Universitat de València
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Evolutionary Genetics

Experimental Evolution and Population Genetics Lab

Dr. Rafael Sanjuán

Ph.D. in Evolutionary Genetics (2005)

Ramón y Cajal Posdoctoral Associate

Institute Cavanilles for Biodiversity and Evolutionary Biology

University of Valencia

E-mail: rafael.sanjuan@uv.es

Tel: +34 963 543 629

Fax: +34 963 543 670

Address:

Institut Cavanilles de Biodiversitat i Biologia Evolutiva

Parc Cientific de la Universitat de València

C/ Catedrático Agustín Escardino n°9

46980 Paterna, Valencia

Spain

 

Presentation

My lab is part of the Evolutionary Genetics Group of the Institute Cavanilles for Biodiversity and Evolutionary Biology. We are located at the Scientific Campus of the University of Valencia (how to find us).

We test population genetics models using experimental evolution and molecular biology tools. I am specially interested in mutation rates, mutational robustness, epistasis, and evolvability.

 

 Model systems

  • Viruses
         Viruses and other microorganisms are excellent models for experimental evolution. Due to their short generation times and elevated population sizes, they evolve fast under lab conditions, which this allows us to do 'real time' evolution. Moreover, the small genomes of viruses facilitate genetic manipulation and the study of the genomic basis of adaptation. I have worked with the following systems:

  1. Vesicular stomatitis virus (VSV), an RNA virus of the family Rhabdoviridae. As most RNA viruses, it has a small genome size (11 kb), a high per-base mutation rate (~10-4-10-5), and low mutational robustness (~40% random nucleotide substitutions kill the virus). In the wild, it is of particular importance to farmers in regions where it can infect livestock. In the lab, it has been extensively used in experimental evolution studies.

  2. Bacteriophages. We are currently working with six phage species, including RNA (Qb, MS2, SP) and ssDNA (FX174, G4, F1) phages. All of them infect the same E. coli strain, which allows us to perform direct comparisons between species.

  3. Hepatitis C virus (HCV). This virus infects an estimated 200 million people worldwide, constituting a major global health concern. I have recently started to analyze HCV sequence datasets in collaboration with Prof. Fernando González-Candelas. We have investigated the effect of the interferon-ribavirin therapy on the mutation rate of HCV. We are also studying the evolutionary constraints imposed by RNA secondary structure.

  • Viroids
         Viroids are plant pathogens constituted by a minimal (200-400 nt) RNA genome. They do not code for any protein and hence rely directly on their  RNA sequence and structure to infect their hosts. I have performed in silico RNA folding with most of the known species of viroids. In collaboration with Dr. Selma Gago, Prof. Santiago Elena, and Prof. Ricardo Flores, we have recently demonstrated that the Chrysanthemum chlorotic mottle viroid (CChMVd) has the highest per-base mutation rate described for any biological entity. This viroid and the other members of the Avsunviroidae family contain hammerhead ribozymes, small RNA motifs that mediate self-cleavage of replicative intermediates and hence, are essential for the replication of the viroid. Based on the principle that the population frequency of nonviable mutations equals the mutation rate, we have screened for mutations at key sites of the hammerheads to obtain a direct estimation of the mutation rate of CChMVd. The famous popular science writer Carl Zimmer has recently featured our paper in Science's blog Origins.

  • Digital evolution
         Digital organisms are self-replicating computer programs that inhabit a virtual world where they reproduce, compete for resources, and evolve according to the same fundamental rules as do natural ones. Despite digital and natural organisms show obvious differences, the former allow us to perform 'experiments' on scales that are beyond reach with any biological entity and to carry out certain genetic manipulations that would be exceedingly laborious on natural organisms. My work with digital organisms has been done in cooperation with Jeff Clune, Prof. Richard Lenski and other members of the the MSU Devolab, as well as with Prof. Santiago Elena (CSIC).

 

 Research tools

  • Experimental evolution
       Experimental evolution offers us a way of testing evolutionary hypotheses under  controlled conditions. As such, it can help us to disentangle the contributions of the different factors involved in evolutionary processes. Cultured viruses, bacteria, yeast, or even higher eukaryotes with sufficiently short generation times can be used as model systems. Serial transfers of these organisms are performed in the lab under a variety of conditions, including different population sizes, environments, transmission modes, or mutation rates. Typically, we estimate biological fitness using growth rate or competition assays, but other relevant parameters, such as virulence, can be measured as well.

