A Model of Elegance
1998; Elsevier BV; Volume: 63; Issue: 4 Linguagem: Inglês
10.1086/302078
ISSN1537-6605
AutoresAlbertha J.M. Walhout, Hideki Endoh, Nicolas Thierry‐Mieg, Wendy Wong, Marc Vidal,
Tópico(s)Light effects on plants
ResumoTo start with we propose to identify every cell in the worm and trace lineages. We shall investigate the constancy of development and study its genetic control by looking for mutants. (Sidney Brenner) Since Sidney Brenner wrote this statement in a visionary research proposal addressed to Max Perutz 35 years ago (Wood Wood, 1988Wood WB The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar, p. xiii), an enormous amount of information has been gathered on the biology of the nematode Caenorhabditis elegans ("the worm"), both fulfilling his predictions and exceeding his original expectations. Researchers have identified every cell in the worm and have described all the lineages by which these cells are formed (Horvitz and Sulston Horvitz and Sulston, 1980Horvitz HR Sulston JE Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans.Genetics. 1980; 96: 435-454PubMed Google Scholar; Sulston et al. Sulston et al., 1983Sulston JE Schierenberg E White JG Thomson JN The embryonic cell lineage of the nematode Caenorhabditis elegans.Dev Biol. 1983; 100: 64-119Crossref PubMed Scopus (2636) Google Scholar; Wood Wood, 1988Wood WB The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Adult hermaphrodites contain 959 somatic nuclei, which are organized into differentiated tissue types such as muscle, intestine, epidermis, and nervous system. The latter is composed of 302 neurons that are connected by ∼7,500 synaptic junctions (Wood Wood, 1988Wood WB The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). The function of many individual cells has been inferred from ablation experiments using laser-beam technology (e.g., see Bargmann and Avery Bargmann and Avery, 1995Bargmann CI Avery L Laser killing of cells in Caenorhabditis elegans.in: Epstein HF Shakes DC Methods in cell biology. Academic Press, Houston1995: 225-250Google Scholar). Genes involved in development and behavior have been identified through the analysis of mutations that affect these functions. The number of genes identified genetically has increased from the original 103 described by Brenner (Brenner, 1974Brenner S The genetics of Caenorhabditis elegans.Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar) to the ∼1,600 complementation groups known at present (Hodgkin Hodgkin, 1997Hodgkin J Skeleton genetic map.in: Riddle DL Blumenthal T Meyer BJ Priess JR C. elegans II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 891-897Google Scholar). A comprehensive genetic map has been compiled and continues to expand. A detailed physical map of overlapping cosmids and YACs covers the genome almost entirely (Coulson et al. Coulson et al., 1995Coulson A Huynh C Kozono Y Shownkeen R The physical map of the Caenorhabditis elegans genome.Methods Cell Biol. 1995; 48: 533-550Crossref PubMed Scopus (29) Google Scholar). Finally, the genome sequence is expected to be completed by the end of this year (Hodgkin et al. Hodgkin et al., 1995Hodgkin J Plasterk RH Waterston RH The nematode Caenorhabditis elegans and its genome.Science. 1995; 270: 410-414Crossref PubMed Scopus (114) Google Scholar). The availability of the complete C. elegans genome sequence will not only benefit worm researchers but will also undoubtedly be useful for the elucidation of human gene function. Thus, in addition to providing fundamental information on how a small, free-living animal develops and responds to its environment, the worm field is now recognized as potentially useful in the understanding of the development and behavior of humans. Again as Brenner put it, "our field has prospered and has come of age; what was once a joke organism…has now become a major experimental system for the study of development" (Wood Wood, 1988Wood WB The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar, p. ix). Not only can the results obtained in C. elegans teach us about the mechanisms that underlie normal development, but they can also be very useful in the study of biochemical mechanisms underlying devastating human diseases. Moreover, the worm system has the potential to become a convenient tool for the design of therapeutic strategies required for the treatment of these diseases. For these reasons, the readers of the Journal might be interested in an overview of C. elegans genetics and genomics and of how worm biology can benefit their research. Here, we place particular emphasis on the benefits of the study of worm gene functions and interactions to the understanding of human biology; on the importance of the genomic information that should soon become available for C. elegans; and