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TECHNICAL ANNEX

1. Mission
  • This database has been constructed with the collaboration of Gifu International Institute of Biotechnology (GiiB) and the Japan Science and Technology Agency (JST) as a part of the Prime Minister's Millennium Projects and the JST Database Development Program. Its mission is to identify, within 3 years, up to 3,000 mtSNPs (mitochondrial single nucleotide polymorphisms) distributed throughout the human mitochondrial genome and to make the information related to these mtSNPs available to the public. This database is linked with the IMS-JST JSNP database, which is one of the Prime Minister's Millennium Projects and was started in July 2000.
2. Data Releases
Subsections of mtSNP database
(1) mtSNP
According to the conventional definition, single nucleotide polymorphisms (SNPs) are nucleotide changes (substitution, deletion, or insertion) that can be detected at frequencies higher than 1%. In this mtSNP database, each group consists of 96 subjects, so that all the nucleotide changes that are recorded in this mtSNP database can be regarded as SNPs. When we compare 2 different groups, certain mtSNPs can be detected only in one group but not in the other. In this case, these mtSNPs can be regarded as group-specific mtSNPs.
(2) mtSAP
mtSNPs in the protein-coding regions can be classified into synonymous SNPs (sSNPs) and nonsynonymous SNPs (nsSNPs). In this mtSNP database, we use the phrase mitochondrial single amino acid polymorphisms (mtSAPs) to mean amino acid replacements caused by nsSNPs.
(3) Grantham value
To evaluate the functional alterations that are caused by a mtSAP, we indicated for each mtSAP the Grantham value that was calculated from the physicochemical differences between the original and altered amino acid residues (Grantham R. Amino acid difference formula to help explain protein evolution. Science 1974; 185: 862-864). The average of the Grantham values of 20*19/2=190 different amino acid replacements is 50. The mtSAPs with Grantham values larger than 50 are regarded as radical amino acid replacements; and those with Grantham values of less than 50, as conservative replacements.
(4) Prediction of effect of mtSAP on the 3-dimensional structure
For further evaluation of the effects of mitochondrial single amino acid polymorphisms (mtSAPs) on the functioning of the mitochondrial respiratory chain, this mtSNP database provides a molecular simulation system that predicts the conformational changes caused by each mtSAP or a group of mtSAPs detected in each individual. At present, the 3-dimensional structures of bovine cytochrome c oxidase and cytochrome bc1 complex (ubiquinol-cytochrome c oxidoreductase) are known. All of the subunits, both mitochondrially encoded and nuclearly encoded, of the bovine enzymes were replaced by those of human enzymes, and then the structures of the human enzymes were calculated by a molecular dynamic simulation system. For each mtSAP or group of mtSAPs that occurred in each individual, the altered structures are predicted by the simulation system in GiiB. These predicted structures will be sent as protein database format (PDB) files by e-mail to each client who has requested them through the 3D Structure section in this mtSNP database.
(5) Location of mtSAP's in the secondary structure of mitochondrially encoded subunits
On the request of each client, locations of mtSAPs on the 2-dimensional structures of subunits encoded by mtDNA are displayed on the web page of the 2D Structure section of this mtSNP database. The 2D structures of NADH dehydrogenase subunits (ND1-6, ND4L), subunits of cytochrome c oxidase (CO1-3), and cytochrome b protein (Cytb) are based on the prediction of their hydrophobic transmembrane domains by the SOSUI system [Classification and Secondary Structure Prediction of Membrane Proteins by Mitaku Group, Department of Biotechnology, Tokyo University of Agriculture and Technology; sosui@proteome.bio.tuat.ac.jp; http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html].
Coordinates of the amino acid residues were predicted by the SOSUI system. For ND1-6 and ND4L, as well as for subunits of ATP synthase (ATP6 and ATP8), the 3-dimensional structures of which are unknown, the amino (N) terminus of each subunit is located in the upper left corner. For the CO1, CO2, and CO3 subunits and the cytochrome b protein, the 3D structures of which are known for bovine enzymes, the orientation and folding of each subunit has been adjusted according to the crystal structure. The upper part corresponds to the inter-membrane space side; and the lower part, to the matrix side.
(6) Location of mtSNP's in the secondary structure of tRNA or rRNA
2D structure of tRNA
The structure of transfer RNA is displayed in the conventional cloverleaf structure by use of the program SstructView (http://smi-web.stanford.edu/projects/helix/sstructview/home.html). To display the location of a given nucleotide substitution, first select one of the tRNA genes in the scroll box and click "OK". Then a list of mtSNPs within the tRNA gene is displayed. Select one (or several) mtSNP by marking the corresponding checkbox, and then click the "OK" box. When the 2D structure of the tRNA molecule is displayed, click the "zoom in" button to enlarge the structure, or click the "zoom out" button to scale down the structure. The selected mtSNP is highlighted in blue color. To identify a nucleotide in the structure, move the cursor over the nucleotide, and then the nucleotide number (from 1 to 59-75 for each tRNA molecule) is displayed.

2D structure of rRNA
The secondary structure of the 12S ribosomal RNA molecule is displayed by use of the program SstructView (http://smi-web.stanford.edu/projects/helix/sstructview/home.html) according to the model of 12S rRNA (http://rrna.uia.ac.be/ssu/index.html) and that of 16S rRNA (http://rrna.uia.ac.be/lsu/index.html) predicted by Dr. Jan Wuyts and Prof. Rupert De Wachter (Department of Biomedical Sciences, Antwerp University [UIA], Belgium). To display the location of a given nucleotide substitution, first select one of the rRNA genes in the scroll box and then click "OK". A list of mtSNPs within the rRNA gene is now displayed. Select one (or several) mtSNP by marking the corresponding checkbox, and then click the "OK" box. When the 2D structure of the rRNA molecule is displayed, click the "zoom in" button to enlarge the structure, or click the "zoom out" button to scale down the structure. The selected mtSNP is highlighted in blue color. To identify the position of a nucleotide in the structure, move the cursor over the nucleotide, and then the nucleotide number is displayed. 
(7) mtSAP in evolution
To examine whether a certain amino acid residue is conserved among species, one can compare a mtSAP with the amino acid sequences of 61 mammalian species. The entire sequences of the mitochondrial genome for these 61 mammalian species are registered in an organelle genome database (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/40674.html).
3. Sequence Information
The entire sequences of mtDNA have been deposited in DDBJ. The sequence data were obtained by Gifu International Institute of Biotechnology (GiiB).
4. Citing mtSNP database
When referring to the mtSNP database in scientific communications, please use the following reference:
   mtSNP: a database of human mitochondrial genome polymorphisms
   Nucleic Acids Research, to be published
5. Disclaimer and Liability
Gifu International Institute of Biotechnology and the Japan Science and Technology Agency make no representations or warranties regarding the content or accuracy of the information and also make no representations or warranties of the marketability or fitness for a particular purpose and accept no responsibility for any consequences of the receipt or use of the information. 
6. Copyright Status
Database and documents available from this website are protected under Japanese and foreign copyright laws. Permission to reproduce this database or documents is required.
7. Contact
If you have any questions or comments relating this website, please contact mtsnp@giib.or.jp 
8. Project Leader
MasashiTanaka, M.D, Ph.D (GiiB)

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