Ancestral gene resurrection

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Ancestral gene resurrection is a technique that belongs to the study of molecular evolution. The method consists in the synthesis of an ancestral gene and expression of the corresponding ancestral protein.[1] The idea of protein resurrection was suggested in 1963 by Pauling and Zuckerkandl.[2] Some early efforts were made in the nineties, led by the laboratory of Steven A. Benner, a biochemist at the Swiss Federal Institute of Technology and later the University of Florida.[3] Thanks to the improvement of algorithms and of better sequencing and synthesis techniques, the method was developed further in the early 2000s to allow the resurrection of much more ancient genes.[4] Over the next decade, ancestral protein resurrection was developed as a strategy to reveal the mechanisms and dynamics of protein evolution, led mainly by Joseph Thornton, an evolutionary biologist at the University of Oregon.[5]

The method[edit]

The ancient protein is inferred by means of phylogenetic methods and a DNA molecule coding for that protein is synthesized. This DNA is expressed in cultured cells or in vitro whereafter the obtained ancestral protein may be tested for its properties. Sequences of extant proteins with the same function obtained from more or less distant related organisms are aligned and the best-fitting evolutionary model is determined. The ancestral protein is reconstructed by maximum likelihood. This virtual sequence of aminoacids allows for the synthesis of oligonucleotides that are subsequently assembled into the gene for the ancestral protein. This gene is amplified by means of PCR and transcribed in vitro or in vivo. The resulting ancestral protein is eventually purified and ready to be tested for its properties.[6][7]

Resurrected proteins[edit]

Also other researchers used this procedure and successfully reconstructed various ancestral proteins. Joseph Thornton resurrected several ancestral hormone receptors (from about 500 Myr ago)[8][9][10] and an ancient ATPase[11] from yeast (800 Myr ago). Some other examples are ancestral visual pigments in vertebrates,[12] enzymes in yeast that break down sugars (800 Myr ago)[13] and enzymes in bacteria that provide resistance to antibiotics (2 - 3 Gyr ago).[14]


These experiments address various important questions in evolutionary biology: does evolution proceed in small steps or in large steps; is evolution reversible; how does complexity evolve. It has been shown that slight mutations in the aminoacid sequence of hormone receptors determine an important change in their preferences for hormones. These changes mean huge steps in the evolution of the endocrine system. Thus very small changes at the molecular level may have enormous consequences. Joe Thornton has also been able to show that evolution is irreversible studying the glucocorticoid receptor. This receptor was changed by seven mutations in a cortisol receptor, but reversing these mutations didn't give the original receptor back. Other earlier neutral mutations acted as a ratchet and made the changes to the receptor irreversible.[15] These different experiments on receptors show that, during their evolution, proteins are greatly differentiated and this explains how complexity may evolve.

A closer look at the different ancestral hormone receptors and the various hormones shows that at the level of interaction between single aminoacid residues and chemical groups of the hormones there exist very small but specific changes. Knowledge about these changes may for example lead to the synthesis of hormonal equivalents capable of mimicking or inhibiting the action of a hormone, which might open possibilities for new therapies.


  1. ^ Thornton J.W. (2004) "Resurrecting ancient genes: experimental analyis of extinct molecules". Nature Reviews Genetics 5, 366-375 doi:10.1038/nrg1324
  2. ^ Pauling, L. & Zuckerkandl, E. Chemical paleogenetics: molecular restoration studies of extinct forms of life. Acta Chem. Scand. 17, S9–S16 (1963)
  3. ^ Jermann TM, Opitz JG, Stackhouse J, Benner SA. Reconstructing the evolutionaryhistory of the artiodactyl ribonuclease superfamily. Nature. 1995 Mar2;374(6517):57-9. PubMed PMID 7532788.
  4. ^ Thornton JW, Need E, Crews D. Resurrecting the ancestral steroid receptor:ancient origin of estrogen signaling. Science. 2003 Sep 19;301(5640):1714-7.PubMed PMID 14500980.
  5. ^ Pearson, Helen (March 21, 2012) "Prehistoric proteins: raising the dead" Nature (London)
  6. ^ Thornton J.W. (2004) "Resurrecting ancient genes: experimental analyis of extinct molecules". Nature Reviews Genetics 5, 366-375 doi:10.1038/nrg1324
  7. ^ Figure 1 from reference 3. "The ancestral gene resurrection strategy"
  8. ^ Thornton JW, Need E, Crews D 2003 "Resurrecting the Ancestral Steroid Receptor: Ancient Origin of Estrogen Signaling" Science Vol. 301 no. 5640 pp. 1714-1717 DOI: 10.1126/science.1086185
  9. ^ Eick GN, Colucci JK, Harms MJ, Orlund EA, Thornton JW (2012). "Evolution of minimal specificity and promiscuity in steroid hormone receptors". PLOS Genetics 8(11): e1003072. doi:10.1371/journal.pgen.1003072
  10. ^ Harms MJ, Eick GN, Goswami D, Colucci JK, Griffin PR, Ortlund EA, Thornton JW.(2013) Biophysical mechanisms for large-effect mutations in the evolution of steroid hormone receptors. Proceedings of the National Academy of Sciences USA. published online June 24
  11. ^ Finnigan G, Hanson-Smith V, Stevens TH, Thornton JW (2012). "Mechanisms for the evolution of increased complexity in a molecular machine." Nature 481:360-4 doi:10.1038/nature10724
  12. ^ Shi, Y. & Yokoyama, S. (2003)Molecular analysis of the evolutionary significance of ultraviolet vision in vertebrates. Proc. Natl Acad. Sci. USA 100, 8308–8313 doi: 10.1073/pnas.1532535100
  13. ^ Voordeckers K, Brown CA, Vanneste K, van der Zande E, Voet A, et al. (2012) Reconstruction of Ancestral Metabolic Enzymes Reveals Molecular Mechanisms Underlying Evolutionary Innovation through Gene Duplication. PLoS Biol 10(12): e1001446. doi:10.1371/journal.pbio.1001446
  14. ^ Risso VA, Jose AG, Mejia-Carmona DF, Gauchier EA, Sanchez-Ruiz JM (2013) "Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian β-Lactamases" J. Am. Chem. Soc. 135 (8), pp 2899–2902 DOI: 10.1021/ja311630a
  15. ^ Bridgham JT, Ortlund EA, Thornton JW. (2009) An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature 461:515-519 doi:10.1038/nature08249