Modeling the evolution of molecular systems from a mechanistic perspective

Trends in Plant Science

Volumn 19, Issue 5, 2014, Pages 292-303

  Gutiérrez, Jayson ^a,b   Maere, Steven ^a,b  

^a DEPARTMENT OF PLANT SYSTEMS BIOLOGY   (Belgium)

^b GHENT UNIVERSITY   (Belgium)

Author keywords

Emergent properties; Evolutionary systems biology; Gene duplication; Genome duplication; Genotype phenotype mapping

Indexed keywords

BIOLOGICAL MODEL; GENE DUPLICATION; GENETICS; GENOTYPE; MOLECULAR EVOLUTION; PHENOTYPE; PLANT; PLANT GENOME; SYSTEMS BIOLOGY;

EVOLUTION, MOLECULAR; GENE DUPLICATION; GENOME, PLANT; GENOTYPE; MODELS, BIOLOGICAL; PHENOTYPE; PLANTS; SYSTEMS BIOLOGY;

EID: 84899934307     PISSN: 13601385     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.tplants.2014.03.004     Document Type: Review

References (159)

1. Ideker T., et al. A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2001, 2:343-372.
2. Milo R., et al. Network motifs: simple building blocks of complex networks. Science 2002, 298:824-827.
3. Barabasi A.L., Oltvai Z.N. Network biology: understanding the cell's functional organization. Nat. Rev. Genet. 2004, 5:101-113.
4. Alm E., Arkin A.P. Biological networks. Curr. Opin. Struct. Biol. 2003, 13:193-202.
5. Alon U. Network motifs: theory and experimental approaches. Nat. Rev. Genet. 2007, 8:450-461.
6. Dobzhansky T. Nothing in biology makes sense except in light of evolution. Am. Biol. Teach. 1973, 35:125-129.
7. Kawecki T.J., et al. Experimental evolution. Trends Ecol. Evol. 2012, 27:547-560.
8. Barrick J.E., Lenski R.E. Genome dynamics during experimental evolution. Nat. Rev. Genet. 2013, 14:827-839.
9. Lynch M., Walsh B. Genetics and Analysis of Quantitative Traits 1998, Sinauer Associates.
10. Lynch M. The Origins of Genome Architecture 2007, Sinauer Associates.
11. Loewe L. A framework for evolutionary systems biology. BMC Syst. Biol. 2009, 3:27.
12. Draghi J.A., et al. Mutational robustness can facilitate adaptation. Nature 2010, 463:353-355.
13. Stewart A.J., et al. Environmental robustness and the adaptability of populations. Evolution 2012, 66:1598-1612.
14. Burns J. The synthetic problem and the genotype-phenotype relation in cellular metabolism. Organization Stability and Process 1970, 47-51. Aldine Publishing Company. C.H. Waddington (Ed.).
15. Goodwin B.C. Development and evolution. J. Theor. Biol. 1982, 97:43-55.
16. Oster G., Alberch P. Evolution and bifurcation of developmental programs. Evolution 1982, 36:444-459.
17. Jaeger J., et al. The inheritance of process: a dynamical systems approach. J. Exp. Zool. B: Mol. Dev. Evol. 2012, 318B:591-612.
18. Gjuvsland A.B., et al. Bridging the genotype-phenotype gap: what does it take?. J. Phys. 2013, 591:2055-2066.
19. Wright S. The roles of mutation, inbreeding, crossbreeding, and selection in evolution. Proceedings of the Sixth International Congress on Genetics 1932, 356-366. D.F. Jones (Ed.).
20. Spirov A., Holloway D. Using evolutionary computations to understand the design and evolution of gene and cell regulatory networks. Methods 2013, 62:39-55.
21. Wagner A. Evolution of gene networks by gene duplications: a mathematical model and its implications on genome organization. Proc. Natl. Acad. Sci. U.S.A. 1994, 91:4387-4391.
22. Siegal M.L., Bergman A. Waddington's canalization revisited: developmental stability and evolution. Proc. Natl. Acad. Sci. U.S.A. 2002, 99:10528-10532.
23. Bergman A., Siegal M.L. Evolutionary capacitance as a general feature of complex gene networks. Nature 2003, 424:549-552.
