Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease.
References
[1]
Smith MT (2010) Advances in Understanding Benzene Health Effects and Susceptibility. Annual review of public health 31: 133–148.
[2]
Snyder R, Hedli C (1996) An overview of benzene metabolism. Environ Health Perspect 104: Suppl 61165–1171.
[3]
Eastmond DA, Smith MT, Irons RD (1987) An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure. Toxicology and applied pharmacology 91: 85–95.
[4]
Smith M (1996) The mechanism of benzene-induced leukemia: a hypothesis and speculations on the causes of leukemia. Environ Health Perspect 104: Suppl 61219–1225.
[5]
Ross D (2005) Functions and distribution of NQO1 in human bone marrow: potential clues to benzene toxicity. Chem Biol Interact 153-154: 137–146.
[6]
Lan Q (2004) Hematotoxicity in Workers Exposed to Low Levels of Benzene. Science 306: 1774–1776.
[7]
Gill DP, Ahmed AE (1981) Covalent binding of [14C]benzene to cellular organelles and bone marrow nucleic acids. Biochem Pharmacol 30: 1127–1131.
[8]
Shen Y, Shen HM, Shi CY, Ong CN (1996) Benzene metabolites enhance reactive oxygen species generation in HL60 human leukemia cells. Hum Exp Toxicol 15: 422–427.
[9]
Gaskell M, McLuckie KIE, Farmer PB (2005) Genotoxicity of the benzene metabolites para-benzoquinone and hydroquinone. Chem Biol Interact 153-154: 267–270.
[10]
Singh MP, Reddy MMK, Mathur N, Saxena DK, Chowdhuri DK (2009) Induction of hsp70, hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed Drosophila melanogaster: role of ROS generation. Toxicology and applied pharmacology 235: 226–243.
[11]
Jemnitz K, Veres Z, Torok G, Toth E, Vereczkey L (2004) Comparative study in the Ames test of benzo[a]pyrene and 2-aminoanthracene metabolic activation using rat hepatic S9 and hepatocytes following in vivo or in vitro induction. Mutagenesis 19: 245–250.
[12]
Hirakawa K, Oikawa S, Hiraku Y, Hirosawa I, Kawanishi S (2002) Catechol and hydroquinone have different redox properties responsible for their differential DNA-damaging ability. Chem Res Toxicol 15: 76–82.
[13]
Gaskell M, McLuckie K, Farmer P (2005) Comparison of the repair of DNA damage induced by the benzene metabolites hydroquinone and p-benzoquinone: a role for hydroquinone in benzene genotoxicity. Carcinogenesis 26: 673–680.
[14]
Brennan RJ, Schiestl RH (1998) Free radicals generated in yeast by the Salmonella test-negative carcinogens benzene, urethane, thiourea and auramine O. Mutat Res 403: 65–73.
[15]
Snyder R (2007) Benzene's toxicity: a consolidated short review of human and animal studies by HA Khan. Hum Exp Toxicol 26: 687–696.
[16]
Giaever G, Chu AM, Ni L, Connelly C, Riles L, et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387–391.
[17]
Birrell GW, Brown JA, Wu HI, Giaever G, Chu AM, et al. (2002) Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents. Proc Natl Acad Sci USA 99: 8778–8783.
[18]
Holland S, Lodwig E, Sideri T, Reader T, Clarke I, et al. (2007) Application of the comprehensive set of heterozygous yeast deletion mutants to elucidate the molecular basis of cellular chromium toxicity. Genome Biol 8: R268.
[19]
Hoon S, St Onge R, Giaever G, Nislow C (2008) Yeast chemical genomics and drug discovery: an update. Trends in Pharmacological Sciences 29: 499–504.
[20]
Jo WJ, Loguinov A, Chang M, Wintz H, Nislow C, et al. (2008) Identification of genes involved in the toxic response of Saccharomyces cerevisiae against iron and copper overload by parallel analysis of deletion mutants. Toxicol Sci 101: 140–151.
[21]
Jo W, Loguinov A, Wintz H, Chang M, Smith A, et al. (2009) Comparative Functional Genomic Analysis Identifies Distinct and Overlapping Sets of Genes Required for Resistance to Monomethylarsonous Acid (MMAIII) and Arsenite (AsIII) in Yeast. Toxicol Sci 111: 424–436.
