{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T18:47:13Z","timestamp":1791226033567,"version":"4.1.0"},"reference-count":75,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5450","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,1,7]]},"abstract":"<jats:p>\n            Protein interaction mapping using large-scale two-hybrid analysis has been proposed as a way to functionally annotate large numbers of uncharacterized proteins predicted by complete genome sequences. This approach was examined in\n            <jats:italic>Caenorhabditis elegans<\/jats:italic>\n            , starting with 27 proteins involved in vulval development. The resulting map reveals both known and new potential interactions and provides a functional annotation for approximately 100 uncharacterized gene products. A protein interaction mapping project is now feasible for\n            <jats:italic>C. elegans<\/jats:italic>\n            on a genome-wide scale and should contribute to the understanding of molecular mechanisms in this organism and in human diseases.\n          <\/jats:p>","DOI":"10.1126\/science.287.5450.116","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:37:46Z","timestamp":1027762666000},"page":"116-122","source":"Crossref","is-referenced-by-count":621,"title":["Protein Interaction Mapping in\n            <i>C. elegans<\/i>\n            Using Proteins Involved in Vulval Development"],"prefix":"10.1126","volume":"287","author":[{"given":"Albertha J. M.","family":"Walhout","sequence":"first","affiliation":[{"name":"Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raffaella","family":"Sordella","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaowei","family":"Lu","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James L.","family":"Hartley","sequence":"additional","affiliation":[{"name":"Life Technologies Inc., Rockville, MD 20850, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary F.","family":"Temple","sequence":"additional","affiliation":[{"name":"Life Technologies Inc., Rockville, MD 20850, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael A.","family":"Brasch","sequence":"additional","affiliation":[{"name":"Life Technologies Inc., Rockville, MD 20850, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolas","family":"Thierry-Mieg","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA."},{"name":"Laboratoire LSR-IMAG, 38402 St-Martin-D'Heres Cedex, France."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Vidal","sequence":"additional","affiliation":[{"name":"Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.277.5331.1453"},{"key":"e_1_3_1_2_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/387s005","volume":"387","author":"Goffeau A.","year":"1997","unstructured":"Goffeau A., et al., Nature (suppl.)387, 1 (1997);","journal-title":"Nature"},{"key":"e_1_3_1_2_4","doi-asserted-by":"crossref","unstructured":"; The C. elegans Sequencing Consortium Science 282 2012 (1998);","DOI":"10.1126\/science.282.5396.2012"},{"key":"e_1_3_1_2_5","doi-asserted-by":"publisher","DOI":"10.1126\/science.282.5389.682"},{"key":"e_1_3_1_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.270.5235.467"},{"key":"e_1_3_1_3_3","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1296-1675"},{"key":"e_1_3_1_3_4","doi-asserted-by":"publisher","DOI":"10.1126\/science.274.5287.610"},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.274.5287.536"},{"key":"e_1_3_1_4_3","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1038\/ng0497-325","volume":"15","author":"Fields S.","year":"1997","unstructured":"Fields S., Nature Genet. 15, 325 (1997) .","journal-title":"Nature Genet."},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1038\/ng0196-72","volume":"12","author":"Bartel P. L.","year":"1996","unstructured":"Bartel P. L., Roecklein J. A., SenGupta D., Fields S., Nature Genet. 12, 72 (1996);","journal-title":"Nature Genet."},{"key":"e_1_3_1_5_3","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/0962-8924(96)40002-2","volume":"6","author":"Evangelista C.","year":"1997","unstructured":"Evangelista C., Lockshon D., Fields S., Trends Cell Biol. 6, 196 (1997).","journal-title":"Trends Cell Biol."},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1038\/ng0797-277"},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1093\/nar\/27.4.919","volume":"27","author":"Vidal M.","year":"1999","unstructured":"Vidal M., Legrain P., Nucleic Acids Res. 27, 919 (1999).","journal-title":"Nucleic Acids Res."},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"7815","DOI":"10.1073\/pnas.96.14.7815","volume":"96","author":"Flores A.","year":"1999","unstructured":"Flores A., et al., Proc. Natl. Acad. Sci. U.S.A. 96, 7815 (1999).