24. Azevedo R.B.R., et al. Sexual reproduction selects for robustness and negative epistasis in artificial gene networks. Nature 2006, 443:87-90.
25. Ciliberti S., et al. Innovation and robustness in complex regulatory gene networks. Proc. Natl. Acad. Sci. U.S.A. 2007, 104:13591-13596.
26. Martin O.C., Wagner A. Multifunctionality and robustness trade-offs in model genetic circuits. Biophys. J. 2008, 94:2927-2937.
27. ten Tusscher K.H., Hogeweg P. The role of genome and gene regulatory network canalization in the evolution of multi-trait polymorphisms and sympatric speciation. BMC Evol. Biol. 2009, 9:159.
28. Espinosa-Soto C., Wagner A. Specialization can drive the evolution of modularity. PLoS Comput. Biol. 2010, 6:e1000719.
29. Macia J., et al. The causes of epistasis in genetic networks. Evolution 2012, 66:586-596.
30. Inoue M., Kaneko K. Cooperative adaptive responses in gene regulatory networks with many degrees of freedom. PLoS Comput. Biol. 2013, 9:e1003001.
31. Kashtan N., Alon U. Spontaneous evolution of modularity and network motifs. Proc. Natl. Acad. Sci. U.S.A. 2005, 102:13773-13778.
32. Payne J.L., Wagner A. Constraint and contingency in multifunctional gene regulatory circuits. PLoS Comput. Biol. 2013, 9:e1003071.
33. Clune J., et al. The evolutionary origins of modularity. Proc. R. Soc. B 2013, 280:20122863.
34. Francois P., Hakim V. Design of genetic networks with specified functions by evolution in silico. Proc. Natl. Acad. Sci. U.S.A. 2004, 101:580-585.
35. Francois P., et al. Deriving structure from evolution: metazoan segmentation. Mol. Syst. Biol. 2007, 3:154.
36. Francois P., Siggia E.D. Predicting embryonic patterning using mutual entropy fitness and in silico evolution. Development 2010, 137:2385-2395.
37. Cotterell J., Sharpe J. An atlas of gene regulatory networks reveals multiple three-gene mechanisms for interpreting morphogen gradients. Mol. Syst. Biol. 2010, 6:425.
38. Rodrigo G., et al. Computational design of synthetic regulatory networks from a genetic library to characterize the designability of dynamical behaviors. Nucleic Acids Res. 2011, 39:e138.
39. ten Tusscher K.H., Hogeweg P. Evolution of networks for body plan patterning: interplay of modularity, robustness and evolvability. PLoS Comput. Biol. 2011, 7:e1002208.
40. Carrera J., et al. Computational design of genomic transcriptional networks with adaptation to varying environments. Proc. Natl. Acad. Sci. U.S.A. 2012, 109:15277-15282.
41. Cuypers T.D., Hogeweg P. Virtual genomes in flux: an interplay of neutrality and adaptability explains genome expansion and streamlining. Genome Biol. Evol. 2012, 4:212-229.
42. Warmflash A., et al. Pareto evolution of gene networks: an algorithm to optimize multiple fitness objectives. Phys. Biol. 2012, 9:056001.
43. Cotterell J., Sharpe J. Mechanistic explanations for restricted evolutionary paths that emerge from gene regulatory networks. PLoS ONE 2013, 8:e61178.
44. Roh K., et al. Evolution of transcription networks in response to temporal fluctuations. Evolution 2013, 67:1091-1104.
45. Cooper M.B., et al. Evolutionary modelling of feed forward loops in gene regulatory networks. Biosystems 2008, 91:231-244.
46. Khatri B.S., et al. Statistical mechanics of convergent evolution in spatial patterning. Proc. Natl. Acad. Sci. U.S.A. 2009, 106:9564-9569.
47. Dasmahapatra S. Model of haplotype and phenotype in the evolution of a duplicated autoregulatory activator. J. Theor. Biol. 2013, 325:83-102.
48. Bullaughey K. Changes in selective effects over time facilitate turnover of enhancer sequences. Genetics 2011, 187:567-582.
49. Pujato M., et al. The underlying molecular and network level mechanisms in the evolution of robustness in gene regulatory networks. PLoS Comput. Biol. 2013, 9:e1002865.