  • Molecular biology
       We use several molecular biology techniques, including DNA and RNA isolation, RT-PCR, sequencing, molecular cloning, site-directed mutagenesis, chemical mutagenesis, and transfection, as well as microbiological and cellular biology routine techniques such as cell culturing, monoclonal antibody production, viral plating and titration. For example, we have used site-directed mutagenesis to estimate directly the fitness effects of single point mutations and epistasis between pairs of mutations in VSV. We also apply site-directed mutagenesis to viroids and phages.

  • Comparative biology
       Comparison between species is the most classical approach in biology. However, this has not very often been combined with experimentation. One of my main research goals is to perform experiments aimed at comparing the basic evolutionary properties of different species, such as their ability to generate genetic variation, adapt to novel environments, tolerate deleterious mutations, or evolve new functional capabilities. I have also applied standard methods of phylogenetic analysis to viral, plant and animal sequence data.

  • Computational biology
       We have used computational tools for phylogenetic analysis, statistical analysis, modeling, prediction of RNA secondary structure and digital evolution, among others. I developed a method for tree topology testing in collaboration with Dr. Borys Wróbel (Polish Academy of Sciences)    .

  • Modeling
      We study theoretical aspects of viral evolution related to lethal mutagenesis, robustness, and mutation rates. We have also developed network models to try to establish a general relationship between epistasis and genomic complexity.

 

 Findings

  • Mutational robustness
         Mutational robustness (also termed genetic robustness), or the ability to tolerate mutations, determines the strength of natural selection and as such, play a central role in evolution.

  1. Using a collection of VSV single-nucleotide mutants obtained by site-directed mutagenesis, we were the first to directly estimate the mutational robustness of an RNA virus (2004). This work established that RNA viruses show remarkably low robustness, ~40% of random point mutations being lethal in the case of VSV.

  2. Due to their high mutation rates and low robustness, RNA viruses are good targets for lethal mutagenesis. I have participated in the development of a population genetics theory of lethal mutagenesis in viruses, in collaboration with Prof. Jim Bull and Dr. Claus Wilke (University of Texas) (2007).

  3. We demonstrated that increased robustness offers a selective advantage in viral populations subjected to chemical mutagenesis (2007). This work also provided support for a prediction of the quasispecies theory known as "the survival of the flattest".

  4. The relationship between robustness and evolvability remains controversial.  We have recently shown (2009) that, in VSV, increased robustness tends to hamper adatptive evolution. We suggest that this result might apply to RNA viruses in general.

  5. I have also studied robustness in viroids. Using RNA folding algorithms, we predicted the  effect of all possible single substitutions on the secondary structure of 29 viroid species (2006). I also developed a one-step site-directed mutagenesis lab protocol for viroids (2007), which has facilitated the creation of mutant collections.

  • Epistasis
         Epistasis (the interaction between genes or loci) is central to several population genetics theories, including those seeking to explain sexual reproduction, ploidy, or speciation. More recently, systems biology has opened new avenues for the study of epistasis.

  1. In a paper co-authored with Prof. Santiago Elena (CSIC), I proposed the existence of a general correlation between  epistasis and genomic complexity and provided support for this correlation using available fitness data from  viruses, prokaryotes, unicellular eukaryotes and higher eukaryotes (2006).

  2. I developed a network model for the correlation between epistasis and genomic complexity in collaboration with Dr. Miguel Nebot (University of Valencia) (2008).

  3. We characterized the distribution of epistasis coefficients between random pairs of mutations using a collection of VSV mutants obtained by site-directed mutagenesis (2004).

  4. We studied epistasis in viroids using RNA folding algorithms. In this work, we provided evidence for the hypothesis that epistasis and robustness are correlated traits (2006).

  5. I estimated selection and epistasis coefficients for a specific RNA secondary structure of the Rous sarcoma virus (2006).

