By amplifying the melanocortin type 1 receptor from the woolly mammoth, we can report the complete nucleotide sequence of a nuclear-encoded gene from an extinct species. We found two alleles and show that one allele produces a functional protein whereas the other one encodes a protein with strongly reduced activity. This finding suggests that mammoths may have been polymorphic in coat color, with both dark- and light-haired individuals co-occurring

Bertranpetit, Jaume

Hofreiter, Michael

Kuznetsova, Tatyana

Lalueza-Fox, Carles

Rabeder, Gernot

Rohland, Nadin

Römpler, Holger

Schöneberg, Torsten

Willerslev, Eske

Römpler, H.

Rohland, N.

Lalueza-Fox, C.

Willerslev, E.

Kuznetsova, T.

Rabeder, G.

Betranpetit, J.

Schöneberg, T.

Hofreiter, M. 

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Nuclear Gene Indicates Coat-Color Polymorphism in Mammoths

Betranpetit, Jaume

Max Planck Society eDoc Server

Udgivelsesdato: 7 July 2006By amplifying the melanocortin type 1 receptor from the woolly mammoth, we can report the complete nucleotide sequence of a nuclear-encoded gene from an extinct species. We found two alleles and show that one allele produces a functional protein whereas the other one encodes a protein with strongly reduced activity. This finding suggests that mammoths may have been polymorphic in coat color, with both dark- and light-haired individuals co-occurring. </sup

Copenhagen University Research Information System

Hofreiter, M.