on the promise of the worm as a model for drug discovery. Defining the mechanism of the action of human genes can be extremely difficult, because of the complexity of human biology and the lack of reliable tools to selectively alter gene function in vivo, not to mention obvious ethical constraints. Since entire protein complexes and biochemical pathways appear to be conserved between humans and relatively simple model organisms, many laboratories have turned to such organisms, with the goal of establishing functional models that can ultimately teach us about human biology. A number of excellent recent reviews describe the experimental features of the worm and detail its virtues as a model organism (Hodgkin et al. Hodgkin et al., 1995Hodgkin J Plasterk RH Waterston RH The nematode Caenorhabditis elegans and its genome.Science. 1995; 270: 410-414Crossref PubMed Scopus (114) Google Scholar; Plasterk Plasterk, 1996Plasterk RHA Postsequence genetics of Caenorhabditis elegans.Genome Res. 1996; 6: 169-175Crossref PubMed Scopus (6) Google Scholar; Ahringer Ahringer, 1997Ahringer J Turn to the worm.Curr Opin Genet Dev. 1997; 7: 410-415Crossref PubMed Scopus (33) Google Scholar; Kuwabara Kuwabara, 1997Kuwabara PE Worming your way through the genome.Trends Genet. 1997; 13: 455-460Abstract Full Text PDF PubMed Scopus (14) Google Scholar). In addition, three books are available in which most of the current biological information and detailed protocols can be found (Wood Wood, 1988Wood WB The nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar; Epstein and Shakes Epstein and Shakes, 1995Epstein HF Shakes DC Wilson L Matsudaira P Caenorhabditis elegans: modern biological analysis of an organism. Vol 48. Academic Press, San Diego1995Google Scholar; Riddle et al. Riddle et al., 1997Riddle DL Blumenthal T Meyer BJ Priess JR C. elegans II. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997Google Scholar). The worm model combines ease of genetic manipulations with the ability to address biological questions related to development, behavior, and cancer. Caenorhabditis elegans genetics is very convenient. For example, worm cultures can be grown easily in the lab, and strains of interest can be stored frozen (Epstein and Shakes Epstein and Shakes, 1995Epstein HF Shakes DC Wilson L Matsudaira P Caenorhabditis elegans: modern biological analysis of an organism. Vol 48. Academic Press, San Diego1995Google Scholar). Since most adult worms develop into self-fertilizing hermaphrodites, it is usually trivial to obtain F2 animals homozygous for a mutation of interest. Moreover, males can also be obtained, which allows the exchange of genetic material by cross-fertilization. Transgenic animals can also be generated by injection of cloned DNA (a gene, a cDNA, a cosmid, or a YAC), along with a dominant marker, into the germ line. The resulting transgenes are transmitted in the form of extrachromosomal DNA arrays. Although this technique does not allow cloning of genes by complementation using cDNA libraries, it is very useful for determination of which of several candidate DNA fragments can rescue the phenotype of loss-of-function mutations (Mello et al. Mello et al., 1991Mello CC Kramer JM Stinchcomb D Ambros V Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2309) Google Scholar; Epstein and Shakes Epstein and Shakes, 1995Epstein HF Shakes DC Wilson L Matsudaira P Caenorhabditis elegans: modern biological analysis of an organism. Vol 48. Academic Press, San Diego1995Google Scholar). Finally, as discussed below, gene function can be analyzed by relatively convenient antisense technologies. Usually the first reaction of a human gene cloner in the seconds following the pile-up and translation of a long-awaited DNA sequence is to "BLAST" the predicted protein sequence against the available databases, with the hope of finding statistically significant homologies. With the complete genomic sequence in hand, it will be possible to determine, in silico, the existence of all potential orthologues in C. elegans ("comparative genomics"). The proportion of positionally cloned human disease genes that show significant homology (P<1×10−10) to potential worm orthologues is currently 63% (Bassett et al. Bassett et al., 1995Bassett Jr, DE Boguski MS Spencer F Reeves R Goebl M Hieter P Comparative genomics, genome cross-referencing and XREFdb.Trends Genet. 1995; 11: 372-373Abstract Full Text PDF PubMed Scopus (63) Google Scholar, Bassett et al., 1997Bassett Jr, DE Boguski MS Spencer F Reeves R Kim S Weaver T Hieter P Genome cross-referencing and XREFdb: implications for the identification and analysis of genes mutated in human disease.Nat Genet. 