Chapter 34 : Cool Tools 5: The ORFeome Project

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This chapter presents the strategy developed to generate a ORFeome collection in a versatile Gateway vector, which allows the transfer of the cloned genes into a variety of Gateway-compatible expression vectors. The development of collections of over-expression strains is facilitated by the pre-establishment of a complete set of cloned open reading frames (ORFs), or ORFeome. Importantly, ORFeomes represent useful resources for the implementation of other approaches used to elucidate gene function apart from gene deletion or overexpression. Individual characterization of mutants on the genome-wide scale can be slow and laborious. In this respect, signature-tagged mutagenesis (STM) provides an attractive alternative. In STM, each mutant is tagged with a different DNA sequence, allowing all tags to be amplified from the DNA of mixed populations of mutants in a single PCR. In the ORFeome, the start codon of the 6,205 ORFs has been included, whereas the stop codon has been excluded to allow insertion of C-terminal tags. The authors have favored C-terminal tagging since tags at the amino termini may possibly interfere with targeting of proteins to the secretory pathway. A panel of libraries of strains expressing tagged proteins should facilitate the systematic execution of high-throughput biochemical and microscopic assays of the proteome. The construction of the ORFeome could pave the way to the generation of the promoterome (library of gene promoters) in order to better characterize expression profiles.

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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Image of FIGURE 1

Workflow of the ORFeome project. Shown is the overall scheme for high-throughput recombinational cloning of the ORFs using the Gateway system. Quality control is monitored and documented using PCR and DNA sequencing. The main steps are briefly summarized on the right in the order they are carried out. doi:10.1128/9781555817176.ch34.f1

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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Image of FIGURE 2

Plasmids used in this work. (A) Schematic map of the CIp10-TETp-GTW destination vector used for overexpression of ORFs. ORFs cloned in a Gateway donor vector can be transferred through Gateway-mediated recombination at and of CIp10-TETp-GTW overexpression vectors. The gene is used for selection of transformants. Derivatives of these overexpression vectors can be targeted to the locus when linearized with StuI or I-SceI. Expression of the cloned ORFs is achieved when cells are grown in the presence of doxycycline, which binds to the tetracycline-dependent transactivator rtTA, allowing transcriptional activation at the Tetp promoter. Each expression plasmid is tagged with a unique 12-bp barcode, enabling growth phenotypes of individual strains to be analyzed in parallel. (B) Structure of the tetracycline-dependent transactivator cassette contained in plasmid pNIMX. Unique restriction sites to excise the entire cassette are indicated. , basal promoter of ; , nourseothricin resistance gene; -adapted reverse tetracycline-dependent transactivator gene. doi:10.1128/9781555817176.ch34.f2