[22]
Jo W, Ren X, Chu F, Aleshin M, Wintz H, et al. (2009) Acetylated H4K16 by MYST1 protects UROtsa cells from arsenic toxicity and is decreased following chronic arsenic exposure. Toxicology and Applied Pharmacology 241: 294–302.
[23]
Patridge EV, Ferry JG (2006) WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. J Bacteriol 188: 3498–3506.
[24]
Varela E, Tien M (2003) Effect of pH and oxalate on hydroquinone-derived hydroxyl radical formation during brown rot wood degradation. Appl Environ Microbiol 69: 6025–6031.
[25]
Lee J, Godon C, Lagniel G, Spector D, Garin J, et al. (1999) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274: 16040–16046.
[26]
Delaunay A, Pflieger D, Barrault MB, Vinh J, Toledano MB (2002) A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111: 471–481.
[27]
Okazaki S, Naganuma A, Kuge S (2005) Peroxiredoxin-mediated redox regulation of the nuclear localization of Yap1, a transcription factor in budding yeast. Antioxid Redox Signal 7: 327–334.
[28]
Veal EA, Ross SJ, Malakasi P, Peacock E, Morgan BA (2003) Ybp1 is required for the hydrogen peroxide-induced oxidation of the Yap1 transcription factor. J Biol Chem 278: 30896–30904.
[29]
Menezes RA, Amaral C, Batista-Nascimento L, Santos C, Ferreira RB, et al. (2008) Contribution of Yap1 towards Saccharomyces cerevisiae adaptation to arsenic-mediated oxidative stress. Biochem J 414: 301–311.
[30]
Akada R, Shimizu Y, Matsushita Y, Kawahata M, Hoshida H, et al. (2001) Use of aYAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeastSaccharomyces cerevisiae. Yeast 19: 17–28.
[31]
Bossier P, Fernandes L, Rocha D, Rodrigues-Pousada C (1993) Overexpression of YAP2, coding for a new yAP protein, and YAP1 in Saccharomyces cerevisiae alleviates growth inhibition caused by 1,10-phenanthroline. J Biol Chem 268: 23640–23645.
[32]
Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, et al. (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes & Development 19: 2816–2826.
[33]
Fernandes L, Rodrigues-Pousada C, Struhl K (1997) Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions. Mol Cell Biol 17: 6982–6993.
[34]
Wysocki R, Fortier P-K, Maciaszczyk E, Thorsen M, Leduc A, et al. (2004) Transcriptional activation of metalloid tolerance genes in Saccharomyces cerevisiae requires the AP-1-like proteins Yap1p and Yap8p. Mol Biol Cell 15: 2049–2060.
[35]
Gulshan K, Rovinsky SA, Coleman ST, Moye-Rowley WS (2005) Oxidant-specific folding of Yap1p regulates both transcriptional activation and nuclear localization. J Biol Chem 280: 40524–40533.
[36]
Vignols F, Bréhélin C, Surdin-Kerjan Y, Thomas D, Meyer Y (2005) A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo. Proc Natl Acad Sci USA 102: 16729–16734.
[37]
Wong C-M, Siu K-L, Jin D-Y (2004) Peroxiredoxin-null yeast cells are hypersensitive to oxidative stress and are genomically unstable. J Biol Chem 279: 23207–23213.
[38]
Biteau B, Labarre J, Toledano MB (2003) ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin. Nature 425: 980–984.
[39]
Cha M-K, Hong S-K, Oh Y-M, Kim I-H (2003) The protein interaction of Saccharomyces cerevisiae cytoplasmic thiol peroxidase II with SFH2p and its in vivo function. J Biol Chem 278: 34952–34958.
[40]
Dougherty D, Garte S, Barchowsky A, Zmuda J, Taioli E (2008) NQO1, MPO, CYP2E1, GSTT1 and GSTM1 polymorphisms and biological effects of benzene exposure--a literature review. Toxicol Lett 182: 7–17.
[41]
Kumar M, Chauhan LKS, Paul BN, Agarwal SK, Goel SK (2009) GSTM1, GSTT1, and GSTP1 polymorphism in north Indian population and its influence on the hydroquinone-induced in vitro genotoxicity. Toxicology Mechanisms and Methods 19: 59–65.