50. Andrecut M., et al. Monte Carlo simulation of a simple gene network yields new evolutionary insights. J. Theor. Biol. 2008, 250:468-474.
51. Tagkopoulos I., et al. Predictive behavior within microbial genetic networks. Science 2008, 320:1313-1317.
52. Karlebach G., Shamir R. Modelling and analysis of gene regulatory networks. Nat. Rev. Mol. Cell Biol. 2008, 9:770-780.
53. Burda Z., et al. Motifs emerge from function in model gene regulatory networks. Proc. Natl. Acad. Sci. U.S.A. 2011, 108:17263-17268.
54. Kashtan N., et al. An analytically solvable model for rapid evolution of modular structure. PLoS Comput. Biol. 2009, 5:e1000355.
55. Kashtan N., et al. Varying environments can speed up evolution. Proc. Natl. Acad. Sci. U.S.A. 2007, 104:13711-13716.
56. Parter M., et al. Facilitated variation: how evolution learns from past environments to generalize to new environments. PLoS Comput. Biol. 2008, 4:e1000206.
57. Crombach A., Hogeweg P. Evolution of evolvability in gene regulatory networks. PLoS Comput. Biol. 2008, 4:e1000112.
58. Draghi J., Wagner G.P. The evolutionary dynamics of evolvability in a gene network model. J. Evol. Biol. 2009, 22:599-611.
59. Hartwell L.H., et al. From molecular to modular cell biology. Nature 1999, 402:C47-C52.
60. Wagner A. Robustness and Evolvability in Living Systems 2007, Princeton University Press.
61. Kashtan N., et al. Extinctions in heterogeneous environments and the evolution of modularity. Evolution 2009, 63:1964-1975.
62. Variano E.A., et al. Networks, dynamics, and modularity. Phys. Rev. Lett. 2004, 92:188701.
63. Calabretta R., et al. Duplication of modules facilitates the evolution of functional specialization. Artif. Life 2000, 6:69-84.
64. Wagner G.P., et al. The road to modularity. Nat. Rev. Genet. 2007, 8:921-931.
65. Lynch M. The frailty of adaptive hypotheses for the origins of organismal complexity. Proc. Natl. Acad. Sci. U.S.A. 2007, 104(Suppl. 1):8597-8604.
66. Wagner A. Does evolutionary plasticity evolve?. Evolution 1996, 50:1008-1023.
67. Wagner A. Genetic redundancy caused by gene duplications and its evolution in networks of transcriptional regulators. Biol. Cybern. 1996, 74:557-567.
68. Lynch M., Conery J.S. The evolutionary fate and consequences of duplicate genes. Science 2000, 290:1151-1155.
69. Maere S., Van de Peer Y. Duplicate retention after small- and large-scale duplications. Evolution after Gene Duplication 2010, 31-56. Wiley. D.A. Liberles, K. Dittmar (Eds.).
70. Meiklejohn C.D., Hartl D.L. A single mode of canalization. Trends Ecol. Evol. 2002, 17:468-473.
71. Waddington C.H. Canalization of development and the inheritance of acquired characters. Science 1942, 150:563-565.
72. Sniegowski P.D., Murphy H.A. Evolvability. Curr. Biol. 2006, 16:R831-R834.
73. Mozhayskiy V., Tagkopoulos I. Guided evolution of in silico microbial populations in complex environments accelerates evolutionary rates through a step-wise adaptation. BMC Bioinformatics 2012, 13:S10.
74. Wagner A. Robustness and evolvability: a paradox resolved. Proc. R. Soc. Lond. B: Biol. Sci. 2008, 275:91-100.
75. Payne J.L., et al. Robustness, evolvability, and the logic of genetic regulation. Artif. Life 2014, 20:111-126.
76. Masel J., Trotter M.V. Robustness and evolvability. Trends Genet. 2010, 26:406-414.
77. Payne J.L., Wagner A. The robustness and evolvability of transcription factor binding sites. Science 2014, 343:875-877.
78. Aldana M., et al. Robustness and evolvability in genetic regulatory networks. J. Theor. Biol. 2007, 245:433-448.