1997; 15: 339-344Crossref PubMed Scopus (70) Google Scholar; also see the XREFdb Website). This proportion drops to 38% for potential fly orthologues, probably because the genome-sequence project is less advanced in that organism. It is 35% for yeast proteins, since the evolutionary divergence is greater (in this case, despite the availability of its entire genome sequence). Until the complete genomic sequence of the worm is released into GenBank, it remains more advantageous to search for worm orthologues directly in the databases available from the two sequencing centers involved in the project (The Sanger Center and the Genome Sequencing Center, University of Washington School of Medicine). Once a potential worm orthologue has been identified, ACeDB (AC . elegansDatabase) (Durbin and Thierry-Mieg Durbin and Thierry-Mieg, 1994Durbin R Thierry-Mieg J The ACeDB genome database.in: Suhai S Computational methods in genome research. Plenum Press, New York1994Google Scholar) can be used to fetch the information necessary to start work with the orthologue corresponding to the gene of choice. For simplicity, ACeDB can be thought of as an organized repository matrix composed of predicted open reading frames (ORFs) in one axis and the relevant information in the other. Just to name a few options, it is possible to find, for each locus, the available information on the genetic-map position and on neighboring genes, the different cosmids and YACs covering the region on the physical map, the genome sequence with the predicted ORFs and predicted intron/exon boundaries, and the availability and sequence of corresponding expressed sequence tags (ESTs) as a confirmation of the intron/exon structure. An excellent summarized version of a "guided tour" in ACeDB is available in a recent review (Kuwabara Kuwabara, 1997Kuwabara PE Worming your way through the genome.Trends Genet. 1997; 13: 455-460Abstract Full Text PDF PubMed Scopus (14) Google Scholar). How can C. elegans be used to elucidate the function of a particular human disease protein (HDP)? One approach consists in finding a potential worm orthologue of HDP (WHD-1) in silico (see above) and analyzing the in vivo phenotype(s) resulting from the abrogation of its function. In addition to providing critical functional information, such phenotype(s) can eventually be used in genetic screens to identify interacting genes. Although convenient, such screens are not trivial and usually require the construction of strains that carry particular alleles; partial loss-of-function alleles are generally preferred, especially when the wild-type gene is essential for development or when one wishes to identify both enhancers and suppressors of an intermediate phenotype. The phenotypes that arise from loss of WHD-1 activity in the worm generally will not resemble the overt human disease phenotypes, but there will often be similarities at the subcellular or biochemical level, which afford insights into the molecular mechanisms of the disease. One example of this is the elucidation of some of the molecular mechanisms involved in the function of the human ras oncogene, by the analysis of the role of its C. elegans orthologue, let-60, in vulva development (Kayne and Sternberg Kayne and Sternberg, 1995Kayne PS Sternberg PW Ras pathways in Caenorhabditis elegans.Curr Opin Genet Dev. 1995; 5: 38-43Crossref PubMed Scopus (60) Google Scholar; Kornfeld Kornfeld, 1997Kornfeld K Vulval development in Caenorhabditis elegans.Trends Genet. 1997; 13: 55-61Abstract Full Text PDF PubMed Scopus (155) Google Scholar). Finding a worm orthologue of HDP can be followed by a number of different approaches. In the most powerful scenario, whd-1 has already been identified by classic forward genetics and subsequently has been cloned and sequenced. Previous identification of a gene by forward genetics means that a particular phenotype(s) has already been associated with the loss of its function (whd-1(lf)). An optimal disease model can be generated if HDP can functionally substitute for WHD-1. For example, the worm sel-12 gene encodes a protein that shares 50% identity with two human presenilins involved in Alzheimer disease. The egg-laying–defective phenotype of sel-12(lf) mutants is rescued by wild-type human presenilin but not by the mutant alleles found in Alzheimer patients (Levitan and Greenwald Levitan and Greenwald, 1995Levitan D Greenwald I Facilitation of lin-12-mediated signaling of sel-12, a C. elegans S182 Alzheimers disease gene.Nature. 