  • Mutation rates
         Mutation is the ultimate source of genetic variation and thus is necessary for evolution. However the relationship between mutation and adaptation is not straightforward. On one hand, the higher the mutation rate, the more beneficial mutations are generated. On the other hand, most mutations with phenotypic effect are deleterious. Thus, the optimal mutation rate probably lies at some intermediate value.

  1. Using a large dataset of molecular clone sequences from an hepatitis C virus outbreak, we have shown that ribavirin (currently used in combination with interferon to treat the infection) increases the mutation rate of the virus in vivo.
  2. We have estimated the mutation rate of Chrysanthemum chlorotic mottle viroid (CChMVd) and shown that it is the highest described in any biological entity (2009).
  3. We have measured the mutation rate of phage FX174 using the Luria-Delbrück fluctuation test (2009). Our estimate, 0.005 mutations per genome per round of copying, is in accordance with Drake's rule, which states that DNA-based mircoorganisms (DNA viruses, bacteria, unicellular eukaryotes) show a constant genomic mutation rate of ~0.003 per round of copying despite large variations in lifestyle and genome complexity.
  4. We also estimated the mutation rate of VSV using the Luria-Delbrück fluctuation test (2005).
  5. We recently provided an upper-limit estimation for the mutation rate of Tobacco etch virus (2009).
  6. We demonstrated for the first time the existence of a fitness cost of replication fidelity in RNA viruses (2005, 2007).
  7. We showed that natural selection fails to optimize mutation rates in rugged adaptive landscapes, using digital organisms (2008).
  • Other findings 
  1. VV enhances VSV adaptability (2008)
  2. Study of VSV adaptability under co-infection and super-infection regimes (2007)
  3. Development of a least-squares statistical test for assessing the confidence of distance-based phylogenetic trees (2005)
  4. Characterization of the effect of ribavirin/interferon treatment on VSV fitness and evolvability (2005)
  5. Comparison of the relative importance of compensatory evolution and reversion in VSV experimental populations undergoing fast fitness recovery (2005)
  6. Identification of trade-offs between fitness-related traits in VSV in cell cultures (2005)
  7. Characterization of the role played by natural selection in the compartmentalization of HIV-1 (2004)
  8. Identification of the role played by gene duplications in the evolution of novel functions (2001)
  9. Description of the fitness effects of transmission mode and genetic bottlenecking on VSV  (2001)

 

 Practical implications

  • Lethal mutagenesis
         Lethal mutagenesis is the extinction of a population caused by the deterministic accumulation of deleterious mutations. Due to their already high spontaneous mutation rates, RNA viruses are good targets for lethal mutagenesis. This has led to the proposal of lethal mutagenesis as a candidate antiviral strategy. The validity of this therapeutic strategy is now being tested in patients (more information).

  • Mutation rate optimization
         Natural selection is short-sighted and hence tends to favor low mutation rates to avoid the immediate costs of deleterious mutations, despite the fact that higher rates would allow populations to better adapt to their environments over the long-term. This finding has implications for the fields of drug development and evolutionary computation.


