFLR ALD BTK AGS AD3 AD4 SOD1 AS PAX6 ATM BTHS BCNS BLM ARSE NPHS1 CFTR

CTNS DFNA1 CADASIL DRPLA DTD DMD GCH1 GK SRY EXTI DcT2 MLH1 MSH2 GLYRA2 XLH

Limffiirdle muscular dystrophy, type 28 LGMD2B

Lissencephaly, X-linked Long QT syndrome Lowe oculocerebrorenal syndrome Machaddoseph's disease Miller-Dieker lissencephaly Multiple endocrine neoplasia, type 2a Myotonic dystrophy Myotubular myopathy 1, X-linked Neurofibromatosis, type 2 Niemann-Pick disease, type C

Pancreatic carcinoma Pendred's syndrome Polycystic kidney disease, type 2 Stargardt's disease Thomsen's disease Tuberous sclerosis 1 Werner's syndrome Wilson's disease

LlSX LQTl OCRL MJD1 PAF RET DM MTM 1 N F2 NPCl

DPC4 PDS PKD2 STGDl CLCl TSCl WRN WND

U11690 M64347 221 876 U78027 X83384 L42110 L44577 KO0065 U84404 M77844 U26455 X92762 U59464 U39817 X83573 AF035835 M28668

AJ222967

L10379 U14528 M18533

L13943 LO8063 S79639 U94835 U07343 U03911 X52009 U49908 MOO7670

U40990 M88162 S75313 L13385 M57464 L19268 U58034 L11353 AF002020

U44378

U50928 U88667 225884 D87438 S69873 L25591

C33D9.1 F58A3.2 T02D1.5 M79.1 F11 C7.4 F35H12.3 F35H12.3 C15Fl.b Y65B4B11 .a F14F3.1 Y48GlC55.a ZK809.2 ZK675.1 T04A11.6 D1014.1 C26G2.1 F21G4.2

C41C4.7 F11 H8.4 F11C7.4 F42A6.7 K12G11.2 F32B4.3b F32G8.6 RllF4.1 F40E10.2 K01G5.6 K01G5.6 T28A8.7 H26D21.2 T10G3.7 T05A8.4 F43G9.6

Y79H2A.11 Y54G9A.3 C16C2.3 F28F8.6 T03F6.5 F58A3.2 KO8B12.5 F53A2.8 CO1 G8.5a F02E8.6

R12B2.1 ZK287.2 Y73F8A. b F12B6.1 E04F6.11 C14H10.3 F18C5.2 Y76A2A.2

9.5e-57(1) 3.6e- 1 2 1 (1 9) 2.7e-253(4) 1.8e-80(8) 6.8e-98(6) 6.1 e-138(2) 4.3e-136(2) 1 .1 e-50(3) 5.1 e-41(2) 3.0e-120(5) 4.5e-43(2) 4.2e-59 1.2e-220(4) 1.9e-92(3) 2.7e-48(1) 6.Oe-60( 1 ) 6.9e-172(9)

1 .Oe-80(1) 8.8e-56(1) 6.7e-121(9) 1.2e-68(1) 4.8e-107 1.3e-143(5) 1 .I e-92(1) 3.3e-182 1.4e-41(1) 6.0e-76(1) 4.7e-76( 1 ) 8.4e-115(1) 1.1 e-99 (3) 5.6e-90(31) 4.0e-109(6) 4.6e-114(1)

1.6e-58(1) 4.9e-167(2) 2.6e-58(1) 3.7e-51(1) 1.2e-149(2) 2.9e-71(16) 6.5e- 1 35(10) 9e-53(1) 3.5e-161(3) 2.0e-118(5)

8.0e-77(3) 6.le-l15(7) 7.4e-88( 1) 1.2e-197(5) 3.3e-163(5)

6.8e-71(4) 2.0e-208( 1)

6.&-42(1)

