41-10 m smallMitochondria are present in the cytoplasm of all eukaryote cells of animals and higher plants and also in some microorganisms (algae, fungi, protozoa).A mitochondrion is a structure formed by two membranes separated by a space, the outer and the inner membrane, which forms these foldings called the cristae.The space enclosed by the inner membrane is the matrix, which appears moderately dense. In the matrix you find strands of DNA, ribosomes or small granules.A mitochondrion can be 1-10 m big and are present in the cytoplasm of all eukaryote cells of animals and higher plants an also in some microorganisms such as fungi.
5Endosymbiont Hypothesis endosymbiont hypothesis: originally proposed in 1883 by Andreas Schimper, but extended by Lynn Margulis in the 1980s.Mitochondrial ribosomal RNA genes and other genes show that the original organism was in the alpha-proteobacterial family (similar to nitrogen-fixing bacteria)Evidence:mitochondria have their own DNA (circular)the inner membrane is more similar to prokaryotic membranes than to eukaryotic. By the hypothesis, the inner membrane was the original prokaryotic membrane and the outer membrane was from the primitive eukaryote that swallowed it.mitochondria make their own ribosomes, which are of the prokaryotic 70S type, not the eukaryotic 80S type.mitochondria are sensitive to many bacterial inhibitors that don’t affect the rest of the eukaryotic cell, such as streptomycin, chloramphenicol, rifampicin.mitochondrial protein synthesis starts with N-formyl methionine, as in the bacteria but unlike eukaryotes.Most of the original bacterial genes have migrated into the nucleus.Eukaryotes that lack mitochondria generally have some mitochondrial genes in their nucleus, evidence that their ancestors had mitochondria that were lost during evolution.
6Mitochondrial Genome Small circular genome >1000 copies/ cell 16569 bp 44% G+CH- Strand GuaninesL- Strand CytosinesD- Loop 7S DNAThe mitochondrial genome is a small circular genome of 16569bp. In one cell more that 1000 copies can be found.44% of the genome is G-C rich.The two single stands are called the H-strand from heavy, because of a high concentration of guanines and the L-stand from light, because of a high concentration of cytosines.A small part of the mtDNA is a tripple DNA strand because of a second replication from the H-strand. This 7S piece of ssDNA is called the D-loop and this is the only non-coding part of the mt genome.
7Mitochondrion plays a role in: Energy production Oxidative phosphorilation (OXPHOS)Maintaining the intracellular homeostasisProtecting the rest of the cell from reactive oxygen species (ROS)Apoptosis important development and diseaseMitochondria play a very big role in energy production. About this I will tell some more in the following slides.Maintaining the intracellular homeostasis of many important metabolites and ions.Protect the rest of the cell from the damaging effects of reactive oxygen species created during the OXPHOS process, by harnessing and inactivate them.Play a central role in necrosis and apoptosis, which are important in normal development and the etiology of many diseases.The many functions of the mitochondria are intricately interconnected.
9Apoptosis ATP Caspase 3 casp9 Bcl2 Apaf1 substrates Mitochondria-the point of no return-to live or to diePro-caspase 3Smac/DiabloXIAPATPCaspase 3casp9AIFBcl2Apaf1AIFsubstratesApoptosisNuclear apoptosis
10Genome StructureThe mitochondrial genome is a circle, 16.6 kb of DNA. A typical bacterial genome is 2-4 Mbp.The two strands are notably different in base composition, leading to one strand being “heavy” (the H strand) and the other light (the L strand).Both strands encode genes, although more are on the H strand.A short region (1121 bp), the D loop (D = “displacement”), is a DNA triple helix: there are 2 overlapping copies of the H strand there.The D loop is also the site where most of replication and transcription is controlled.Genes are tightly packed, with almost no non-coding DNA outside of the D loop. In one case, two genes overlap: they share 43 bp, using different reading frames. Human mitochondrial genes contain no introns, although introns are found in the mitochondria of other groups (plants, for instance).