 List of publications

  • Cuevas J.M., Duffy S., Sanjuán R. (2009). Point muation rate of bacteriophage FX174. Genetics in press.
  • Cuevas J.M., Moya A., Sanjuán R. (2009). A genetic background with low mutational robustness is associated with increased adaptability to a novel host in an RNA virus. J. Evol. Biol. in press.
  • Domingo-Calap P., Sentandreu V., Bracho A., González-Candelas F., Moya A., Sanjuán R. (2009). Unequal distribution of RT-PCR artifacts along the E1-E2 region of hepatitis C virus. J. Virol. Methods 161: 136-140.
  • Cuevas J.M., Domingo-Calap P., Pereira-Gómez M., Sanjuán R. (2009). Experimental evolution and population genetics of RNA viruses.  The Open Evolution Journal 3: 9-16.
  • Cuevas J.M., González-Candelas F., Moya A., Sanjuán R. (2009), Effect of ribavirin on the mutation rate and spectrum of hepatitis C virus in vivo. J. Virol. 83: 5760-5764.
  • Cuevas J.M., Sanjuán R. (2009). Evolución experimental: evolución en tiempo real. Apuntes de Ciencia y Tecnología 30: 30-34.
  • Sanjuán R., Agudelo-Romero P., Elena S.F. (2009). Upper-limit mutation rate estimation for a plant virus. Biol. Lett. 5: 394-396.
  • Gago S., Elena S.F., Flores R., Sanjuán R. (2009). Extremely high mutation rate of a hammerhead viroid. Science 323: 1308.
  • Elena S.F., Sanjuán R. (2008) The effect of genetic robustness on evolvability in digital organisms. BMC Evol. Biol. 8: 284.
  • Clune J., Misevic D., Ofria C., Lenski R., Elena S.F., Sanjuán R. (2008). Selection fails to optimize mutation rates in rugged fitness landscapes. PLoS Comput. Biol., 4: e1000187.
  • Carrillo F.Y., Sanjuán R., Moya A., Cuevas J.M. (2008). Enhanced adaptation of vesicular stomatitis virus in cells infected with vaccinia virus. Infect. Genet. Evol., 8: 614-620.
  • Sanjuán R., Nebot M.R. (2008) A network model for the correlation between epistasis and genomic complexity. PLoS ONE, 3: e2663.
  • Bull J.J., Sanjuán R., and Wilke C.O. Lethal mutagenesis (2008) in: Origin and Evolution of Viruses (Eds: E. Domingo, C. Parrish, J. Holland), pp. 207-218 Elsevier.
  • Sanjuán R. Quasispecies and experimental evolution of RNA viruses. (2008) in: Enciclopedia of Virology (Eds: B.W.J. Mahy, M.H.V. Van Regenmortel), pp. 359-365 Elsevier (Oxford)
  • Elena S.F., Agudelo-Romero P., Carrasco P., Codoñer F.M., Martín S., Torres C., and Sanjuán R. (2008). Experimental evolution of plant RNA viruses. Heredity, 100: 478-483.
  • Serrani J.C., Sanjuán R., Fos M., García-Martínez J.L. (2007). Gibberellin regulation of fruit-set and growth in tomato. Plant Physiol., 145: 246-257.
  • Elena S.F. and Sanjuán R. (2007). Virus evolution: insights from an experimental approach. Annu. Rev. Ecol. Evol. Syst., 38: 27-52.
  • Sanjuán R. and Daròs J.A. (2007). Site-directed mutagenesis of viroid dimeric cDNA. J. Virol. Methods, 145: 71-75.
  • Sanjuán R., Cuevas J.M., Furió V., Holmes E.C., and Moya A. (2007). Selection for robustness in mutagenized RNA viruses. PLOS Genet., 15: e93.
  • Bordería A.V., Codoñer F.M., and Sanjuán R. (2007) Selection promotes organ compartmentalization in HIV-1: evidence form gag and pol genes. Evolution, 61: 272-279.
  • Bull J.J., Sanjuán R., and Wilke C.O. (2007) Theory of lethal mutagenesis for viruses. J. Virol., 81: 2930-2939.
  • Furió V., Moya A., and Sanjuán R. (2007). The cost of replication fidelity in human immunodeficiency virus type 1. Proc. Biol. Sci., 274: 225-230.
  • Carrillo F.Y., Sanjuán R., Moya A., and Cuevas F.M. (2007). The effect of co- and superinfection on the adaptive dynamics of vesicular stomatitis virus. Infect. Genet. Evol., 7: 69-73.
  • Czarna A., Sanjuán R., González-Candelas F., and Wróbel B. (2006). Topology testing of phylogenies using least squares methods. BMC Evol. Biol., 6: 105.
  • Sanjuán R. and Elena S.F. (2006). Epistasis correlates to genomic complexity. Proc. Natl. Acad. Sci. USA, 103: 14402-14405.