Pleckstrin homology domain FGF receptor, egl-75 ABC transporters Tyrosine protein kinase, abl- 1 Contains EGF-like repeats Presenilin, sel-72 Presenilin, sel- 12 Superoxide dismutase Ubiquitin protein ligase Paired homeobox protein, vab-3 PI3 kinase Putative acetyltransferase Patched membrane protein ReqQ DNA helicase Arysulfatase Immunoglobulin/tibronectin type 111 domain CAMP-dependent chloride channeVABC

Novel membrane protein Similar to formin, cyk-1 Contains EGF-like repeats RNA-binding protein Sulphate transporter Dystrophin GTP cyclohydrolase I Glycerol kinase HMG box protein Novel protein Novel protein DNA mismatch repair protein MutS DNA repair protein Glutamate-gated chloride channel M13 peptidase (zinc metalloprotease) Novel putatative transmembrane protein,

homology to fer- 1 Putative Ca-dependent signalling protein Ion transport protein Inositol-l,4,5-triphosphate 5-phosphatase Novel protein WD domain, G-protein 0-subunit repeats x 4 FGF receptor, egl- 75 Serinehhreonine protein kinase (myotonin) W E zinc finger Erzinsdmoesinshadixins membrane proteins Homology to patched membrane

Putatibe TGF-p signal transducer, sma-4 Sulphate permease Novel membrane protein, pkd-2 ABC transporter Choride channel protein Novel protein DEAD box DNA helicase Copper transporter

transporter

receptor and others, npc- 7


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antiamyloidic therapy for Alzheimer’s disease if the mechanisms of regulation are conserved in humans. For example mutants in sel-10, a homologue of the cdc4 gene involved in cell cycle control, affect sel-12 activity. It is more than likely that research into many more human diseases will be greatly aided by following similar strategies where there is a model organism orthologue with which to work (see Table 1 for such candidates).

However, C. eleguns can also be used to study -

human disease genes or disease processes without the need for C. eleguns orthologues. For example insight into polyglutamine (polyQ) disorders, such as Huntington’s disease, have been gained by expressing the human huntingtin fragments in C. eleguns (64). This work has suggested that the neurodegeneration may occur through programmed cell death as the effect of human huntingtin on C. eleguns was reduced in ced-3 mutant worms (ced-3 encodes a caspase required for apoptosis). In addition, the C. eleguns neurones where human huntingtin was expressed are possibly functionally impaired before the detection of any neurodegeneration. Both these observations could be relevant to human pathology and aetiology in Huntington’s disease, which provides a good example of how the versatility of C. eleguns and the huge amount of data that we already have on this organism can be exploited. It may be possible to identify new genes interacting with disease genes by such methods. Even if the disease gene itself is not highly conserved, as is the case for human huntingtin, genes that interact with it in C. eleguns may be conserved in humans. The potential of such research is most exciting.

C. eleguns could also prove useful for drug screening. Screens that look for mutants showing increased sensitivity, resistance or behavioural changes can identify genes that are affected by the drugs, involved in their metabolism or in genetic pathways connected to such genes. This has already been carried out successfully with cholinesterase inhibitor (65). C. eleguns has also been used to identify virulence genes in the human opportunistic pathogen Pseudomonus ueruginosu. Strain PA14 was shown to kill C. eleguns, requiring many of the virulence genes previously identified in mouse. This assay was then used to isolate novel mutants with reduced virulence in C. eleguns, some of which were subsequently shown to have reduced virulence in mice. This assay could allow the entire genome of this gram-negative bacteria to be screened for pathogenesis-related genes (66).

C. elegans: a model for parasitic nematodes

If C. eleguns is a model for human disease genes, it should be a good model for genes of interest in

parasitic nematodes (10, 12). Nematode parasites of humans, animals and plants cause premature death, chronic sickness, loss of productive labour and widespread malnutrition and impose a multibillion dollar load on developed and developing countries. Hence it is important that C. eleguns is used to achieve a better understanding of other nematodes to enable control strategies to be developed for alleviating effects on human populations. Recent molecular phylogeny work within the phylum Nematoda has provided an essential framework of the relationships between the major taxa (67,68). This has been especially important for allowing clarification of the relationships between species of medical importance and their relationship to C. eleguns (Fig 2).

