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Substitutions in woolly mammoth hemoglobin confer biochemical properties adaptive for cold tolerance

Nature Genetics volume 42, pages 536540 (2010) | Download Citation


We have genetically retrieved, resurrected and performed detailed structure-function analyses on authentic woolly mammoth hemoglobin to reveal for the first time both the evolutionary origins and the structural underpinnings of a key adaptive physiochemical trait in an extinct species. Hemoglobin binds and carries O2; however, its ability to offload O2 to respiring cells is hampered at low temperatures, as heme deoxygenation is inherently endothermic (that is, hemoglobin-O2 affinity increases as temperature decreases). We identify amino acid substitutions with large phenotypic effect on the chimeric β/δ-globin subunit of mammoth hemoglobin that provide a unique solution to this problem and thereby minimize energetically costly heat loss. This biochemical specialization may have been involved in the exploitation of high-latitude environments by this African-derived elephantid lineage during the Pleistocene period. This powerful new approach to directly analyze the genetic and structural basis of physiological adaptations in an extinct species adds an important new dimension to the study of natural selection.

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  1. 1.

    , , & Planetary biology— paleontological, geological, and molecular histories of life. Science 296, 864–868 (2002).

  2. 2.

    , , & Reconstructing the evolutionary history of the artiodactyl ribonuclease superfamily. Nature 374, 57–59 (1995).

  3. 3.

    , , & Molecular evolution of the ribonuclease superfamily. Prog. Biophys. Mol. Biol. 51, 165–192 (1988).

  4. 4.

    et al. Proboscidean mitogenomics: chronology and mode of elephant evolution using mastodon as outgroup. PLoS Biol. 5, e207 (2007).

  5. 5.

    , , & The pattern and process of mammoth evolution in Eurasia. Quat. Int. 126–128, 49–64 (2005).

  6. 6.

    The impact of Quaternary Ice Ages on mammalian evolution. Phil. Trans. R. Soc. Lond. B 359, 221–241 (2004).

  7. 7.

    & Mammoths: Giants of the Ice Age (University of California Press, Berkeley, California, USA, 2007).

  8. 8.

    , , , & Sebaceous glands of the woolly mammoth, Mammuthus primigenius Blum: histological evidence. Dokl. Biol. Sci. 398, 382–384 (2004).

  9. 9.

    et al. Nuclear gene indicates coat-color polymorphism in mammoths. Science 313, 62 (2006).

  10. 10.

    et al. Sequencing the nuclear genome of the extinct woolly mammoth. Nature 456, 387–390 (2008).

  11. 11.

    , , , & Hemoglobins, XLVIII: the primary structure of hemoglobin of the Indian elephant (Elephas maximus, Proboscidea): beta2=Asn. Hoppe-Seyler's Z. Physiol. Chem. 363, 683–691 (1982).

  12. 12.

    , , , & Phosphate-haemoglobin interaction. The primary structure of the haemoglobin of the African elephant (Loxodonta africana, Proboscidea): asparagine in position 2 of the beta-chain. Hoppe-Seyler's Z. Physiol. Chem. 365, 743–749 (1984).

  13. 13.

    , , & Origin and ascendency of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals. Mol. Biol. Evol. 26, 1469–1478 (2009).

  14. 14.

    Species adaptation in a protein molecule. Mol. Biol. Evol. 1, 1–28 (1983).

  15. 15.

    , , & Involvement of Glu G3(101)beta in the function of hemoglobin. Comparative O2 equilibrium studies of human mutant hemoglobins. J. Biol. Chem. 260, 5919–5924 (1985).

  16. 16.

    et al. Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers. Protein Sci. 2, 1320–1330 (1993).

  17. 17.

    & Hemoglobin variants with altered oxygen affinity. Hemoglobin 4, 243–261 (1980).

  18. 18.

    , & Hb F-La Grange or α2γ2101(G3)Glu→Lys; 75Ile; 136Gly: A high oxygen affinity fetal haemoglobin variant observed in a causcasian newborn. Biochim. Biophys. Acta 789, 224–228 (2004).

  19. 19.

    , , & Hemoglobin function under extreme life conditions. Eur. J. Biochem. 223, 309–317 (1994).

  20. 20.

    , , , & From the Arctic to fetal life: physiological importance and structural basis of an 'additional' chloride-binding site in haemoglobin. Biochem. J. 380, 889–896 (2004).