  • Sanjuán R., Forment J., and Elena S.F. (2006). In silico predicted robustness of viroids RNA secondary structures. II. Interaction between mutation pairs. Mol. Biol. Evol., 23: 2123-2130.
  •  Sanjuán R., Forment J., and Elena S.F. (2006). In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations. Mol. Biol. Evol., 23: 1427-1436.
  • Sanjuán R. (2006) Quantifying antagonistic epistasis in a multifunctional RNA secondary structure of the Rous sarcoma virus. J. Gen. Virol., 87: 1595-1602.
  • Elena SF., Carrasco P., Daròs J.A., and Sanjuán R. (2006). Mechanisms of genetic robustness in RNA viruses. EMBO R. 7, 168-173.
  • Furió V., Moya A., and Sanjuán R. (2005). The cost of replication fidelity in an RNA virus. Proc. Natl. Acad. Sci. USA, 102, 10233-10237.
  • Elena S.F. and Sanjuán R. (2005). On the adaptive value of high mutation rates in RNA viruses: separating causes from consequences. J. Virol., 79, 11555-11558.
  • Sanjuán R., Cuevas J.M., Moya A., and Elena S.F. (2005). Epistasis and the adaptability of an RNA virus. Genetics, 170, 1001-1008.
  • Elena S.F. and Sanjuán R. (2005). RNA viruses as complex adaptive systems. Biosystems, 81, 31-41.
  • Sanjuán R. and Wróbel B. (2005). Weighted least-squares likelihood ratio test for branch testing in phylogenies reconstructed from distance methods. Syst. Biol., 54: 218-229.
  • Cuevas J.M., Sanjuán R., Moya A., and Elena S.F. (2005). Mode of selection and experimental evolution of antiviral drugs resistance in vesicular stomatitis virus. Infect. Genet. Evol., 5, 55-65.
  • Cuevas J.M., Moya A., and Sanjuán R. (2005). Following the very initial growth of biological RNA viral clones. J Gen. Virol., 86: 435-443.
  • Sanjuán R., Moya A., and Elena S.F. (2004). The contribution of epistasis to the architecture of fitness in an RNA virus. Proc. Natl. Acad. Sci. USA, 101: 15376-15379.
  • Sanjuán R., Moya A., and Elena S.F. (2004). The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc. Natl. Acad. Sci. USA, 101: 8396-8401.
  • Sanjuán R., Codoner F.M., Moya A., and Elena S.F. (2004). Natural selection and the organ-specific differentiation of HIV-1 V3 hypervariable region. Evolution, 58: 1185-1194.
  • Elena S.F. and Sanjuán R. (2003). Evolution: Climb every mountain? Science, 302: 2074-2075.
  • Elena S.F., Codoner F.M., Cuevas J.M., and Sanjuán R. (2003). Adaptive dynamics during experimental evolution of RNA viruses. Biology International, 44: 75-78.
  • Elena S.F., Codoner F.M., and Sanjuán R. (2003). Intraclonal variation in RNA viruses: generation, maintenance and consequences. Biol. J. Linn. Soc., 79: 17-26.
  • Elena S.F., Sanjuán R., Borderia A.V., and Turner P.E. (2002). Differential effects of horizontal and vertical transmission in the fitness of an RNA virus: A reanalysis. Infect. Genet. Evol., 1: 307-309.
  • Sanjuán R. and Marín I. (2001). Tracing the origin of the compensasome: evolutionary history of DEAH helicase and MYST acetyltransferase gene families. Mol. Biol Evol., 18: 330-343.
  • Elena S.F., Sanjuán R., Borderia A.V., and Turner P.E. (2001). Transmission bottlenecks and the evolution of fitness in rapidly evolving RNA viruses. Infect. Genet. Evol.: 1: 41-48.

 

 Lab people

Position Name E-mail
Postdoc José Manuel Cuevas cuevastuv.es
Ph.D. Student Victoria Furió victoria.furiouv.es
Ph.D. Student Pilar Domingo-Calap pilar.domingouv.es
Graduate Student Joan Peris hoeman5beta@hotmail.com
Technician Conxa Hueso  

 

UNDERGRADUATE STUDENTS

Name E-mail
Marianoel Pereira marianoe@alumni.uv.es
Francisco M. Cortés Sánchez francor3@alumni.uv.es
José Aguilar Rodríguez arojo@alumni.uv.es

 

PAST MEMBERS

Temesgen Woldezion (Student of the MHIRT program), July-August 2008.

Loles Catalán (Technician), December 2008 - July 2009.


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           Last modified  October 1, 2009