79. Torres-Sosa C., et al. Criticality is an emergent property of genetic networks that exhibit evolvability. PLoS Comput. Biol. 2012, 8:e1002669.
80. Steiner C.F. Environmental noise, genetic diversity and the evolution of evolvability and robustness in model gene networks. PLoS ONE 2012, 10.1371/journal.pone.0052204.
81. Pechenick D.A., et al. The influence of assortativity on the robustness and evolvability of gene regulatory networks upon gene birth. J. Theor. Biol. 2013, 330:26-36.
82. Martin S.L., Husband B.C. Whole genome duplication affects evolvability of flowering time in an autotetraploid plant. PLoS ONE 2012, 10.1371/journal.pone.0044784.
83. Ohno S. Evolution by Gene Duplication 1970, Springer-Verlag.
84. Poelwijk F.J., et al. Empirical fitness landscapes reveal accessible evolutionary paths. Nature 2007, 445:383-386.
85. de Visser J.A.G.M., et al. The causes of epistasis. Proc. Biol. Sci. 2011, 278:3617-3624.
86. Breen M.S., et al. Epistasis as the primary factor in molecular evolution. Nature 2012, 490:535-538.
87. Poelwijk F.J., et al. Reciprocal sign epistasis is a necessary condition for multi-peaked fitness landscapes. J. Theor. Biol. 2011, 272:141-144.
88. Covert A.W., et al. Experiments on the role of deleterious mutations as stepping stones in adaptive evolution. Proc. Natl. Acad. Sci. U.S.A. 2013, 110:E3171-E3178.
89. Weinreich D.M., Chao L. Rapid evolutionary escape by large populations from local fitness peaks is likely in nature. Evolution 2005, 59:1175-1182.
90. Chen J.C., Conrad M. A multilevel neuromolecular architecture that uses the extradimensional bypass principle to facilitate evolutionary learning. Phys. D 1994, 75:417-437.
91. Conrad M. Towards high evolvability dynamics. Evolutionary Systems 1998, 33-43. Kluwer Academic Publishers. G. Van de Vijver (Ed.).
92. Cariani P.A. Extradimensional bypass. Biosystems 2002, 64:47-53.
93. Gavrilets S. Evolution and speciation on holey adaptive landscapes. Trends Ecol. Evol. 1997, 12:307-312.
94. Gavrilets S. A dynamical theory of speciation on holey adaptive landscapes. Am. Nat. 1999, 154:1-22.
95. Gordon R. Evolution escapes rugged fitness landscapes by gene or genome doubling: the blessing of higher dimensionality. Comput. Chem. 1994, 18:325-331.
96. Poelwijk F.J., et al. Tradeoffs and optimality in the evolution of gene regulation. Cell 2011, 146:462-470.
97. Shoval O., et al. Evolutionary trade-offs, Pareto optimality, and the geometry of phenotype space. Science 2012, 336:1157-1160.
98. Dragosits M., et al. Evolutionary potential, cross-stress behavior and the genetic basis of acquired stress resistance in Escherichia coli. Mol. Syst. Biol. 2013, 9:643.
99. Hittinger C.T., Carroll S.B. Gene duplication and the adaptive evolution of a classic genetic switch. Nature 2007, 449. 677-681.
100. Des Marais D.L., Rausher M.D. Escape from adaptive conflict after duplication in an anthocyanin pathway gene. Nature 2008, 454:762-765.
101. Voordeckers K., et al. Reconstruction of ancestral metabolic enzymes reveals molecular mechanisms underlying evolutionary innovation through gene duplication. PLoS Biol. 2012, 10:e1001446.
102. Huang R.Q., et al. Enzyme functional evolution through improved catalysis of ancestrally nonpreferred substrates. Proc. Natl. Acad. Sci. U.S.A. 2012, 109:2966-2971.
103. Sikosek T., et al. Escape from adaptive conflict follows from weak functional trade-offs and mutational robustness. Proc. Natl. Acad. Sci. U.S.A. 2012, 109:14888-14893.
104. Chapman E.J., Estelle M. Mechanism of auxin-regulated gene expression in plants. Annu. Rev. Genet. 2009, 43:265-285.