Citation: Legrand M, Munro C, d’Enfert C. 2012. Cool Tools 5: The ORFeome Project, p 505-510. In Calderone R, Clancy C (ed), and Candidiasis, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817176.ch34
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1. Aguiar, J. C.,, J. LaBaer,, P. L. Blair,, V. Y. Shamailova,, M. Koundinya,, J. A. Russell,, F. Huang,, W. Mar,, R. M. Anthony,, A. Witney,, S. R. Caruana,, L. Brizuela,, J. B. Sacci, Jr.,, S. L. Hoffman, and, D. J. Carucci. 2004. High-throughput generation of P. falciparum functional molecules by recombinational cloning. Genome Res. 14: 20762082.
2. Camacho, L. R.,, D. Ensergueix,, E. Perez,, B. Gicquel, and, C. Guilhot. 1999. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34: 257267.
3. Chua, G.,, Q. D. Morris,, R. Sopko,, M. D. Robinson,, O. Ryan,, E. T. Chan,, B. J. Frey,, B. J. Andrews,, C. Boone, and, T. R. Hughes. 2006. Identifying transcription factor functions and targets by phenotypic activation. Proc. Natl. Acad. Sci. USA 103: 1204512050.
4. Cox, J. S.,, B. Chen,, M. McNeil, and, W. R. Jacobs, Jr. 1999. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402: 7983.
5. Decourty, L.,, C. Saveanu,, K. Zemam,, F. Hantraye,, E. Frachon,, J. C. Rousselle,, M. Fromont-Racine, and, A. Jacquier. 2008. Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles. Proc. Natl. Acad. Sci. USA 105: 58215826.
6. Dephoure, N.,, R. W. Howson,, J. D. Blethrow,, K. M. Shokat, and, E. K. O’Shea. 2005. Combining chemical genetics and proteomics to identify protein kinase substrates. Proc. Natl. Acad. Sci. USA 102: 1794017945.
7. Deutschbauer, A. M.,, D. F. Jaramillo,, M. Proctor,, J. Kumm,, M. E. Hillenmeyer,, R. W. Davis,, C. Nislow, and, G. Giaever. 2005. Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast. Genetics 169: 19151925.
8. Dricot, A.,, J. F. Rual,, P. Lamesch,, N. Bertin,, D. Dupuy,, T. Hao,, C. Lambert,, R. Hallez,, J. M. Delroisse,, J. Vandenhaute,, I. Lopez-Goni,, I. Moriyon,, J. M. Garcia-Lobo,, F. J. Sangari,, A. P. Macmillan,, S. J. Cutler,, A. M. What-more,, S. Bozak,, R. Sequerra,, L. Doucette-Stamm,, M. Vidal,, D. E. Hill,, J. J. Letesson, and, X. De Bolle. 2004. Generation of the Brucella melitensis ORFeome version 1.1. Genome Res. 14: 22012206.
9. Dupuy, D.,, Q. R. Li,, B. Deplancke,, M. Boxem,, T. Hao,, P. Lamesch,, R. Sequerra,, S. Bosak,, L. Doucette-Stamm,, I. A. Hope,, D. E. Hill,, A. J. Walhout, and, M. Vidal. 2004. A first version of the Caenorhabditis elegans Promoterome. Genome Res. 14: 21692175.
10. Enjalbert, B.,, A. Rachini,, G. Vediyappan,, D. Pietrella,, R. Spaccapelo,, A. Vecchiarelli,, A. J. Brown, and, C. d’Enfert. 2009. A multifunctional, synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections. Infect. Immun. 77: 48474858.
11. Fields, S., and, O. Song. 1989. A novel genetic system to detect protein-protein interactions. Nature 340: 245246.
12. Gelperin, D. M.,, M. A. White,, M. L. Wilkinson,, Y. Kon,, L. A. Kung,, K. J. Wise,, N. Lopez-Hoyo,, L. Jiang,, S. Piccirillo,, H. Yu,, M. Gerstein,, M. E. Dumont,, E. M. Phizicky,, M. Snyder, and, E. J. Grayhack. 2005. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19: 28162826.
13. Hillenmeyer, M. E.,, E. Fung,, J. Wildenhain,, S. E. Pierce,, S. Hoon,, W. Lee,, M. Proctor,, R. P. St. Onge,, M. Tyers,, D. Koller,, R. B. Altman,, R. W. Davis,, C. Nislow, and, G. Giaever. 2008. The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320: 362365.
14. Hudson, J. R., Jr.,, E. P. Dawson,, K. L. Rushing,, C. H. Jackson,, D. Lockshon,, D. Conover,, C. Lanciault,, J. R. Harris,, S. J. Simmons,, R. Rothstein, and, S. Fields. 1997. The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. Genome Res. 7: 11691173.
15. Huh, W. K.,, J. V. Falvo,, L. C. Gerke,, A. S. Carroll,, R. W. Howson,, J. S. Weissman, and, E. K. O’Shea. 2003. Global analysis of protein localization in budding yeast. Nature 425: 686691.
16. Jones, T.,, N. A. Federspiel,, H. Chibana,, J. Dungan,, S. Kalman,, B. B. Magee,, G. Newport,, Y. R. Thorstenson,, N. Agabian,, P. T. Magee,, R. W. Davis, and, S. Scherer. 2004. The diploid genome sequence of Candida albicans. Proc. Natl. Acad. Sci. USA 101: 73297334.
17. Lamesch, P.,, N. Li,, S. Milstein,, C. Fan,, T. Hao,, G. Szabo,, Z. Hu,, K. Venkatesan,, G. Bethel,, P. Martin,, J. Rogers,, S. Lawlor,, S. McLaren,, A. Dricot,, H. Borick,, M. E. Cusick,, J. Vandenhaute,, I. Dunham,, D. E. Hill, and, M. Vidal. 2007. hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes. Genomics 89: 307315.
18. Martzen, M. R.,, S. M. McCraith,, S. L. Spinelli,, F. M. Torres,, S. Fields,, E. J. Grayhack, and, E. M. Phizicky. 1999. A biochemical genomics approach for identifying genes by the activity of their products. Science 286: 11531155.