1995; 377: 351-354Crossref PubMed Scopus (613) Google Scholar; Levitan et al. Levitan et al., 1996Levitan D Toyle TG Brousseau D Lee MK Thinakaran G Slunt GG Sisodia SS et al.Assessment of normal and mutant human presenilin function in C. elegans.Proc Natl Acad Sci USA. 1996; 93: 14940-14944Crossref PubMed Scopus (337) Google Scholar). It should be stressed that a negative result in such rescue experiments is not sufficient to rule out the possibility of a functional conservation between HDP and WDP. In a second scenario, whd-1 is already known through classic forward genetics, but the gene remains uncloned (of 1,600 complementation groups identified, ∼500 have been cloned). In some cases, the physical position of whd-1 might point to a few mutations located in the corresponding area of the genetic map and representing potential whd-1 loci. The functional rescue, by wild-type whd-1, of one of these mutations then provides evidence that the whd-1 gene corresponds to a particular complementation group. Although potentially powerful, this approach can be complicated in regions of the genetic map that are characterized by a high density of potential loci. In the third, and most likely, scenario, whd-1 has not been identified by forward genetics, and there is virtually no functional information available. In this case, phenotypes associated with the loss of whd-1 will have to be uncovered. A technique called "RNA-mediated interference" (RNAi) is very powerful in this respect. Antisense, sense, or double-stranded whd-1 RNA injected into the germ-line syncytium of an adult hermaphrodite is often found to impede whd-1 function in the progeny (Guo and Kemphues Guo and Kemphues, 1995Guo S Kemphues KJ par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed.Cell. 1995; 81: 611-620Abstract Full Text PDF PubMed Scopus (854) Google Scholar; Rocheleau et al. Rocheleau et al., 1997Rocheleau CE Downs WD Lin R Wittmann C Bei Y Cha YH Ali M et al.Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos.Cell. 1997; 90: 707-716Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar; Fire et al. Fire et al., 1998Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (10934) Google Scholar). The effect is likely specific, since the RNAi phenotype often resembles that of loss-of-function mutations. The effect of RNAi is not limited to embryos but is transmitted through larval development and adulthood, for several generations. In general, however, the effect of RNAi cannot be propagated for many generations, so subsequent genetic analyses, such as enhancer and suppressor screens, are not possible. Such screens depend on the availability of whd-1 null or partial loss-of-function alleles. Two methods are available to create whd-1 null alleles. The first method is based on the ability to isolate a worm strain with the Tc1 transposable element inserted within whd-1 and subsequent screening by PCR for imprecise excision leading to a deletion in whd-1 (Zwaal et al. Zwaal et al., 1993Zwaal RR Broeks A van Meurs J Groenen JT Plasterk RH Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank.Proc Natl Acad Sci USA. 1993; 90: 7431-7435Crossref PubMed Scopus (230) Google Scholar). Although this method was very promising, it is now being replaced by a more efficient method, based on the use of a chemical mutagen to create libraries of deletion alleles (Jansen et al. Jansen et al., 1997Jansen G Hazendonk E Thijssen KL Plasterk RH Reverse genetics by chemical mutagenesis in Caenorhabditis elegans.Nat Genet. 1997; 17: 119-121Crossref PubMed Scopus (229) Google Scholar). Again in this case, strains containing deletions in whd-1 are identified by a PCR reaction. The number of genes that have been deleted this way increases rapidly. If neither RNAi nor the deletion of whd-1 confers a detectable phenotype, then a few more tools based on the localization of expression of whd-1 are available to aide in the formulation of hypotheses about the function of the gene. Whole-mount techniques are available for RNA in situ hybridization that are useful for the localization of expression down to single cells. More conveniently, transgenic animals can be generated that express β-galactosidase or green fluorescent protein (GFP) under the control of a whd-1 promoter, or as a fusion with the whd-1 gene product. Gene expression, as detected by the reporter construct, can then be used to screen for phenotypes in a more subtle way (Chalfie et al. Chalfie et al., 1994Chalfie M Tu Y Euskirchen G Ward WW Prasher DC Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5279) Google Scholar; Troemel et al. Troemel et al., 1995Troemel ER Chou JH Dwyer ND Colbert HA Bargmann CI Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans.Cell. 1995; 83: 207-218Abstract Full Text PDF PubMed Scopus (517) Google Scholar) If the human disease is caused by a gain-of-function mutation in HDP, one or more worm orthologues might also be identified and tested as described above. However, a more direct approach is to generate transgenic animals that overexpress the mutant dominant gain-of-function version of HDP and to analyze the resulting phenotype. In principle, these animals can also be used in screens to identify genetic interactors and to elucidate HDP function. Despite the power of genetics and cell biology available in model organisms such as the worm, the number of genes with a function assigned is still relatively small, compared with the invested efforts. The ∼500 cloned C. elegans genes that correspond to genetically identified complementation groups represent ∼4% of the expected 13,000 genes. Hence, it seems crucial to design complementary approaches to increase the speed of gene-function discovery. The current explosion of complete genome sequences should facilitate such global approaches (Lander Lander, 1996Lander ES The new genomics: global views of biology.Science. 