105. Vernoux T., et al. The auxin signalling network translates dynamic input into robust patterning at the shoot apex. Mol. Syst. Biol. 2011, 7:508.
106. Finet C., et al. Evolution of the ARF gene family in land plants: old domains, new tricks. Mol. Biol. Evol. 2013, 30:45-56.
107. Guilfoyle T.J., Hagen G. Auxin response factors. Curr. Opin. Plant Biol. 2007, 10:453-460.
108. Sauer M., et al. Auxin: simply complicated. J. Exp. Bot. 2013, 64:2565-2577.
109. Inze D., De Veylder L. Cell cycle regulation in plant development. Annu. Rev. Genet. 2006, 40:77-105.
110. De Veylder L., et al. The ins and outs of the plant cell cycle. Nat. Rev. Mol. Cell Biol. 2007, 8:655-665.
111. Komaki S., Sugimoto K. Control of the plant cell cycle by developmental and environmental cues. Plant Cell Physiol. 2012, 53:953-964.
112. Smaczniak C., et al. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies. Development 2012, 139:3081-3098.
113. Becker A., Theissen G. The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol. Phylogenet. Evol. 2003, 29:464-489.
114. Pabon-Mora N., et al. Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development. Plant Physiol. 2012, 158:1685-1704.
115. Zahn L.M., et al. The evolution of the SEPALLATA subfamily of MADS-box genes: a preangiosperm origin with multiple duplications throughout angiosperm history. Genetics 2005, 169:2209-2223.
116. Veron A.S., et al. Evidence of interaction network evolution by whole-genome duplications: a case study in MADS-box proteins. Mol. Biol. Evol. 2007, 24:670-678.
117. Mondragon-Palomino M., Theissen G. MADS about the evolution of orchid flowers. Trends Plant Sci. 2008, 13:51-59.
118. Khan M.R., et al. MPF2-like-A MADS-box genes control the inflated calyx syndrome in Withania (Solanaceae): roles of Darwinian selection. Mol. Biol. Evol. 2009, 26:2463-2473.
119. Chang Y.Y., et al. Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiol. 2010, 152:837-853.
120. Mondragon-Palomino M., Theissen G. Conserved differential expression of paralogous DEFICIENS- and GLOBOSA-like MADS-box genes in the flowers of Orchidaceae: refining the 'orchid code'. Plant J. 2011, 66:1008-1019.
121. Lee H.L., Irish V.F. Gene duplication and loss in a MADS box gene transcription factor circuit. Mol. Biol. Evol. 2011, 28:3367-3380.
122. Geuten K., et al. Robustness and evolvability in the B-system of flower development. Ann. Bot. 2011, 107:1545-1556.
123. Vekemans D., et al. Gamma paleohexaploidy in the stem lineage of core eudicots: significance for MADS-box gene and species diversification. Mol. Biol. Evol. 2012, 29:3793-3806.
124. Ruelens P., et al. FLOWERING LOCUS C in monocots and the tandem origin of angiosperm-specific MADS-box genes. Nat. Commun. 2013, 10.1038/ncomms3280.
125. Remington D.L., et al. Contrasting modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol. 2004, 135:1738-1752.
126. Vandesteene L., et al. Expansive evolution of the TREHALOSE-6-PHOSPHATE PHOSPHATASE gene family in Arabidopsis. Plant Physiol. 2012, 160:884-896.
127. Jiao Y., et al. Ancestral polyploidy in seed plants and angiosperms. Nature 2011, 473:97-100.
128. Van de Peer Y., et al. The flowering world: a tale of duplications. Trends Plant Sci. 2009, 14:680-688.
129. Soltis D.E., et al. Polyploidy and angiosperm diversification. Am. J. Bot. 2009, 96:336-348.
130. Veitia R.A. Paralogs in polyploids: one for all and all for one?. Plant Cell 2005, 17:4-11.
131. Birchler J.A., Veitia R.A. The gene balance hypothesis: implications for gene regulation, quantitative traits and evolution. New Phytol. 2010, 186:54-62.