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_9_2","unstructured":"Because many developmental and behavioral mechanisms are relatively conserved ["},{"key":"e_1_3_1_9_3","doi-asserted-by":"publisher","DOI":"10.1126\/science.282.5396.2033"},{"key":"e_1_3_1_9_4","doi-asserted-by":"publisher","DOI":"10.1126\/science.282.5396.2028"},{"key":"e_1_3_1_9_5","unstructured":"] information obtained from a C. elegans protein interaction map may be useful to formulate hypotheses about human biology. In C. elegans the validity of potential protein-protein interactions can be tested using genetic methods such as conventional epistatic analyses and RNA mediated interference (RNAi) ["},{"key":"e_1_3_1_9_6","doi-asserted-by":"publisher","DOI":"10.1038\/35888"},{"key":"e_1_3_1_9_7","unstructured":"]. Importantly these analyses can be performed in the context of a developing animal with a fixed number of recognizable cells [W. B. Wood The Nematode Caenorhabditis elegans (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1988)]."},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1086\/302078","volume":"63","author":"Walhout A. J. M.","year":"1998","unstructured":"Walhout A. J. M., Endoh H., Thierry-Mieg N., Wong W., Vidal M., Am. J. Hum. Genet. 63, 955 (1998).","journal-title":"Am. J. Hum. Genet."},{"key":"e_1_3_1_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-9525(97)01005-6"},{"key":"e_1_3_1_11_3","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1016\/S0168-9525(98)01592-3","volume":"14","author":"Sternberg P. W.","year":"1998","unstructured":"Sternberg P. W., Han M., Trends Genet. 14, 466 (1998) .","journal-title":"Trends Genet."},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/123.1.109"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81722-5"},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"3449","DOI":"10.1242\/dev.126.15.3449","volume":"126","author":"Thomas J. H.","year":"1999","unstructured":"Thomas J. H., Horvitz H. R., Development 126, 3449 (1999).","journal-title":"Development"},{"key":"e_1_3_1_15_2","unstructured":"J. L. Hartley G. F. Temple M. A. Brasch in preparation. RC is also referred to as Gateway cloning. Recently another recombination-based cloning system referred to as \u201cunivector-plasmid fusion system\u201d has been described ["},{"key":"e_1_3_1_15_3","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-9822(07)00560-X"},{"key":"e_1_3_1_15_4","unstructured":"]. This system is based on Cre- lox site-specific recombination. This system can also be used to conveniently transfer DNA inserts into different vectors. It is yet unclear how the standardization and versatility of this system will compare to RC. Details of the RC\u2013two-hybrid system will be described elsewhere (M. A. Rhodes and M. A. Brasch in preparation)."},{"key":"e_1_3_1_16_2","unstructured":"A. J. M. Walhout et al. Methods Enzymol. in press."},{"key":"e_1_3_1_17_2","unstructured":"Sequencing of 10 independent clones corresponding to two ORFs ( lin-53 and lin-37 ) revealed approximately one mutation per 10 000 nucleotides presumably originating from misincorporations during the PCR reactions (A. J. M. Walhout R. Sordella M. Vidal data not shown). The remaining steps described here were carried out with pools of approximately 10 Entry clones per vORF. Because the average size of vORFs is about 1.5 kb we estimate that approximately 9 of 10 Entry clones generated in the BP cloning step correspond to wild-type vORFs. For a subset of vORFs protein immunoblot analysis showed that full-length DB-vORFs are expressed in yeast cells from the DB destination vectors. The insert size of each vORF Entry clone was verified using PCR analysis. We also demonstrated that DB-vORF Expression clones can encode full-length proteins using COOH-terminal fusions to green fluorescent protein (GFP) (Fig. 1A). It is noteworthy that both the cloning and the last two verification strategies can be performed on 96-well plates and thus are compatible with high-throughput procedures."},{"key":"e_1_3_1_18_2","unstructured":"M. Vidal in The Yeast Two-Hybrid System P. Bartel and S. Fields Eds. (Oxford Univ. Press New York 1997) pp. 109\u2013147."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","first-page":"12980","DOI":"10.1073\/pnas.91.26.12980","volume":"91","author":"Finley R. L.","year":"1994","unstructured":"Finley R. L., Brent R., Proc. Natl. Acad. Sci. U.S.A. 91, 12980 (1994).