1996; 274: 536-539Crossref PubMed Scopus (861) Google Scholar; Fields Fields, 1997Fields S The future is function.Nat Genet. 1997; 15: 325-327Crossref PubMed Scopus (59) Google Scholar). With this in mind, several labs, including our own, are specializing in the generation of resources and functional information on a genomewide scale (a field that is often referred to as "functional genomics"). Supporting evidence for the necessity of such projects can be found in the genomic sequencing projects of several model organisms, which have taught us several lessons. First, it is very cost-efficient to centralize the efforts of repetitive experiments such as sequencing (Hodgkin et al. Hodgkin et al., 1995Hodgkin J Plasterk RH Waterston RH The nematode Caenorhabditis elegans and its genome.Science. 1995; 270: 410-414Crossref PubMed Scopus (114) Google Scholar). Since functional studies can also be somewhat repetitive (see below), the same principle should apply to functional genomics projects. Second, novel interesting and somewhat unexpected aspects of biology have emerged from genomewide projects. For example, the complete sequencing of the yeast genome has revealed that it derives from a duplication (Wolfe and Shields Wolfe and Shields, 1997Wolfe KH Shields C Molecular evidence for an ancient duplication of the entire yeast genome.Nature. 1997; 387: 708-713Crossref PubMed Scopus (1329) Google Scholar). It is possible that new principles might similarly emerge from large-scale functional genomics analyses. Finally, standardized, or even automated, methods and computer tools that generate, analyze, or access sequence data might be applicable to large-scale projects aimed at the elucidation of gene function. Importantly, functional genomics projects in model organisms will make available both materials and information of great potential biological or clinical interest. Much of the information revealed by time-consuming experiments in the worm might soon be retrieved by no more than a click of a computer mouse. The complete genome sequence of all six chromosomes in C. elegans will lead to ∼13,000 ORFs, predicted by a program called Genefinder. It has been estimated that 99.9%), Genefinder correctly predicts the intron/exon junctions in ∼50% of the cases. However, a large-scale project of EST sequencing is expected to lead to definitive answers on the genomic structure and expression of many predicted ORFs. For example, it is possible that computer-predicted ORFs might not be expressed in vivo and therefore might not represent genuine genes, whereas other sequences that could not be recognized by Genefinder might indeed be expressed. In addition, genes may have an intron/exon structure different than that predicted by the program. The EST information currently available can be accessed in ACeDB. The yeast model system, with the first eukaryotic genome sequenced (Goffeau et al. Goffeau et al., 1996Goffeau A Barrell BG Bussey H Davis RW Dujon B Feldmann H Galibert F et al.Life with 6000 genes.Science. 1996; 274 (563-567): 546Crossref PubMed Scopus (3062) Google Scholar), already provides a great example for the development of functional genomics projects: (i) genomewide expression analysis using DNA arrays representing every single predicted yeast ORF is possible (Schena et al. Schena et al., 1995Schena M Shalon D Davis RW Brown PO Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Science. 1995; 270: 467-470Crossref PubMed Scopus (7353) Google Scholar); (ii) a consortium is making progress in deleting every single predicted ORF (e.g., see Smith et al. Smith et al., 1996Smith V Chou KN Lashkari D Botstein D Brown PO Functional analysis of the genes of yeast chromosome V by genetic footprinting.Science. 1996; 274: 2069-2074Crossref PubMed Scopus (197) Google Scholar); and (iii) a protein-interaction map is underway (Evangelista et al. Evangelista et al., 1997Evangelista C Lockshon D Fields S The yeast two-hybrid system: prospects for protein linkage maps.Trends Cell Biol. 1997; 6: 196-199Abstract Full Text PDF Scopus (42) Google Scholar; Fromont-Racine et al. Fromont-Racine et al., 1997Fromont-Racine M Rain JC Legrain P Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens.Nat Genet. 