  21. 21.

    & Temperature of skin in the arctic as a regulator of heat. J. Appl. Physiol. 7, 355–364 (1955).

  22. 22.

    et al. Allosteric modulation by tertiary structure in mammalian hemoglobins. Introduction of the functional characteristics of bovine hemoglobin into human hemoglobin by five amino acid substitutions. J. Biol. Chem. 270, 30588–30592 (1995).

  23. 23.

    et al. Production of unmodified human adult hemoglobin in Escherichia coli. Proc. Natl. Acad. Sci. USA 90, 8108–8112 (1993).

  24. 24.

    Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function. J. Appl. Physiol. 72, 1611–1615 (1992).

  25. 25.

    , , , & 1.25 Å resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms. J. Mol. Biol. 360, 690–701 (2006).

  26. 26.

    , , & X-ray diffraction study of di and tetra-ligated T-state hemoglobin from high salt crystals. J. Mol. Biol. 227, 480–492 (1992).

  27. 27.

    et al. Mutagenic dissection of hemoglobin cooperativity: Effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers. Prot. Str. Funct. Gen. 14, 333–350 (1992).

  28. 28.

    et al. Multiplex amplification of ancient DNA. Nat. Protoc. 1, 720–728 (2006).

  29. 29.

    et al. Genetic structure and extinction of the woolly mammoth, Mammuthus primigenius. Curr. Biol. 17, 1072–1075 (2007).

  30. 30.

    , , & A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J. Comput. Chem. 25, 1656–1676 (2004).

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We thank T. Kuznetsova for the mammoth samples, W. Korver for providing Asian and African elephant blood and A. Bang, T.C. Tam, N. Ho, J. Hare, J. da Silva and M. Pagel for technical assistance. Financial support was provided by the National Sciences and Engineering Research Council (NSERC) of Canada (K.L.C. and J.S.), Winnipeg Foundation (K.L.C.), University of Manitoba Research Grant Program (K.L.C.), Japan Society for the Promotion of Science (J.R.H.T.), Max Planck Society (M.H. and N.R.), US National Institutes of Health grant R01GM-084614; C.H.), Danish Natural Science Research Council and the Carlsberg Foundation (R.E.W.) and the Australian Research Council (A.C. and L.N.W.). J.E.E.R., J.W.H. and A.V.S. were supported by NSERC Undergraduate Research Awards, and A.M.S. was supported by a University of Manitoba Graduate Fellowship.

Author information

Author notes

    • Nadin Rohland

    Present address: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Michael Hofreiter

    Present address: Department of Biology, University of York, York, UK.

    • Roy E Weber
    •  & Alan Cooper

    These authors contributed equally to this work.


  1. Department of Biological Sciences, University of Manitoba, Winnipeg, Canada.

    • Kevin L Campbell
    • , Jason E E Roberts
    • , Angela M Sloan
    • , Anthony V Signore
    •  & Jesse W Howatt
  2. Australian Centre for Ancient DNA, School of Earth and Environmental Sciences, University of Adelaide, Adelaide, Australia.

    • Laura N Watson
    • , Jeremy J Austin
    •  & Alan Cooper
  3. Department of Chemistry, University of Manitoba, Winnipeg, Canada.

    • Jörg Stetefeld
  4. Protein Design Laboratory, Yokohama City University, Yokohama, Japan.

    • Jeremy R H Tame
  5. Junior Research Group Molecular Ecology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

    • Nadin Rohland
    •  & Michael Hofreiter
  6. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.

    • Tong-Jian Shen
    •  & Chien Ho
  7. Zoophysiology, Institute of Biological Sciences, University of Aarhus, Aarhus, Denmark.

    • Roy E Weber


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K.L.C. conceived the research. K.L.C., J.S., M.H., J.J.A., T.-J.S., C.H., R.E.W. and A.C. designed the experiments. K.L.C., J.E.E.R., L.N.W., A.M.S., A.V.S., J.W.H., N.R., T.-J.S., R.E.W. and J.J.A. conducted the experiments. K.L.C., J.S., A.V.S., J.R.H.T., R.E.W. and A.C. analyzed the data. K.L.C. and A.C. drafted the manuscript, and K.L.C., M.H., J.R.H.T., C.H., R.E.W. and A.C. contributed to the final manuscript writing and its revisions.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Kevin L Campbell or Alan Cooper.

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