[42]
Gardner RG, Swarbrick GM, Bays NW, Cronin SR, Wilhovsky S, et al. (2000) Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p. J Cell Biol 151: 69–82.
[43]
Chen Y, Feldman DE, Deng C, Brown JA, De Giacomo AF, et al. (2005) Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae. Mol Cancer Res 3: 669–677.
[44]
Sul D, Shim I, Im H, Won N, Kim H, et al. (2005) Benzene increases the ratio of arachidonic acids to docosahexaenoic acids and inhibits the de novo synthesis of ceramide in the rat liver. J Appl Toxicol 25: 74–81.
[45]
Girao H, Geli M-I, Idrissi F-Z (2008) Actin in the endocytic pathway: from yeast to mammals. FEBS Lett 582: 2112–2119.
[46]
Yamaguchi-Iwai Y, Dancis A, Klausner R (1995) AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J 14: 1231–1239.
[47]
De Freitas J, Kim J, Poynton H, Su T, Wintz H, et al. (2004) Exploratory and confirmatory gene expression profiling of mac1Delta. J Biol Chem 279: 4450–4458.
[48]
Eide DJ, Bridgham JT, Zhao Z, Mattoon JR (1993) The vacuolar H(+)-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism. Mol Gen Genet 241: 447–456.
[49]
Szczypka MS, Zhu Z, Silar P, Thiele DJ (1997) Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription. Yeast 13: 1423–1435.
[50]
Kaplan CD, Kaplan J (2009) Iron acquisition and transcriptional regulation. Chem Rev 109: 4536–4552.
[51]
Singh V, Ahmad S, Rao G (1994) Prooxidant and antioxidant properties of iron-hydroquinone and iron-1,2,4-benzenetriol complex. Implications for benzene toxicity. Toxicology 89: 25–33.
[52]
Sánchez P, Gálvez N, Colacio E, Mi？ones E, Domínguez-Vera JM (2005) Catechol releases iron(III) from ferritin by direct chelation without iron(II) production. Dalton Trans. pp. 811–813.
[53]
Tan S-X, Teo M, Lam YT, Dawes IW, Perrone GG (2009) Cu, Zn superoxide dismutase and NADP(H) homeostasis are required for tolerance of endoplasmic reticulum stress in Saccharomyces cerevisiae. Mol Biol Cell 20: 1493–1508.
[54]
Irons RD, Neptun DA (1980) Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization. Arch Toxicol 45: 297–305.
[55]
Pfeiffer E, Metzler M (1996) Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro. Chem Biol Interact 102: 37–53.
[56]
Zhang L, Robertson ML, Kolachana P, Davison AJ, Smith MT (1993) Benzene metabolite, 1,2,4-benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells. Environ Mol Mutagen 21: 339–348.
[57]
Shiga T, Suzuki H, Yamamoto A, Yamamoto H, Yamamoto K (2010) Hydroquinone, a Benzene Metabolite, Induces Hog1-dependent Stress Response Signaling and Causes Aneuploidy in Saccharomyces cerevisiae. JRR 51: 405–415.
[58]
Garg TK, Chang JY (2003) Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12, 14-PGJ2. BMC Ophthalmol 3: 5.
[59]
Pons M, Cousins SW, Csaky KG, Striker G, Marin-Casta？o ME (2010) Cigarette Smoke-Related Hydroquinone Induces Filamentous Actin Reorganization and Heat Shock Protein 27 Phosphorylation through p38 and Extracellular Signal-Regulated Kinase 1/2 in Retinal Pigment Epithelium. Implications for Age-Related Macular Degeneration. The American journal of pathology 177: 1198–1213.
[60]
Grandori R, Carey J (1994) Six new candidate members of the alpha/beta twisted open-sheet family detected by sequence similarity to flavodoxin. Protein Sci 3: 2185–2193.
[61]
Andrade SLA, Patridge EV, Ferry JG, Einsle O (2007) Crystal structure of the NADH:quinone oxidoreductase WrbA from Escherichia coli. J Bacteriol 189: 9101–9107.
[62]
Carey J, Brynda J, Wolfová J, Grandori R, Gustavsson T, et al. (2007) WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H:quinone oxidoreductases. Protein Sci 16: 2301–2305.
[63]
Deller S, Macheroux P, Sollner S (2008) Flavin-dependent quinone reductases. Cell Mol Life Sci 65: 141–160.
