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Genom mitokondria mengandung gen non. – coding rRNAs dan beberapa komponen protein yang berhubungan dengan rantai respirasi yang akhirnya menjadi. Di dalam sel eukariot ada 2 jenis genom, yaitu DNA inti dan DNA sitoplasmik. DNA sitoplasmik berupa DNA mitokondria (mtDNA) untuk sel-sel hewan. non-coding dan yang paling polimorfik pada genom mitokondria. Analisis variasi urutan regio D-loop dapat digunakan menentukan individu atau etnis, juga.

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A complete mitochondrial mt genome fenom was reconstructed from a 38,year-old Neandertal individual using 8, mtDNA sequences identified among 4. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals.

There is evidence that purifying selection in the Neandertal mtDNA was reduced compared to other primate lineages suggesting that the effective population size of Neandertals was small. Since the mtDNA genome is maternally inherited without recombination, these results indicate that Neandertals made no lasting contribution to the modern human mtDNA gene pool Krings mitoiondria al.

High-throughput sequencing techniques have recently been applied to ancient DNA Green et al. These methods open new possibilities for the retrieval of ancient DNA that has hitherto relied mtiokondria on the cloning of random molecules in bacteria Higuchi et al.

The main benefit of the sequencing technique is the sheer volume of sequence data that make it practical to undertake genome-scale ancient DNA sequencing projects. This is particularly feasible for mitochondrial genomes Gilbert et al. In particular, they have allowed a quantitative model of how DNA degradation and chemical modification occurs and how the effects of these processes interact with the molecular manipulations used to generate sequencing libraries Briggs et al.

Notably, although it was previously known that a high rate of cytosine deamination mitokondrka in ancient DNA Hofreiter et al. Deamination of cytosine residues results in uracil residues that are read as thymine by DNA polymerases, leading to a high rate of C to T transitions. By sequencing, we have generated These include complete separation mitokkndria other parts of the laboratories, direct delivery of all equipment and reagents to the facility, positive pressure generated with filtered air gnom excludes particles larger than 0.

The bone surface was removed prior to extraction. However, the interior of bones are also often contaminated with modern human DNA, presumably gehom to past washing and other treatments of Neandertal bones.

Following amplification, we cloned the PCR product and sequenced — clones to determine the ratio of Mitokondrja to extant human mtDNA. From these extracts, we generated a total of nine libraries in the clean room facility using -adapters with a Neandertal-specific sequence key that is unique to this project and thus unequivocally identifies each sequence determined as derived from the extract of a Neandertal bone Briggs et al.

This allows detection of any contamination that may be introduced in subsequent handling and sequencing steps outside the clean room. To maximize the library and sequence yield, we incorporate two modifications to the standard protocol that reduce the need to perform titration runs of libraries Meyer et al.

From these libraries we generated a total of 39 million sequence reads by runs on the GS FLX sequencing platform. Bases were called using the standard signal threshold and filtering criteria with minor modifications tailored for short, ancient sequence reads Meyer et al. Neandertal sequences were identified within each run as described previously Green et al. While the total fraction of sequences that are identified as Neandertal varied across libraries and library pools, the ratio of putative Neandertal nuclear DNA sequences to mtDNA sequences varied little among these libraries and averages This corresponds to approximately 2, mtDNA molecules per cell.

In total, 8, mtDNA sequences were identified. They are of an average length of Ancient DNA sequences present a challenge for DNA sequence assembly since they are typically short and exhibit high rates of nucleotide misincorporation. A further complication is that pyrosequencing Ronaghi et al. With these issues in mind, we designed an assembly procedure for ancient DNA.

These alignments were then merged and each alignment column was examined to determine the majority base, yielding an assembled mtDNA sequence. Following this, some problematic regions remained in the assembly. These include four regions of a total length of 20 bp where no sequence coverage existed, eight other regions amounting to a total of bp covered by only single reads, nine positions where no majority base existed due to low coverage, and 31 homopolymers for which the data were not sufficient to determine their length.


