All thirteen amino acid substitutions in the eight proteins were found to be compatible with nonsynonymous SNVs described above and reported previously 6, 15 (Fig.
Of the proteins identified via LC-MS/MS as significant identities with the African elephant proteins, we selected one extracellular matrix protein (laminin subunit gamma-1), five serum proteins (fatty acid binding protein 4, serum albumin, serotransferrin, complement factor H and fibrinogen alpha chain) and two intracellular proteins (aminopeptidase and glutathione S-transferase) for further comparative analysis because all of them showed high sequence coverage. To further consolidate evidence of the identification of mammoth proteins, we performed comparative analyses between genomic sequences and our proteomic data. The gene ontology analysis identified GO terms related to bone marrow functions such as osteoblast differentiation ( p = 4.80E-04) in bone marrow samples, and those related to muscle functions such as muscle sliding ( p = 7.10E-14) in muscle samples (Supplementary Table S3). 2b), indicating the identification of tissue-specific proteins. Among 869 proteins, 408 were identified from the bone marrow sample, whereas 677 were from the muscle sample and only 216 proteins were common to both samples (Fig. As a result, 41.8% of the proteins were identified in the order Proboscidea, into which the woolly mammoth is classified. africana (41%), followed by Elephas maximus (Asian elephant) and Mammut americanum (American mastodon), and five mammoth proteins were identified (Fig. Of them, the greatest number of matches was seen in L. Consequently, we identified 869 distinct proteins. The data obtained by LC-MS/MS analyses of proteins extracted from the mammoth samples were searched against UniProt mammalian protein database including all proteins predicted from the Loxodonta africana (African elephant) genome and 134 UniProt mammoth sequences. We then performed proteomic analyses to gain information about the repertoire and modifications of proteins. These SNVs showed a significant overlap with those of the five elephantid specimens (Total SNVs: 30%, Coding SNVs: 50%, Nonsynonymous SNVs: 43%) (Fig. Variant calling using SAMtools resulted in 27,182,201 SNVs in the Yuka mammoth genome against the African elephant reference genome. Next, we focused on single sequence nucleotide variants (SNVs) by comparing them with those of the five elephantids (M4 and M25 woolly mammoths and three Asian elephants) 6. 1b), which contains mammoth specimens found in the large area across the Holarctic 12, 13, 14.
Phylogenetic analysis indicates that the Yuka mammoth mitochondrial genome fell into the clade I (Fig. The average mapping rate of reads was 51.7%, and 83.0% of the elephant reference genome was covered by more than one sequence read, yielding 23-fold coverage of the reference genome. We aligned 1,446,220,624 sequencing reads to the African elephant genome (Loxafr3.0) and successfully mapped 747,034,525 reads (Supplementary Table S1). Genomic DNA libraries of the Yuka mammoth remains were constructed using Phusion polymerase, which provides efficient amplification with high fidelity, excluding post-mortem damage (C-to-T or G-to-A substitutions) (Supplementary Fig. The authenticity of our tissue samples after such a long frozen period was confirmed by whole-genome sequencing. Radiocarbon dating suggested that Yuka mammoth was 28,140 (☒30) years old. 1a) 11 led us to study the biological activities of mammoth nuclei. In the present study, the combination of NT and less-invasive live-cell imaging, previously developed by us 10, and excavation of other remains of the woolly mammoth from the Siberian permafrost, named ‘Yuka’ (Fig.
Our initial attempt of NT using 15,000-year-old mammoth tissues resulted in no nuclear reorganisation in mouse oocytes 9, possibly owing to the technological limitations at that time and the inappropriate state of the frozen mammoth tissues. Meanwhile, the investigation of biological activities of nuclei isolated from the remains using means of nuclear transfer (NT) approach is still in progress. Moreover, proteomic analyses have shown the presence of proteins in the remains 7, 8. Fundamental studies on woolly mammoth ( Mammuthus primigenius) genes, including whole genome analyses 1, 2, 3, led to the reconstitution of mammoth haemoglobin with cold tolerance 4 and to an understanding of the expression of mammoth-specific coat colour 5 and temperature-sensitive channels 6. Ancient species carry invaluable information about the genetic basis of adaptive evolution and factors related to extinction.