1997; 16: 277-282Crossref PubMed Scopus (685) Google Scholar). Similar projects are now underway in C. elegans. Projects to delete every single predicted ORF or aimed at the identification of every ORF essential for worm development and using RNAi are in the planning stage (A. Coulson, R. Plasterk, and A. Hyman, personal communication), and a C. elegans cDNA array, which should be useful for the creation of expression maps, is also under development (S. Kim, personal communication). Other projects are underway to determine in what cell and at what stage of development each ORF is expressed (e.g., see Tabara et al. Tabara et al., 1996Tabara H Motohashi T Kohara Y A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans.Nucleic Acids Res. 1996; 24: 2119-2124Crossref PubMed Scopus (91) Google Scholar). Because protein-protein interactions are important for most biological functions, we have initiated a large-scale project to generate a protein-interaction map for C. elegans. Protein-interaction maps are defined here as publicly available databases in which information on potential protein-protein interactions can be found. Like other functional genomics resources, protein-interaction maps could produce functional information available at the click of a mouse. Together with additional information such as cellular localization or time of expression, they would lead to the formulation of hypotheses that could be tested in the relevant biological system. The two-hybrid system has been proved useful for the identification of protein-protein interactions (for a recent review, see Vidal and Legrain Vidal and Legrain, 1998Vidal M, Legrain P (1998) Yeast forward and reverse "n" hybrid systems. Nucleic Acids Res (in press)Google Scholar). However, the system can also be complicated by high numbers of false positives and can be limited by the lack of direct connections with biology. As shown in the sidebar we are addressing these two issues by integrating "forward" and "reverse" versions of the two-hybrid strategy with classical genetics available in the nematode. A protein-interaction map for C. elegans should nicely complement the information generated by a yeast protein-interaction mapping project. Although valuable in itself—and, in some ways, more tractable—the yeast protein-interaction project cannot be expected to elucidate interactions among proteins that arose during animal evolution. Furthermore, the technical challenges posed both by the worm's larger genome and by the greater prevalence of introns in C. elegans genes compared with yeast genes make the worm project a closer model for some future endeavor, to map human protein interactions.The Two-Hybrid System: Forward and ReverseThe two-hybrid system makes use of the fact that regulatory transcription factors are generally composed of two separable domains: a sequence-specific DNA-binding domain (DB) and a transactivation domain (AD) (Keegan et al. Keegan et al., 1986Keegan L Gill G Ptashne M Separation of DNA binding from the transcription- activating function of a eukaryotic regulatory protein.Science. 1986; 231: 699-704Crossref PubMed Scopus (352) Google Scholar). Neither DB nor AD activates transcription unless they are held together (Triezenberg et al. Triezenberg et al., 1988Triezenberg SJ Kingsbury RC McKnight SL Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression.Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (571) Google Scholar), so, when two proteins (X and Y) are expressed as hybrid proteins, one of which is fused to DB (DB-X) and the other of which is fused to AD (AD-Y), a physical interaction between X and Y can be identified through the reconstitution of a functional transcription factor (Fields and Song Fields and Song, 1989Fields S Song O A novel genetic system to detect protein-protein interactions.Nature. 1989; 340: 245-246Crossref PubMed Scopus (4695) Google Scholar). In "forward" two-hybrid screens, one selects for protein-protein interactions, using a positive selectable reporter gene which must be activated to allow cell growth under specific conditions. When applied to a large-scale project, such as the generation of a comprehensive protein-interaction map, high rates of false-positives, as observed in many conventional two-hybrid screens, can obscure genuine interactions. However, common causes of false positives have been eliminated by optimizing the assay in advance (see, e.g., Vidal and Legrain Vidal and Legrain, 1998Vidal M, Legrain P (1998) Yeast forward and reverse "n" hybrid systems. Nucleic Acids Res (in press)Google Scholar).Beyond the need to optimize the forward two-hybrid
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