Estimates of the number of major human nematode infections are currently running at over 4 billion, including around 500 million attributed to non- nematodes (11). Some of the nematodes responsible for these infections are closely related to C. eleguns, while other species are more distant (Fig 2). This will undoubtedly effect the utility of C. eleguns as a model for these parasites. For example, the animal parasitic Strongylida (including the human hookworms Ancylo- stoma and Necutor) are robustly placed within the Rhabditida, and C. eleguns is likely to be an excellent model for these important pathogens as they are reasonably closely related. However, other parasite groups are more distant, and it is not yet clear how useful C. eleguns will be in these situations.

One issue for exploiting C. eleguns as a model for parasitic nematodes is a need for knowledge about parasite genomes and genes of interest. The C. eleguns genome project provides data about nematode-specific genes (those without homologues in other phyla), but not about parasitism-specific genes, as it is a free- living nematode. The WHO-sponsored Filarial Genome Project (FGP) has addressed this issue for a group of important human parasites (69). The project has generated genomics data in the form of expressed sequence tags (ESTs) from the filarial parasite Brugiu muluyi. In addition to being a causative agent of human filariasis and elephantiasis, Brugiu muluyi is the current model for the other filarial nematodes because it can be maintained in the laboratory by using jird (gerbil) hosts. More than 19 000 ESTs representing 5000-6000 genes have been generated from cDNA libraries from different stages of the life cycle (70). This approach to gene identification has proved highly successful and has provided the parasitology community with a large number of novel vaccine candidates and drug targets (71) as well as insights into parasite biology. Similar gene identification projects are now underway for the other major groups of nematodes important to humans (72).

There are a number of ways in which C. eleguns can be used as a valuable model for parasitic



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Figure 2. Phlyogenetic structure of the Nematoda revealed by analysis of full-length small subunit rDNA sequences. There are important human pathogens in four of the five major groups (I-V). The numbers after the species name (where present) indicate the number of estimated current infections in millions (1 1). Enterobius (pinworm) is a cosmopolitan infection in developed and developing countries, and it is likely to infect most people. (Adapted from (62, 63))

nematodes by exploiting all the tools already described. Firstly, a comparison of gene sequence data sets, where available, can identify nematode-specific protein domains, which could be exploited as antihelminth drug targets (10). Studying gene function is not possible in parasite nematodes because the nature of the life cycle makes mutation analysis and genetics unworkable; however, C. elegans can be exploited for this purpose. Functional analysis of C. elegans orthologs of interesting parasite genes can be undertaken as easily as that of human disease genes. In addition, C. elegans can be used as a heterologous system to study functional aspects of parasite genes through transgenesis (72, 73). Finally, interesting genes identified in the C. elegans genome may be used as probes to isolate parasitic counterparts either by hybridization, degenerate polymerase chain reaction (PCR) or by ‘in silico’ methods where gene data sets are available for other nematodes (AA Aboobaker, DB Guiliano and ML Blaxter, unpublished observations, 1999).

Conclusion

In many ways the full extent of the medical significance of C. elegans as a model organism is only just becoming apparent. The C. elegans genome project has successfully handled the data produced by sequencing a genome eight times the size that of the budding yeast Saccharomyces cerevisiae and has made it available to the research community in such a way as to maximize its usefulness (6, 7). When complete, the Human Genome Project will have produced 30 times more sequence data. This may well have the greatest impact on medical science ever (17). The C. elegans project provides a good model for how to analyse and present such crucial information to an impatient research community.

As we have discussed, C. elegans is an advantageous system for investigating the functions and interactions of both human disease genes and those identified in parasitic nematodes. The simplicity of the C. elegans system greatly enhances the ease of experimentation,


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and yet its position as a metazoan with most major cell and tissue types means that much of what is found will be universally applicable. One caveat, however, is that some genes may not be conserved and may function and interact differently. This is likely to be especially true of genes with divergent sequences.

Over the next few years C. elegans will hopefully continue to make an impact on our understanding of

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