Yuri K. Moliaka

Complete Mitochondrial Genome and Phylogeny of Pleistocene MammothMammuthus primigenius

Phylogenetic relationships between the extinct woolly mammoth(Mammuthus primigenius), and the Asian(Elephas maximus) and African savanna(Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch—the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as ~1,600–1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests thatM. primigenius andE. maximus are sister species that diverged soon after their common ancestor split from theL. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population ofM. primigenius throughout the late Pleistocene.

Genotype Analysis Identifies the Cause of the “Royal Disease”

DNA from historical specimens reveals the mutation causing the hemophilia that afflicted the royal families of Europe. The “royal disease,” a blood disorder transmitted from Queen Victoria to European royal families, is a striking example of X-linked recessive inheritance. Although the disease is widely recognized to be a form of the blood clotting disorder hemophilia, its molecular basis has never been identified, and the royal disease is now likely extinct. We identified the likely disease-causing mutation by applying genomic methodologies (multiplex target amplification and massively parallel sequencing) to historical specimens from the Romanov branch of the royal family. The mutation occurs in F9, a gene on the X chromosome that encodes blood coagulation factor IX, and is predicted to alter RNA splicing and to lead to production of a truncated form of factor IX. Thus, the royal disease is the severe form of hemophilia, also known as hemophilia B or Christmas disease.

Genomic identification in the historical case of the Nicholas II royal family

Accurate unambiguous identification of ancient or historical specimens can potentially be achieved by DNA analysis. The controversy surrounding the fate of the last Russian Emperor, Nicholas II, and his family has persisted, in part, because the bodies of 2 children, Prince Alexei and 1 of his sisters, have not been found. A grave discovered in 1991 contained remains putatively identified as those of the Russian Royal family. However, not all family members were represented. Here, we report the results of genomic analyses of new specimens, the human remains of 2 burned skeletons exhumed from a grave discovered in July 2007, and the results of a comprehensive genomic analysis of remains from the 1991 discovery. Additionally, ≈117 years old archival blood specimens from Nicholas II were obtained and genotyped, which provided critical material for the specific determination of individual identities and kinship identifications. Results of genotypic analyses of damaged historical specimens were evaluated alongside samples from descendants of both paternal and maternal lineages of the European Royal families, and the results conclusively demonstrate that the recently found remains belong to children of Nicholas II: Prince Alexei and his sister. The results of our studies provide unequivocal evidence that the remains of Nicholas II and his entire family, including all 5 children, have been identified. We demonstrate that convergent analysis of complete mitochondrial genome sequences combined with nuclear DNA profiles is an efficient and conclusive method for individual and kinship identification of specimens obtained from old historic relics.

Impas 1 possesses endoproteolytic activity against multipass membrane protein substrate cleaving the presenilin 1 holoprotein

Presenilins (PS1 and PS2) are supposed to be unusual aspartic proteases and components of the γ‐secretase complex regulating cleavage of type I proteins. Multiple mutations in PS1 are a major cause of familial early‐onset Alzheimers disease (AD). We and others recently identified PS‐related families of proteins (IMPAS/PSH/signal peptide peptidases (SPP)). The functions of these proteins are yet to be determined. We found that intramembrane protease‐associated or intramembrane protease aspartic protein Impas 1 (IMP1)/SPP induces intramembranous cleavage of PS1 holoprotein in cultured cells coexpressing these proteins. Mutations in evolutionary invariant sites in hIMP1 or specific γ‐secretase inhibitors abolish the hIMP1‐mediated endoproteolysis of PS1. In contrast, neither AD‐like mutations in hIMP1 nor in PS1 substrate abridge the PS1 cleavage. The data suggest that IMP1 is a bi‐aspartic polytopic protease capable of cleaving transmembrane precursor proteins. These data, to our knowledge, are a first observation that a multipass transmembrane protein or the integral protease per se may be a primary substrate for an intramembranous proteolysis.

O2-05-07

not available. PL3-02 CHOLINESTERASES IN NEURODEGENERATION: HAVE WE MISSED SOMETHING? Roger Lane, Novartis Neuroscience Clinical Development and Medical Affairs, East Hanover, NJ, USA. Contact e-mail: roger.lane@novartis.com Background: In addition to co-regulatory esterase functions in terminating acetylcholine (ACh)-mediated neurotransmission, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) proteins which exist in varS197 Plenary: PL3: Interventions and Treatments