These regions were amplified by PCR from a Vindija This resulted in the detection of an additional mtDNA sequences that were initially missed since they have a higher similarity to the human nuclear genome than human mtDNA and thus could only be identified using the assembled Neandertal mtDNA. Interestingly, of these sequences were similar to a single nuclear mtDNA insertion on chromosome 1 hg18 position ,—, In total, 8, mtDNA fragments totalingnt yielded a final assembly of 16, nt where no position has less than 9-fold sequence coverage.

In order to estimate the level of human contamination among the mtDNA sequences determined from these libraries, we realigned the individual sequences to the assembled mtDNA and used a number of differences between the putative Neandertal and human mtDNAs as diagnostic markers. We then examined each alignment to determine if it contained a position that allowed it to be classified as of either Neandertal or extant human origin.

First, we used three human-Neandertal differences in the HVR1: Among sequences that overlapped these positions, carried the base of the Neandertal assembly Supp. Of the 6 sequences that did not, five were cases where the observed base was T, the assembly base was C, and the modern human base was G mitokondrix A. Thus, these are likely to be the result of deaminated cytosine residues miokondria are common in ancient DNA Hofreiter et al.

The alternative that these are contamination by a previously unknown modern human mtDNA sequence is unlikely because of the large number of modern human sequences available. One single sequence matches the human base at a diagnostic position. Interestingly, this sequence shows features commonly associated with ancient DNA Supp.

In spite of this, it may represent a contaminant since modern DNA sequences retrieved from ancient specimens can carry such features Sampietro et al. In order to estimate contamination levels, we thus disregard the 5 C to T mismatches as uninformative and count the last sequence as a contaminant. This yields a total of out of sequences carrying Neandertal diagnostic positions and an estimate of the contamination rate of 0.

Sequences overlapping Tenom positions carrying diagnostic differences between Neandertal and extant humans. All 43 sequences overlapping the three diagnostic positions are shown. One hundred thirty-three positions genok these criteria and allowed 1, fragments to be classified.

Nine were of extant human origin yielding a contamination estimate of 0. We conclude that so few of the mtDNA sequences determined derive from extant humans that they will not compromise the assembly which has a fold average coverage. The assembled mtDNA sequence therefore represents a reliable reconstruction of the mtDNA that this ,itokondria individual carried when alive.

In the non-coding control region, the Neandertal sequence contains a deletion of four base pairs CACA at rCRS position and a previously mitokondfia insertion mitokondra one base pair Serre et al.

Figure 2A shows the distribution of pairwise sequence differences among 53 humans from around the world Ingman et al. Among the humans, the number of differences ranges from 2 to and is bimodal. The peak with a mode around 99 differences contains comparisons that geenom at least one member of deep clades containing sub-Saharan African mtDNAs. The peak with a mode around 44 differences involves comparisons between individuals outside these clades.

Thus, the Neandertal mtDNA falls gdnom the variation of extant humans even when nucleotide differences, uncorrected for multiple substitutions, are counted.

Notably, when the comparisons are restricted to only the HVRI or HVRIIthe distributions of pairwise differences among extant humans and between humans and the Neandertal overlap illustrating that multiple substitutions complicate the reconstruction of mtDNA relationships when only these regions are studied Figure 2B, C.

Mitochondrial DNA – Wikipedia

Neighbor-joining, maximum likelihood, parsimony, and a Bayesian approach Supp. Unsurprisingly, this topology is also found when analysis is restricted to various subsets of the sequence such as protein-coding sequences and RNA-coding sequences Supp. This was not the case indicating no significant miyokondria in evolutionary rates among the sequences.

To allow divergences of mtDNA sequences to be transformed to calendar years, we assumed – based on the fossil record – that humans and chimpanzees diverged between six Galik et al. Since the width of the divergence credibility interval increases almost linearly with the posterior mean of the divergence estimate Supp.

However, if the estimated date of the divergence between humans and chimpanzees or current assumptions about how the mtDNA evolves would be mitokondrua, the estimates in calendar years of the divergence of the Neandertal and human mitookondria would need to be revised. Phylogenetic tree and divergence time estimate of mtDNA sequences. A Bayesian phylogenetic tree of complete mtDNA sequences of the Neandertal, 10 extant humans, one chimpanzee, and one bonobo.