19. Matsuyama, A.,, R. Arai,, Y. Yashiroda,, A. Shirai,, A. Kamata,, S. Sekido,, Y. Kobayashi,, A. Hashimoto,, M. Hamamoto,, Y. Hiraoka,, S. Horinouchi, and, M. Yoshida. 2006. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol. 24: 841847.
20. Mazurkiewicz, P.,, C. M. Tang,, C. Boone, and, D. W. Holden. 2006. Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat. Rev. Genet. 7: 929939.
21. Murad, A. M.,, P. R. Lee,, I. D. Broadbent,, C. J. Barelle, and, A. J. Brown. 2000. CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16: 325327.
22. Noble, S. M.,, S. French,, L. A. Kohn,, V. Chen, and, A. D. Johnson. 2010. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat. Genet. 42: 590598.
23. Oh, J.,, E. Fung,, U. Schlecht,, R. W. Davis,, G. Giaever,, R. P. St. Onge,, A. Deutschbauer, and, C. Nislow. 2010. Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection. PLoS Pathog. 6: e1001140.
24. Park, Y. N., and, J. Morschhauser. 2005. Tetracycline-inducible gene expression and gene deletion in Candida albicans. Eukaryot. Cell 4: 13281342.
25. Pellet, J.,, L. Tafforeau,, M. Lucas-Hourani,, V. Navratil,, L. Meyniel,, G. Achaz,, A. Guironnet-Paquet,, A. Aublin-Gex,, G. Caignard,, P. Cassonnet,, A. Chaboud,, T. Chantier,, A. Deloire,, C. Demeret,, M. Le Breton,, G. Neveu,, L. Jacotot,, P. Vaglio,, S. Delmotte,, C. Gautier,, C. Combet,, G. Deleage,, M. Favre,, F. Tangy,, Y. Jacob,, P. Andre,, V. Lotteau,, C. Rabourdin-Combe, and, P. O. Vidalain. 2010. ViralORFeome: an integrated database to generate a versatile collection of viral ORFs. Nucleic Acids Res. 38: D371–D378.
26. Rajagopala, S. V.,, N. Yamamoto,, A. E. Zweifel,, T. Nakamichi,, H. K. Huang,, J. D. Mendez-Rios,, J. Franca-Koh,, M. P. Boorgula,, K. Fujita,, K. Suzuki,, J. C. Hu,, B. L. Wanner,, H. Mori, and, P. Uetz. 2010. The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology. BMC Genomics 11: 470.
27. Reboul, J.,, P. Vaglio,, J. F. Rual,, P. Lamesch,, M. Martinez,, C. M. Armstrong,, S. Li,, L. Jacotot,, N. Bertin,, R. Janky,, T. Moore,, J. R. Hudson, Jr.,, J. L. Hartley,, M. A. Brasch,, J. Vandenhaute,, S. Boulton,, G. A. Endress,, S. Jenna,, E. Chevet,, V. Papasotiropoulos,, P. P. Tolias,, J. Ptacek,, M. Snyder,, R. Huang,, M. R. Chance,, H. Lee,, L. Doucette-Stamm,, D. E. Hill, and, M. Vidal. 2003. C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat. Genet. 34: 3541.
28. Roemer, T.,, B. Jiang,, J. Davison,, T. Ketela,, K. Veillette,, A. Breton,, F. Tandia,, A. Linteau,, S. Sillaots,, C. Marta,, N. Martel,, S. Veronneau,, S. Lemieux,, S. Kauffman,, J. Becker,, R. Storms,, C. Boone, and, H. Bussey. 2003. Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery. Mol. Microbiol. 50: 167181.
29. Shea, J. E.,, M. Hensel,, C. Gleeson, and, D. W. Holden. 1996. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 93: 25932597.
30. Smith, A. M.,, L. E. Heisler,, J. Mellor,, F. Kaper,, M. J. Thompson,, M. Chee,, F. P. Roth,, G. Giaever, and, C. Nislow. 2009. Quantitative phenotyping via deep barcode sequencing. Genome Res. 19: 18361842.
31. Sopko, R.,, D. Huang,, N. Preston,, G. Chua,, B. Papp,, K. Kafadar,, M. Snyder,, S. G. Oliver,, M. Cyert,, T. R. Hughes,, C. Boone, and, B. Andrews. 2006. Mapping pathways and phenotypes by systematic gene overexpression. Mol. Cell 21: 319330.
32. Stevenson, L. F.,, B. K. Kennedy, and, E. Harlow. 2001. A large-scale overexpression screen in Saccharomyces cerevisiae identifies previously uncharacterized cell cycle genes. Proc. Natl. Acad. Sci. USA 98: 39463951.
33. Stynen, B.,, P. Van Dijck, and, H. Tournu. 2010. A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans. Nucleic Acids Res. 38: e184.
34. van het Hoog, M.,, T. J. Rast,, M. Martchenko,, S. Grin-dle,, D. Dignard,, H. Hogues,, C. Cuomo,, M. Berriman,, S. Scherer,, B. B. Magee,, M. Whiteway,, H. Chibana,, A. Nantel, and, P. T. Magee. 2007. Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes. Genome Biol. 8: R52.
35. Walhout, A. J.,, G. F. Temple,, M. A. Brasch,, J. L. Hartley,, M. A. Lorson,, S. van den Heuvel, and, M. Vidal. 2000. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328: 575592.
36. Xu, D.,, B. Jiang,, T. Ketela,, S. Lemieux,, K. Veillette,, N. Martel,, J. Davison,, S. Sillaots,, S. Trosok,, C. Bachewich,, H. Bussey,, P. Youngman, and, T. Roemer. 2007. Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans. PLoS Pathog. 3: e92.
37. Zhu, H.,, M. Bilgin,, R. Bangham,, D. Hall,, A. Casamayor,, P. Bertone,, N. Lan,, R. Jansen,, S. Bidlingmaier,, T. Houfek,, T. Mitchell,, P. Miller,, R. A. Dean,, M. Gerstein, and, M. Snyder. 2001. Global analysis of protein activities using proteome chips. Science 293: 21012105.

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