[64]
Wan J, Shi J, Hui L, Wu D, Jin X, et al. (2002) Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning. Environ Health Perspect 110: 1213–1218.
[65]
Akileswaran L, Brock BJ, Cereghino JL, Gold MH (1999) 1,4-benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression. Appl Environ Microbiol 65: 415–421.
[66]
Sollner S, Nebauer R, Ehammer H, Prem A, Deller S, et al. (2007) Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification. FEBS J 274: 1328–1339.
[67]
Zappa F, Ward T, Butler J, Pedrinis E, McGown A (2001) Overexpression of NAD(P)H:quinone oxidoreductase 1 in human reproductive system. J Histochem Cytochem 49: 1187–1188.
[68]
Seow HA, Penketh PG, Belcourt MF, Tomasz M, Rockwell S, et al. (2004) Nuclear overexpression of NAD(P)H:quinone oxidoreductase 1 in Chinese hamster ovary cells increases the cytotoxicity of mitomycin C under aerobic and hypoxic conditions. J Biol Chem 279: 31606–31612.
[69]
Gopalakrishna R, Chen ZH, Gundimeda U (1994) Tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase C and influence invasion and metastasis of lung carcinoma cells. Proc Natl Acad Sci USA 91: 12233–12237.
[70]
Gillis B, Gavin IM, Arbieva Z, King ST, Jayaraman S, et al. (2007) Identification of human cell responses to benzene and benzene metabolites. Genomics 90: 324–333.
[71]
Ren X, Lim S, Smith MT, Zhang L (2009) Werner syndrome protein, WRN, protects cells from DNA damage induced by the benzene metabolite hydroquinone. Toxicol Sci 107: 367–375.
[72]
Barreto G, Madureira D, Capani F, Aon-Bertolino L, Saraceno E, et al. (2009) The role of catechols and free radicals in benzene toxicity: An oxidative DNA damage pathway. Environ Mol Mutagen 50: 771–780.
[73]
Badham HJ, Winn LM (2010) In Utero Exposure to Benzene Disrupts Fetal Hematopoietic Progenitor Cell Growth via Reactive Oxygen Species. Toxicol Sci 113: 207–215.
[74]
Thorpe GW, Fong CS, Alic N, Higgins VJ, Dawes IW (2004) Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes. Proc Natl Acad Sci USA 101: 6564–6569.
[75]
Toledano MB, Kumar C, Le Moan N, Spector D, Tacnet F (2007) The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis. FEBS Lett 581: 3598–3607.
[76]
Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, Macisaac KD, et al. (2004) Transcriptional regulatory code of a eukaryotic genome. Nature 431: 99–104.
[77]
Yang X, Talibi D, Weber S, Poisson G, Raymond M (2001) Functional isolation of the Candida albicans FCR3 gene encoding a bZip transcription factor homologous to Saccharomyces cerevisiae Yap3p. Yeast 18: 1217–1225.
[78]
Subba Rao G, Bachhawat AK, Gupta CM (2002) Two-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p. Funct Integr Genomics 1: 357–366.
[79]
Venters BJ, Wachi S, Mavrich TN, Andersen BE, Jena P, et al. (2011) A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces. Molecular Cell 41: 480–492.
[80]
Milgrom E, Diab H, Middleton F, Kane PM (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J Biol Chem 282: 7125–7136.
[81]
Py B, Barras F (2010) Building Fe-S proteins: bacterial strategies. Nat Rev Micro 8: 436–446.
[82]
Schilke B, Voisine C, Beinert H, Craig E (1999) Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 96: 10206–10211.
[83]
Zhang L, Smith MT, Bandy B, Tamaki SJ, Davison AJ (1994) Role of quinones, active oxygen species and metals in the genotoxicity of 1,2,4-benzenetriol, a metabolite of benzene. In: Nohl H, Esterbauer H, Rice-Evans C, editors. Free Radicals in the Environment, Medicine and Toxicology. London: Richelieu Press. pp. 521–562.
[84]
Lindsey R, Bender R, Osheroff N (2005) Stimulation of topoisomerase II-mediated DNA cleavage by benzene metabolites. Chem Biol Interact 153-154: 197–205.