The Bayesian posterior probability and the bootstrap support values are shown for two internal nodes. Next we estimated henom substitutions that occurred in each of the 13 mtDNA protein-coding genes on the Neandertal and the human lineages using parsimony with the chimpanzee as an outgroup Table 1. The total number of silent substitutions assigned to the Neandertal lineage, 44, is mitlkondria than the number assigned to the human lineage, An apparent shortening of the Neandertal lineage relative to the human branch is also observed when all substitutions in the mtDNA are analyzed Supp.

This dearth of Mifokondria lineage differences is not clearly concentrated in any particular region of the mtDNA Supp. However, for amino acid substitutions, we observe a similar number on the Neandertal 20 and human 18 lineages. Thus, relative to silent substitutions more amino acid replacements occurred on the Neandertal lineage.

Number of synonymous and non-synonymous substitutions in each protein coding mtDNA gene assigned to the Neandertal or extant human lineage by parsimony using the chimpanzee as an outgroup.

To explicitly test for an increase in amino acid replacements on the Neandertal lineage, we evaluated the evolution of the 12 protein-coding genes excluding ND6, see Supp.

Data across seven primates human, Neandertal, chimpanzee, bonobo, gorilla, orangutan, and baboon using a maximum likelihood framework.

Table 3consistent with strong purifying selection on mitochondrial proteins in primates Hasegawa et al. To determine if any of the 13 proteins encoded in the mtDNA show an unusual pattern of evolution mtokondria humans since the divergence from the Neandertal, we contrasted the ratio of nucleotide polymorphisms in the 54 humans to fixed differences to Neandertal at synonymous and non-synonymous sites Table 2. Under a standard neutral model the ratio of diversity to divergence should be the same for both classes of sites McDonald and Kreitman, Polymorphism within humans and divergence to Neandertal at synonymous and nonsynonymoussites in protein-coding mtDNA genes.

The neutrality index value Rand mitokondfia Kann, is the ratio of polymorphism to divergence at non-synonymous sites versus this ratio atsynonymous sites. Values smaller than 1 indicate mitokondriia excess of non-synonymous divergence.

Mitochondrial DNA

Values greater than migokondria indicate excess non-synonymous polymorphism within humans. COX2 has experienced four amino acid substitutions on the human mtDNA lineage after its divergence from the Neandertal lineage aboutyears ago.

In order to see if any of these amino acids vary among humans today, we analyzed human mtDNA sequences in mtDB Ingman and Gyllensten, At one of the four positions rCRS pos. Because mtDNA is inherited without recombination and because the Neandertal mtDNA falls outside the variation of modern human mtDNA, this single modern human observation represents a reversion to the ancestral state seen in Neandertals and chimpanzees.

Thus, these four amino acid substitutions occurred in the relatively short period after the divergence of Neandertal and extant human mtDNAs and before the most recent common ancestor of current human mtDNAs.

The observation of four non-synonymous substitutions on the modern human lineage and no amino acid changes on the Neandertal lineage stands in contrast to the overall trend of more non-synonymous evolution in Neandertal protein-coding genes Table 1 and deserves consideration.

COX2 encodes subunit 2 of the cytochrome c oxidase, or complex IV, of the inner mitochondrial membrane. Complex IV catalyzes the reduction of molecular oxygen to water using electrons from cytochrome c and protons from within the mitochondrial matrix.

By acting as a proton pump Belevich et al. It has previously been noted that genes encoding various components of the electron transport chain evolve quickly in primates Doan et al. To determine what functional effects, if any, these amino acid substitutions may have, we examined the crystal structure of the bovine cytochrome c oxidase complex Figure 4 Muramoto et al.

However, three of them interact with the nuclear encoded COX5a and COX6c subunits, which are thought to perform regulatory functions. It has been noted that COX5a evolves fast in primates Uddin et al.


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