OceanRep

Nuclear Gene Indicates Coat-ColorPolymorphism in MammothsHolger Ro¨mpler,1 Nadin Rohland,2 Carles Lalueza-Fox,3 Eske Willerslev,4 Tatyana Kuznetsova,5Gernot Rabeder,6 Jaume Bertranpetit,7 Torsten Scho¨neberg,1 Michael Hofreiter2*Because more than 99% of all species thathave ever lived on Earth are extinct, thegenetic basis of most phenotypic traits thathave evolved during life_s history cannot be studieddirectly. Although it is possible to approximatelyreconstruct the gene sequences of extant species_common ancestors (1), many extinct species rep-resent end points of evolution, and their gene se-quences cannot be investigated by using thisapproach. For late Pleistocene species, ancientDNA techniques should in principle allow analysesof extinct genes. However, despite progress in theanalysis of ancient DNA, to date only short frag-ments of nuclear genes could be amplified fromPleistocene specimens (2, 3).We have combined two methods recently in-troduced in ancient DNA research, large-scaleextraction and multiplex polymerase chain reaction(PCR), to amplify 1236 base pairs (bp), including thecomplete coding sequence, of the single-exon nuclearmelanocortin type 1 receptor (Mc1r) gene from anÈ43,000-year-old mammoth (Mammuthus primige-nius) bone from Siberia. The product of the Mc1rgene is a key protein in the determination of haircolor in both humans andother animals (4), pro-viding an opportunity toconnect genotype and phe-notype in extinct taxa. Forexample, reduced activityof MC1R has been shownto result in red hair in hu-mans and cows and red oryellow hair inmice, horses,and dogs (4). Dark- andlight-colored mammothhair has been recoveredfrompermafrostmummies,giving circumstantial evi-dence that mutations mayoccur in one of the genesinvolved in determinationofhair color.Weamplifiedthe mammoth Mc1r genein 23 primary fragmentsvarying in length from101 to 138 bp. Three po-sitions were found to bepolymorphic in the in-dividual sequenced, allresulting in amino acidchanges EThr21YAla21(Thr21Ala), Arg67Cys, andArg301Ser; positions relative to the elephant Mc1rsequence^ (Fig. 1A) (5). Because template damagemay affect ancient DNA sequences (6), we se-quenced each polymorphic position from at leastthree independent PCRs and, by using single-nucleotide polymorphism (SNP) typing, from addi-tional specimens. The identification of homozygousindividuals from these additional mammoth sam-ples revealed most likely allelic constitutions ofThr21-Arg67-Arg301 (allele 1) and Ala21-Cys67-Ser301 (allele 2), respectively (5). Whereas Thr21 ishighly variable amongmammalianMC1R orthologs,substitutions of Arg67 and Arg301 (Fig. 1A) affecthighly conserved residues. To investigate whether thetwo alleles show a functional difference, we con-structedMc1r expression vectors that are identical tothe inferred mammoth alleles on the amino acidlevel. The product of allele 1 shows high basalactivity and robust responses to two agonists,aMSH(Fig. 1B) and NDP-aMSH (5), in a concentration-dependent manner. In contrast, the product of allele2 shows an almost complete loss in basal activityand an È65% reduction in efficacy (Emax) to bothagonists. Functional testing of each position variablein the mammoth individually and all possible com-binations of the three mammoth-specific aminoacids revealed the Arg67Cys substitution as themain cause for MC1R dysfunction (Fig. 1B) (5).Pleistocenemammoths harbored both a fully anda partially activeMC1R variant. Given an otherwisefunctional pathway for hair coloration, the reductionin activity of the Arg67Cys variant would besufficient to result in substantially lighter hair color.Intriguingly, light-colored populations of the beachmouse (Peromyscus polionotus leucocephalus) car-ry an Mc1r allele that contains an Arg-to-Cys sub-stitution at the homologous sequence position, andfunctional tests and crossing experiments haverevealed both a reduction in basal and induced ac-tivity highly similar to that observed for the mam-moth MC1R protein and a strong associationbetween this amino acid polymorphism and adaptivecoat color phenotype (7). These findings stronglyargue for a phenotypic effect of this mutation also inthe mammoth. Thus, it is possible that mammothpopulations were polymorphic with regard to haircolor, harboring both dark- and light-haired individ-uals. Although it is currently impossible to deter-mine whether hair color was adaptive in mammoth,our study shows that functional analyses of nucleargenes are possible from late Pleistocene species. Thisopens up the possibility of studying a wide range ofextinct species_ features invisible in the fossil record.References and Notes1. J. W. Thornton, Nat. Rev. Genet. 5, 366 (2004).2. A. Greenwood, C. Capelli, G. Possnert, S. Pa¨a¨bo, Mol. Biol.Evol. 16, 1466 (1999).3. H. N. Poinar et al., Science 311, 392 (2006).4. J. L. Rees, Annu. Rev. Genet. 37, 67 (2003).5. Materials and methods are available as supportingmaterial on Science Online.6. M. Hofreiter et al., Nucleic Acids Res. 29, 4793 (2001).7. H. E. Hoekstra, R. J. Hirschmann, R. A. Bundey, P. A.Insel, J. P. Crossland, Science 313, 101 (2006).8. We thank the members of our research groups and severalanonymous reviewers for comments that improved themanuscript. This work was funded by the Max PlanckGesellschaft, Bundesministerium fu¨r Bildung, Wissenschaft,Forschung und Technologie and DeutscheForschungsgemeinschaft. Sequences have been deposited inGenBank under accession nos. DQ648859 to DQ648866.Supporting Online Materialwww.sciencemag.org/cgi/content/full/313/5783/62/DC1Materials and MethodsTables S1 and S2References20 April 2006; accepted 30 May 200610.1126/science.1128994BREVIA1Molecular Biochemistry, Institute of Biochemistry, Medical Faculty,University of Leipzig, 04103 Leipzig, Germany. 2Department ofEvolutionary Genetics, Max Planck Institute for EvolutionaryAnthropology, 04103 Leipzig, Germany. 3Departament deBiologia Animal, Facultat de Biologia, Universitat de Barcelona,08028 Barcelona, Spain. 4Ancient DNA and Evolution Group,Centre for Ancient Genetics, Niels Bohr Institute and BiologicalInstitute, University of Copenhagen, 2100 Copenhagen, Denmark.5Department of Paleontology, Faculty of Geology, Moscow StateUniversity, 119992 Moscow, Russia. 6Department of Paleontology,University of Vienna, 1090 Vienna, Austria. 7Unitat de BiologiaEvolutiva, Facultat de Cie`ncies de la Salut i de la Vida, UniversitatPompeu Fabra, 08003 Barcelona, Spain.*To whom correspondence should be addressed. E-mail:hofreite@eva.mpg.deFig. 1. (A) Localization of the fixed and polymorphic positions in mammothMc1r. The seven transmembrane domain architecture of MC1R is depicted.Compared with the Mc1r sequence of the African elephant, the approximatepositions of the fixed silent position and polymorphic positions in the twomammoth alleles are circled. (B) Partial loss of function in mammoth MC1Rvariants. For functional characterization of the wild type (allele 1), Arg67Cys andThr21Ala–Arg67Cys–Arg301Ser (allele 2) constructs of the Mc1r gene were tran-siently transfected into COS-7 cells and tested for agonist-induced cyclic aden-osine monophosphate (cAMP) accumulation. Green fluorescent protein (GFP)plasmid–transfected cells served as controls. Intracellular cAMP was measured inresponse to increasing concentrations of a-MSH as described (5). Shown is themean T SEM of four independent experiments, each performed in triplicate.7 JULY 2006 VOL 313 SCIENCE www.sciencemag.org62

Nuclear gene indicates coat-color polymorphism in mammoths

Nuclear gene indicates coat-color polymorphism in mammoths

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