132. Maere S., et al. Modeling gene and genome duplications in eukaryotes. Proc. Natl. Acad. Sci. U.S.A. 2005, 102:5454-5459.
133. Freeling M. Bias in plant gene content following different sorts of duplication: tandem, whole-genome, segmental, or by transposition. Annu. Rev. Plant Biol. 2009, 60:433-453.
134. Van de Peer Y., et al. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 2009, 10:725-732.
135. Fawcett J.A., et al. Significance and biological consequences of polyploidization in land plant evolution. Plant Genome Diversity 2013, 277-293. Springer-Verlag. J. Greilhuber (Ed.).
136. De Bodt S., et al. Genome duplication and the origin of angiosperms. Trends Ecol. Evol. 2005, 20:591-597.
137. Freeling M., Thomas B.C. Gene-balanced duplications, like tetraploidy, provide predictable drive to increase morphological complexity. Genome Res. 2006, 16:805-814.
138. Conant G.C., Wolfe K.H. Turning a hobby into a job: how duplicated genes find new functions. Nat. Rev. Genet. 2008, 9:938-950.
139. Fawcett J.A., et al. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proc. Natl. Acad. Sci. U.S.A. 2009, 106:5737-5742.
140. Vanneste K., et al. Tangled up in two: a burst of genome duplications at the end of the Cretaceous and the consequences for plant evolution. Proc. Biol. Sci. 2014, (in press).
141. Chen Z.J. Genomic and epigenetic insights into the molecular bases of heterosis. Nat. Rev. Genet. 2013, 14:471-482.
142. Chao D.Y., et al. Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis. Science 2013, 341:658-659.
143. Conant G.C., Wolfe K.H. Increased glycolytic flux as an outcome of whole-genome duplication in yeast. Mol. Syst. Biol. 2007, 3:129.
144. van Hoek M.J.A., Hogeweg P. Metabolic adaptation after whole genome duplication. Mol. Biol. Evol. 2009, 26:2441-2453.
145. Hahn M.W. Distinguishing among evolutionary models for the maintenance of gene duplicates. J. Hered. 2009, 100:605-617.
146. Sole R.V., Valverde S. Spontaneous emergence of modularity in cellular networks. J. R. Soc. Interface 2008, 5:129-133.
147. Neyfakh A.A., et al. A system for studying evolution of life-like virtual organisms. Biol. Direct 2006, 1:23.
148. Tsuda M.E., Kawata M. Evolution of gene regulatory networks by fluctuating selection and intrinsic constraints. PLoS Comput. Biol. 2010, 6:e1000873.
149. Prud'homme B., et al. Emerging principles of regulatory evolution. Proc. Natl. Acad. Sci. U.S.A. 2007, 104:8605-8612.
150. Davidson E.H. Evolutionary bioscience as regulatory systems biology. Dev. Biol. 2011, 357:35-40.
151. Hoekstra H.E., Coyne J.A. The locus of evolution: evo devo and the genetics of adaptation. Evolution 2007, 61:995-1016.
152. Poelwijk F.J., et al. Evolutionary potential of a duplicated repressor-operator pair: simulating pathways using mutation data. PLoS Comput. Biol. 2006, 2:e58.
153. Bintu L., et al. Transcriptional regulation by the numbers: models. Curr. Opin. Genet. Dev. 2005, 15:116-124.
154. Janssens H., et al. Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene. Nat. Genet. 2006, 38:1159-1165.
155. Segal E., et al. Predicting expression patterns from regulatory sequence in Drosophila segmentation. Nature 2008, 451:535-540.
156. Martinez C.A., et al. A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design. Methods 2013, 62:91-98.
157. Ramsey J. Polyploidy and ecological adaptation in wild yarrow. Proc. Natl. Acad. Sci. U.S.A. 2011, 108:7096-7101.
158. Hermsen R., et al. Transcriptional regulation by competing transcription factor modules. PLoS Comput. Biol. 2006, 2:1552-1560.
159. Snitkin E.S., Segre D. Epistatic interaction maps relative to multiple metabolic phenotypes. PLoS Genet. 2011, 7:e1001294.