[85]
Oikawa S, Hirosawa I, Hirakawa K, Kawanishi S (2001) Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol. Carcinogenesis 22: 1239–1245.
[86]
Eastmond DA, Mondrala ST, Hasegawa L (2005) Topoisomerase II inhibition by myeloperoxidase-activated hydroquinone: a potential mechanism underlying the genotoxic and carcinogenic effects of benzene. Chem Biol Interact 153-154: 207–216.
[87]
Buthbumrung N, Mahidol C, Navasumrit P, Promvijit J, Hunsonti P, et al. (2008) Oxidative DNA damage and influence of genetic polymorphisms among urban and rural schoolchildren exposed to benzene. Chem Biol Interact 172: 185–194.
[88]
Winn L (2003) Homologous recombination initiated by benzene metabolites: a potential role of oxidative stress. Toxicol Sci 72: 143–149.
[89]
Delaney JC, Smeester L, Wong C, Frick LE, Taghizadeh K, et al. (2005) AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo. Nat Struct Mol Biol 12: 855–860.
[90]
Lan Q, Zhang L, Shen M, Jo WJ, Vermeulen R, et al. (2009) Large-scale evaluation of candidate genes identifies associations between DNA repair and genomic maintenance and development of benzene hematotoxicity. Carcinogenesis 30: 50–58.
[91]
Galvan N, Lim S, Zmugg S, Smith M, Zhang L (2008) Depletion of WRN enhances DNA damage in HeLa cells exposed to the benzene metabolite, hydroquinone. Mutat Res 649: 54–61.
[92]
Lyu BN, Ismailov SB, Ismailov B, Lyu MB (2008) Mitochondrial concept of leukemogenesis: key role of oxygen-peroxide effects. Theor Biol Med Model 5: 23.
[93]
Mewes HW, Frishman D, Güldener U, Mannhaupt G, Mayer K, et al. (2002) MIPS: a database for genomes and protein sequences. Nucleic Acids Res 30: 31–34.
[94]
Robinson M, Grigull J, Mohammad N, Hughes T (2002) FunSpec: a web-based cluster interpreter for yeast. BMC Bioinformatics 3: 35.
[95]
Ballman KV, Grill DE, Oberg AL, Therneau TM (2004) Faster cyclic loess: normalizing RNA arrays via linear models. Bioinformatics 20: 2778–2786.
[96]
Team R (2008) R: A language and environment for statistical computing. R Foundation for Statistical Computing Vienna Austria.
[97]
Gerlovina I (2010) Bioinformatic Approaches to Parallel Deletion Analysis in Yeast: An Example Studying Environmental Exposure. Unpublished Master's Thesis, University of California, Berkeley.
[98]
Thomas R, Gohlke JM, Stopper GF, Parham FM, Portier CJ (2009) Choosing the right path: enhancement of biologically relevant sets of genes or proteins using pathway structure. Genome Biol 10: R44.
[99]
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, et al. (2008) KEGG for linking genomes to life and the environment. Nucleic Acids Research 36: D480–484.
[100]
Kruskal WH, Wallis WA (1952) Use of ranks in one-criterion variance analysis. Journal of the American statistical Association 47: 583–621.
[101]
Gosling J, Joy B, Steele G, Bracha G (2000) The Java language specification Second Edition. Addison-Wesley Professional.
[102]
Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863–14868.
[103]
Von Mering C, Jensen LJ, Kuhn M, Chaffron S, Doerks T, et al. (2007) STRING 7--recent developments in the integration and prediction of protein interactions. Nucleic Acids Research 35: D358–362.
[104]
Pinheiro J, Bates D, DebRoy S, Sarkar D, R Core Team (2009) nlme: Linear and Nonlinear Mixed Effects Models. R package version 31-96:
[105]
Pollard KS, Gilbert HN, Ge Y, Taylor S, Dudoit S (2010) multtest: Resampling-based multiple hypothesis testing. R package version 2514:
[106]
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, et al. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498–2504.
[107]
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29.
[108]
Alexa A, Rahnenfuhrer J (2009) topGO: Enrichment analysis for Gene Ontology. R package version 1162:
[109]
Chivian D, Kim DE, Malmstr？m L, Bradley P, Robertson T, et al. (2003) Automated prediction of CASP-5 structures using the Robetta server. Proteins 53: Suppl 6524–533.