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_20_2","unstructured":"The interaction between LET-23(RTK) and SEM-5(Grb2) is dependent upon tyrosine phosphorylation ["},{"key":"e_1_3_1_20_3","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1016\/0092-8674(92)90167-B","volume":"70","author":"Lowenstein E. J.","year":"1992","unstructured":"Lowenstein E. J., et al., Cell 70, 431 (1992);","journal-title":"Cell"},{"key":"e_1_3_1_20_4","unstructured":"] which is seemingly absent in yeast cells. False negatives can also occur from the fact that the two-hybrid system is based upon a transcriptional activation event that occurs in the nucleus. Hence interactions involving HDAC its recruiting proteins or transcriptional repressors such as LIN-1 may result in transcriptional repression in yeast cells and thus be incompatible with transcriptional activation for example LIN-1(Ets-like)\/LIN-31(Forkhead\/HNF) ["},{"key":"e_1_3_1_20_5","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81186-1"},{"key":"e_1_3_1_20_6","unstructured":"] and LIN-35(pRB)\/LIN-53(RbAp48) (12). Proteins with transmembrane domains might not be exported correctly to the yeast nucleus as DB- or AD-fusions for example SEL-12(presenilin)\/LIN-12(Notch) and SEL-12(presenilin)\/SEL-10 ["},{"key":"e_1_3_1_20_7","doi-asserted-by":"crossref","first-page":"3263","DOI":"10.1073\/pnas.96.6.3263","volume":"96","author":"Ray W. J.","year":"1999","unstructured":"Ray W. J., et al., Proc. Natl. Acad. Sci. U.S.A. 96, 3263 (1999);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_20_8","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.26.15787"},{"key":"e_1_3_1_20_9","unstructured":"]. In addition the failure to detect an interaction can sometimes be attributed to the fact that in the matrix setting shown here full-length vORFs rather than discrete domains were expressed in fusion with DB and AD. For example several discrete domains of E2F family members exhibit a stronger two-hybrid read-out with pRB than the corresponding full-length proteins ["},{"key":"e_1_3_1_20_10","doi-asserted-by":"crossref","first-page":"10321","DOI":"10.1073\/pnas.93.19.10321","volume":"93","author":"Vidal M.","year":"1996","unstructured":"Vidal M., Braun P., Chen E., Boeke J. D., Harlow E., Proc. Natl. Acad. Sci. U.S.A. 93, 10321 (1996);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_20_11","unstructured":"]. Finally the version of the two-hybrid system used here might affect the read-out of particular interactions. Hence the LET-23(RTK)\/LIN-7 interaction which requires the COOH-terminal end of the receptor ["},{"key":"e_1_3_1_20_12","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81096-X"},{"key":"e_1_3_1_20_13","unstructured":"] might not be detectable due to the B2-encoded peptide resulting from RC. It should be noted that the evidence for the interactions reported in the literature is sometimes limited to other versions of the yeast two-hybrid system."},{"key":"e_1_3_1_21_2","unstructured":"We have developed an improved version of the two-hybrid system (17). Three reporter genes expressed from different Gal4p-inducible promoters are used to increase the selectivity of the assay. In addition single-copy DB- and AD-vectors are used to obtain relatively low expression levels of the two-hybrid proteins thus improving the specificity of the selection. This version gives rise to decreased rates of biologically irrelevant interactions (6) ["},{"key":"e_1_3_1_21_3","doi-asserted-by":"crossref","first-page":"2403","DOI":"10.1073\/pnas.92.6.2403","volume":"92","author":"Sardet C.","year":"1995","unstructured":"Sardet C., et al., Proc. Natl. Acad. Sci. U.S.A. 92, 2403 (1995);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_21_4","doi-asserted-by":"crossref","first-page":"11960","DOI":"10.1073\/pnas.92.26.11960","volume":"92","author":"Englert C.","year":"1995","unstructured":"Englert C., et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11960 (1995);","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_21_5","doi-asserted-by":"publisher","DOI":"10.1101\/gad.10.10.1206"},{"key":"e_1_3_1_21_6","doi-asserted-by":"crossref","first-page":"15299","DOI":"10.1073\/pnas.93.26.15299","volume":"93","author":"Kim S. S.","year":"1996","unstructured":"Kim S. S., et al., Proc. Natl. Acad. Sci. 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Virol."},{"key":"e_1_3_1_21_10","doi-asserted-by":"crossref","first-page":"1128","DOI":"10.1101\/gr.9.11.1128","volume":"9","author":"Walhout A. J. M.","year":"1999","unstructured":"Walhout A. J. M., Vidal M., Genome Res. 9, 1128 (1999);","journal-title":"Genome Res."},{"key":"e_1_3_1_21_11","unstructured":"]. We have also developed a semi-automated version of the two-hybrid system in which most steps are carried out on 96-well plates to increase the throughput of the assay (A. J. M. Walhout et al. data not shown) . The ISTs were retransformed into fresh DB-vORF-containing yeast cells and retested for two-hybrid readouts. This step eliminates false positives probably resulting from cis - and trans -acting mutations that can influence reporter gene activity (17)."},{"key":"e_1_3_1_22_2","unstructured":"R. Durbin and J. Thierry-Mieg in Computational Methods in Genome Research S. Suhai Ed. (Plenum New York 1994)."