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AMPLIFICATON OF THE MITOCONDRIAL CONTROL REGION

AMPLIFICATON OF THE MITOCONDRIAL CONTROL REGION

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AMPLIFICATON OF THE MITOCONDRIAL CONTROL REGION

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  1. AMPLIFICATON OF THE MITOCONDRIAL CONTROL REGION A tool for studying genetic origins

  2. Human DNA • What makes up human DNA? • DNA is bound to proteins in 46 chromosomes found in the nucleus of almost every cell in your body. • The chromosomes hold the vast bulk of genetic information that you've inherited from your parents.

  3. Genomic DNA is not the only DNA • Every human cell has a "second" genome, found in the cell's energy-generating organelle, • The mitochondrion

  4. Genomic DNA is not the only DNA • Mitochondria lie outside the nucleus, but still within the cell • Mitochondria are tiny structures that help cells in a number of ways, including producing the energy that cells need.

  5. Genomic DNA is not the only DNA • 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.

  6. Genomic DNA is not the only DNA • The primitive host cell, had an organized nucleus, • Provided a ready source of energy-rich nutrients to the mitochondrion, • The mitochondrion provided the cell with a means to extract energy using oxygen..

  7. Genomic DNA is not the only DNA • This attribute became key to survival. • The earliest atmosphere was composed primarily of hydrogen. • Oxygen began to accumulate with the advent of photosynthesis. • The primitive atmosphere shifted from reducing to oxidizing.

  8. Genomic DNA is not the only DNA • Like mitochondria, chloroplasts have their own genome • These were also once free-living bacteria. • Early plant cells evolved by serial endosymbiosis: successively engulfing two sorts of bacteria to obtain mitochondria and chloroplasts

  9. Genomic DNA is not the only DNA • Each mitochondrion—there are about 1,700 in every human cell—includes an identical loop of DNA about 16,000 base pairs long containing 37 genes.

  10. Genomic DNA is not the only DNA • In fact, each mitochondrion has several copies of its own genome, and ….. • There are several hundred to several thousand mitochondria per cell.

  11. Genomic DNA is not the only DNA • Nuclear DNA consists of three billion base pairs and an estimated 70,000 genes. • Each cell contains only two copies of each of the nuclear genes (one on each of the paired chromosomes) • But there are thousands of copies of a given mt gene per cell.

  12. Genomic DNA is not the only DNA • 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 • 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

  13. Characteristics of mtDNA are like bacteria • Mitochondria and bacteria share several genomic features that demonstrate their common ancestry. • Like bacterial chromosomes and plasmids, the mt genome is a circular molecule. • Mt and bacterial genomes generally have little noncoding DNA. Genes are in sequence (one after another) and they do not have introns (intervening sequences)

  14. Nuclear DNA vs. mt DNA • The mt (and bacterial) genome is a circular molecule to protect this DNA from exonucleases, which digest free ends of linear DNA molecules • Genes are tightly packed together on the chromosome, with few intergenic regions between genes and few introns within genes. By contrast, "end caps" of repetitive DNA sequences, the telomeres, protect the linear chromosomes of eukaryotic organisms. Eukaryotic genes are widely dispersed on chromosomes and have numerous introns.

  15. Nuclear DNA vs. mt DNA • mt DNA is 16,000 base pairs long containing 37 genes. • Nuclear DNA consists of three billion base pairs and an estimated 70,000 genes

  16. Mt DNA • The free-living ancestor of mitochondria were thought to have had a complement of at least 850 genes. • Over time, genes for functions that could be provided by the host were lost. • 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.

  17. Mt DNA • All of the 37 mt genes are involved in the production of energy and its storage in ATP. • Thirteen of these genes encode proteins involved in oxidative phosphorylation.

  18. mt DNA • 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.

  19. mt DNA • Genes take up the majority of the mt genome. • A noncoding region of approximately 1,200 nucleotides spans both sides of the arbitrary "0" position of the mt genome and goes by three terms: • control region, • D-loop, and • hypervariable region.

  20. mt DNA • 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.

  21. mt DNA • 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.

  22. mt DNA • The DNA sequence of the control region is termed hypervariable, because it accumulates point mutations at approximately 10 times the rate of nuclear DNA. • It will harbor several SNP that can be used to identify an individual’s genetic origins and trace inheritance patterns

  23. How does the Control Region Mutate? • 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 source of potent mutagens called oxygen free radicals.

  24. What is mutation? • In biology, mutations are changes to the nucleotidesequence of the genetic material of an organism. • Mutations can be caused by copying errors in the genetic material during cell division, • by exposure to ultraviolet or ionizing radiation, • chemical mutagens, or viruses, or • can be induced by the organism, itself, by cellular processes such as hypermutation

  25. mt DNA • 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.

