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Mitochondrial (mt) Point Mutations are single nucleotide polymorphisms (SNPs) detected in the ring-shaped chromosome found in the cell's energy-producing organelle. Mutations are common in mt DNA, in large part due to exposure to oxygen free radicals generated as a by-product of respiration. Mitochondrial mutations can be used to retrace the common maternal lineage of modern humans and to determine our relationship to the extinct hominid Neandertal.
Introduction
Every human cell has a "second" genome, found in the cell's energy-generating organelle, the mitochondrion. In fact, each mitochondrion has several copies of its own genome, and there are several hundred to several thousand mitochondria per cell. This means that the mitochondrial (mt) genome is highly amplified. While each cell contains only two copies of a given nuclear gene (one on each of the paired chromosomes), there are thousands of copies of a given mt gene per cell. Because of this high copy number, it is possible to obtain a mt DNA type from the equivalent of a single cell's worth of mt DNA. Thus, mt DNA is the genetic system of choice in cases where tissue samples are very old, very small, or badly degraded by heat and humidity.
Under good circumstances - working from fresh cell samples - mt DNA is the easiest human DNA to amplify by PCR. This experiment examines a 440-nucleotide sequence from the noncoding region of mt genome. Hand cycling is a realistic alternative to automated thermal cyclers, and the high yield of amplified product can be visualized in an agarose gel with a variety of stains.
Because each student is amplifying the same region, the gel electrophoresis results will also be the same for each. However, amplified student samples may be submitted to our Sequencing Service, which will generate student mt DNA sequences and post the results on our Sequence Server. Comparison of control region sequences reveals that most people have a unique pattern of single nucleotide polymorphisms (SNPs). These sequence differences, in turn, are the basis for far-ranging investigations on human DNA diversity and the evolution of hominids.
T H E O R Y
Evolution of the Mitochondrial Genome
There is strong evidence that mitochondria once existed as free-living bacteria, which were taken up by primitive ancestors of eukaryotic cells in an arrangement termed endosymbiosis. The primitive host cell, which had an organized nucleus, provided a ready source of energy-rich nutrients to the mitochondrion, and the mitochondrion provided the cell with a means to extract energy using oxygen. This attribute became key to survival, as the primitive atmosphere shifted from reducing to oxidizing. Although the earliest atmosphere was composed primarily of hydrogen, oxygen began to accumulate with the advent of photosynthesis. Like mitochondria, chloroplasts have their own genome and were once free-living bacteria. Thus, early plant cells evolved by serial endosymbiosis: successively engulfing two sorts of bacteria to obtain mitochondria and chloroplasts.
The human mt genome, like those of other eukaryotes, has been vastly reduced through evolutionary time. The free-living ancestor of mitochondria, perhaps similar to a Rickettsia, must have had a complement of at least 850 genes. Over time, genes for functions that could be provided by the host were lost. Also, some genes needed for respiration were transferred to the nucleus. Over millions of years of evolutionary time, this reduction resulted in the small mt chromosome found in eukaryotes.
Besides having similar dimensions, mitochondria and bacteria share several genomic features that demonstrate their common ancestry. Like bacterial chromosomes and plasmids, the mt genome is a circular molecule. A circular configuration was the first mechanism to provide protection from exonucleases, which digest free ends of linear DNA molecules. By contrast, "end caps" of repetitive DNA sequences, the telomeres, protect the linear chromosomes of eukaryotic organisms. Mt and bacterial genomes, also, generally have little noncoding DNA. Genes are usually tightly packed together on the chromosome, with few intergenic regions between genes and few introns within genes. These features are contrary to eukaryotic genes, which are widely dispersed on chromosomes and have numerous introns.
Structure of the Mitochondrial Genome
The entire DNA sequence of the human mt genome - 16,569 nucleotides - was determined in 1981, well in advance of the Human Genome Project. The mt genome contains 37 genes, all of which are involved in the production of energy and its storage in ATP. Thirteen of these genes encode proteins involved in oxidative phosphorylation. The remaining genes encode tranfer RNAs (22 genes) and ribosomal RNAs (2 genes) that translate the proteins' genes within the mitochrondrion. Mammalian mt genes use a slightly different genetic code than nuclear genes, where UGA = tryptophan, AUA = methionine, and AGA and AGG = stop.
Genes take up the majority of the mt genome. However, a noncoding region of approximately 1,200 nucleotides spans both sides of the arbitrary "0" position of the mt genome and goes by three confusing terms: control region, D-loop, and hypervariable region. Control region refers to the fact that this region contains the signals that control RNA and DNA synthesis. A single promoter on each DNA strand initiates transcription in each direction, and a single origin initiates replication of each strand. D-loop refers to the early phase of replication, when the first newly-synthesized strand displaces one of the parental strands, forming a "bubble" or loop. The DNA sequence of the control region is termed hypervariable, because it accumulates point mutations at approximately 10 times the rate of nuclear DNA.
Oxygen Free Radicals and Mitochondrial Mutation
The control region is relatively tolerant of a high mutation rate, because binding sites for DNA and RNA polymerase are defined by only short nucleotide sequences. The high mutation rate of mt DNA is almost certainly due to the fact that the mt genome is located in close proximity to the respiratory machinery of the cell - a known source of potent mutagens called oxygen free radicals.
Oxygen free radicals are a natural byproduct of respiration. Electrons formed during the oxidation of glucose are passed along the electron transport chain, a series of electron-accepting molecules embedded in the mt membrane. Protons created during electron transfer ultimately are used to drive the synthesis of ATP. In the final step of transfer, electrons are combined with oxygen and protons to produce water.
However, faulty electron transfer at any point in the electron transport chain, results in an electron being accepted by atomic oxygen(O2). The superoxide free radical created (O2. -) has a single unpaired electron (designated by the "dot" in the chemical formula), which seeks to react with an electron source to make a stable electron pair. Under physiological conditions, electrons "leak" from the electron transport chain, converting about 1-3% of oxygen molecules into superoxide.
The cell has evolved a two-step mechanism to disable oxygen free radicals. In the first step, superoxide free radical is simultaneously reduced and oxidized (dismutated) to form hydrogen peroxide and oxygen (reaction 1 below). This is accomplished by superoxide dismutase, a so-called metabol