},{"key":"e_1_3_1_23_2","unstructured":""},{"key":"e_1_3_1_24_2","unstructured":"www.proteome.com\/databases\/index.htm"},{"key":"e_1_3_1_25_2","unstructured":""},{"key":"e_1_3_1_26_2","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/S0092-8674(00)81227-1","volume":"94","author":"Sieburth D. S.","year":"1998","unstructured":"Sieburth D. S., Sun Q., Han M., Cell 94, 119 (1998).","journal-title":"Cell"},{"key":"e_1_3_1_27_2","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1016\/S0092-8674(00)80098-7","volume":"86","author":"Bai C.","year":"1996","unstructured":"Bai C., et al., Cell 86, 263 (1996).","journal-title":"Cell"},{"key":"e_1_3_1_28_2","first-page":"7483","volume":"14","author":"Kikuchi A.","year":"1994","unstructured":"Kikuchi A., Demo S. D., Ye Z. H., Chen Y. W., Williams L. T., Mol. Cell. Biol. 14, 7483 (1994).","journal-title":"Mol. Cell. Biol."},{"key":"e_1_3_1_29_2","doi-asserted-by":"crossref","first-page":"2483","DOI":"10.1242\/dev.126.11.2483","volume":"126","author":"Solari F.","year":"1999","unstructured":"Solari F., Bateman A., Ahringer J., Development 126, 2483 (1999).","journal-title":"Development"},{"key":"e_1_3_1_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(00)80299-3"},{"key":"e_1_3_1_31_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.278.5338.631"},{"key":"e_1_3_1_32_2","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/138.3.609"},{"key":"e_1_3_1_32_3","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(94)90579-7"},{"key":"e_1_3_1_33_2","unstructured":"For yet unknown reasons several proteins notoriously behave as promiscuous interactors in the context of the two-hybrid system. Because such promiscuous interactors would bias the clustering analysis they should systematically be removed from the analysis . In the AD-wrmcDNA library this is the case for AD-PAL-1 AD-UNC-15 AD-F33G12.5 and AD-F54D5.5 which were found as potential ISTs for several vORF baits but also for unrelated baits such as DB-LIN-5 DB-LIN-6 DB-MEX-3 DB-OSM-10 and DB-PCT-1 (A. J. M. Walhout M. Lorson S. van den Heuvel N. Huang C. Hunter A. Sugimoto A. Hart M. Vidal unpublished data)."},{"key":"e_1_3_1_34_2","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1093\/genetics\/137.4.987","volume":"137","author":"Clark S. G.","year":"1994","unstructured":"Clark S. G., Lu X., Horvitz H. R., Genetics 137, 987 (1994);","journal-title":"Genetics"},{"key":"e_1_3_1_34_3","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1091\/mbc.5.4.395","volume":"5","author":"Huang L. S.","year":"1994","unstructured":"Huang L. S., Tzou P., Sternberg P. W., Mol. Biol. Cell 5, 395 (1994);","journal-title":"Mol. Biol. Cell"},{"key":"e_1_3_1_34_4","unstructured":"; X. Lu and H. R. Horvitz in preparation."},{"key":"e_1_3_1_35_2","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1038\/348169a0","volume":"348","author":"Herman R. K.","year":"1990","unstructured":"Herman R. K., Hedgecock E. M., Nature 348, 169 (1990).","journal-title":"Nature"},{"key":"e_1_3_1_36_2","unstructured":"In summary the two-hybrid readouts are excellent for LIN-53(RbAp48)\/LIN-37 (interaction was detected multiple times in both DB-X\/AD-Y and DB-Y\/AD-X orientations with full-length proteins in the matrix scores positive for three reporter genes and belongs to clusters) and the available biological information on both proteins is consistent with an interaction (both proteins are required for a synMuv function and co-localize). Because no interolog has been identified yet for this interaction it is possible that its study in C. elegans might reveal new features of pRB function in humans."},{"key":"e_1_3_1_37_2","unstructured":"A. J. M. Walhout H. Endoh M. Vidal data not shown."},{"key":"e_1_3_1_38_2","unstructured":"We thank N. Dyson F. Holstege L. Matthews and S. van den Heuvel for comments on the manuscript; M. Chalfie A. Coulson E. Harlow H. R. Horvitz S. Kim E. Lander M. Murray R. Plasterk J. and D. Thierry-Mieg and S. van den Heuvel for support; J. Hitti L. Doucette-Stamm and B. Guild (Genome Therapeutics Corp. Waltham MA) for their help with sequencing; H. Endoh H. R. Horvitz S. Kim M. Lorson and S. van den Heuvel for reagents or unpublished information or both; and C. Ngwu for generous help with the design of our Web site. Supported by grants 1 RO1 HG01715-01 (National Human Genome Research Institute) and 1 R21 CA81658 A 01 (National Cancer Institute) awarded to M.V."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.287.5450.116","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T09:52:40Z","timestamp":1705139560000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.287.5450.116"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,1,7]]},"references-count":75,"journal-issue":{"issue":"5450","published-print":{"date-parts":[[2000,1,7]]}},"alternative-id":["10.1126\/science.287.5450.116"],"URL":"https:\/\/doi.org\/10.1126\/science.287.5450.116","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,1,7]]}}}