  26. mt DNA • In the final step of transfer, electrons are combined with oxygen and protons to produce water.

  27. Oxidative Phosphorylation can damage DNA • 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. • Electrons can "leak" from the electron transport chain, converting about 1-3% of oxygen molecules into superoxide.

  28. Oxidative Phosphorylation can damage DNA • The cell has evolved a two-step mechanism to disable oxygen free radicals..

  29. In the first step, • Superoxide free radical is simultaneously reduced and oxidized (dismutated) to form hydrogen peroxide and oxygen . • 2O2- + 2H+ ------------ H2O2 + O2 superoxide dismutase • This is accomplished by superoxide dismutase, a so-called metabolic enzyme. Although hydrogen peroxide is also a reactive oxygen species, it is much less reactive than superoxide

  30. In the second step, • Hydrogen peroxide is converted into water and oxygen by catalase enzymes (reaction 2). • H2O2 ------- H2O + O2 • catalase • Hydrogen peroxide readily diffuses out of the mitochondria, and its level in the cytoplasm may provide the cell a means to monitor the efficiency of respiration.

  31. Oxidative Phosphorylation can damage DNA • The most mutagenic of the reactive oxygen species, hydroxyl (.OH) free radical, is generated when peroxide readily reacts with ferrous iron (Fe2+) or other transition metal ions to produce hydroxyl radical (reaction 3). • H2O2 ------- Fe2+ + OH- + . OH + Fe3+ • Ferric iron (Fe3+) can accept an electron from superoxide, cycling it back to the ferrous state and making it available to react with another peroxide molecule. • O2 - + Fe3+ ---- O2 + Fe2+

  32. Oxidative Phosphorylation can damage DNA • Even trace amounts of iron ion can potentially catalyze the formation of large amounts of hydroxyl free radical. • These chemical groups react with all types of biologically important molecules - nucleic acids, proteins, sugars, and lipids - producing radicals that undergo further reactions. • DNA radicals can react with protein radicals (in histones) to form crosslinks that interfere with chromatin unfolding, DNA repair, replication, and transcription.

  33. Oxidative Phosphorylation can damage DNA Free radicals attack all positions of the deoxyribose sugar, leading to single- and double-stranded breaks in DNA. Hydroxyl radicals also deaminate nucleotides, leading to point mutations or SNPs - notably C>T, G>C and G>T changes.

  34. DNA damage by oxygen free radicals suggests an accelerating degradation of mt function over time. • There evidence for "mitochondrial theory of aging." • mt genes have been implicated in a number of degenerative diseases - including Alzheimer disease, mitochondrial myopathy, Kearns-Sayre syndrome, CPEO (chronic progressive external phthalmoplegia), Leigh syndrome, Pearson syndrome, dystonia, and diabetes.

  35. mt DNA is inherited maternally • Whenever an egg cell is fertilized, nuclear chromosomes from a sperm cell enter the egg and combine with the egg's nuclear DNA, producing a mixture of both parents' genetic code. • The mtDNA from the sperm cell, however, is left behind, outside of the egg cell.

  36. mt DNA is inherited maternally • So the fertilized egg contains a mixture of the father and mother's nuclear DNA and an exact copy of the mother's mtDNA, but none of the father's mtDNA. • The result is that mtDNA is passed on only along the maternal line. • All of the mtDNA in the cells of a person's body are copies of his or her mother's mtDNA, and all of the mother's mtDNA is a copy of her mother's, and so on. • No matter how far back you go, mtDNA is always inherited only from the mother.

  37. mt DNA is inherited maternally • If you went back six generations in your own family tree, you'd see that your nuclear DNA is inherited from 32 men and 32 women[1]. • Your mtDNA, on the other hand, would have come from only one of those 32 women.

  38. mt DNA is inherited maternally • Review: • Mitochondria have DNA independent of the nuclear DNA • There are many copies of the mt DNA in every cell • Similar to bacterial DNA • Has an area called the D-loop-that is hypervariable (many SNP’s) • Mitochondria are only inherited maternally • Information: • http://www.pbs.org/wgbh/nova/neanderthals/mtdna.html • http://www.geneticorigins.org/mito/mitoframeset.htm

  39. Using mt DNA Under good circumstances - working from fresh cell samples - mt DNA is the easiest human DNA to amplify by PCR. The experiment we will perform examines a 440-nucleotide sequence from the noncoding region of mt genome. Each student will amplifying the same region, the gel electrophoresis results will also be the same for each. However, amplified student samples will be submitted to a Sequencing Service, which will generate student mt DNA sequences 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.

  40. Sample results!