11The Human Mitochondrial Genome copies/mitochondrionCircular~ 16 kb (some plants ~100 kb!)Crowded (~40 genes)13 genes involved in oxidative phosphorylation + other genes (DNA pol, rDNAs, tRNAs)Most proteins in mitochondria are imported from cytoplasm100,000 copies of mitochondrial DNA in ovum
12Organization of the human genome Limited autonomy of mt genomes mt encoded nuclearNADH dehydrog 7 subunits >41 subunitsSuccinate CoQ red 0 subunits 4 subunitsCytochrome b-c1 comp 1 subunit 10 subunitsCytochrome C oxidase 3 subunits 10 subunitsATP synthase complex 2 subunits 14 subunitstRNA components 22 tRNAs nonerRNA components 2 components noneRibosomal proteins none ~80Other mt proteins none mtDNA pol, RNA pol etc.
14The Human Mitochondrial Genome expression unlike nucleus genome… Transcription controlled by nuclear proteins:3 promoters-* H1: H-strand; complete transcription of one strand of mtDNA* L: L-strand; complete transcription of light strand of mtDNA* H2: Synthesis of 2 rRNAsTranscripts then procesed into individual genes prior to translation
15Coding- Non-coding 37 genes 28 genes H- strand 9 genes L- strand 24 genes specify a mature RNA product2 mitochondrial rRNA molecules (23S and 16S)As I said before the mt genome is completely coding sequence except the D-loop.The mtDNA codes for 37 genes from which 28 on the H-strand and 9 on the L-strand.From these 37 genes 24 specify a mature RNA product.2 encode mt rRNA molecules22 encode tRNA molecules22 tRNA molecules13 genes specify polypeptides
17Mitochondrial Genetic code is somewhat different… Human Mito StandardAGA Ter ArgAGG Ter ArgAUA, AUU Met IleUGA Trp TerUGA encodes trp at low efficiency in E. coliPlastid genetic code: GUG, UUG, AUU, CUG can initiate translation
18Mitochondrial inheritance pattern - uniparental maternal in animals Paternal inheritance in gymnosperms, some angiosperms
19Mitochondrial disease (1) Incidence from 1: to 1:4000Affecting most energy demanding tissuesCentral nervous systemHeartSkeletal muscleThis is not always the caseMitochondrial diabetesLiver and kidney diseasePearson syndromeSpecific, but highly variable clinical features with various gene defectsMitochondrial disease has an incidence from 1: to 1:4000.They affect most energy demanding tissues, like central nervous system, heart skeletal muscle.But this is not always the case. For example in mitochondrial diabetes and liver and kidney disease.Mitochondrial diseases are specific, but can have highly variable clinical features with various gene defects.
20Clinical presentation of OXPHOS defects Unexplained combination of neuromuscular and/ or non-neuromuscular symptomsProgressive courseInvolvement of seemingly unrelated tissues or organsClinical symptoms either isolated or in combination, may occur at any stageFrequent feature; increasing number of organs involved in the course of the diseaseWhile initial symptoms usually persist and gradually worsen, they may occasionally improve or even disappear, as other organs become involvedOxidative phosphorilation defects give rice to the most common symptoms in mitochondrial disease. Clinical presentation of these defects are:Unexplained combinations of neuromuscular and/ or non neuromuscular symptomsProgressive courseInvolvement of seemingly unrelated tissues and organs.*Clinical symptoms either isolated or in combination, may occure at any stage*Frequent feature; increasing number of organs involved in the course of the disease.*While initial symptoms usually presist and gradually worsen, they may occasionally improve or even disappear, as other organs become involved
21Particular Genetical Processes HeteroplasmyMixed population of normal and mutant mitochondrialgenomes in one cellRelaxed replicationmtDNA is degradated and replicates continuously,even in non dividing cellsAn other particular process is hetroplasmy. Which means that in one cell a mixed population of as well normal as mutant mtDNA is present.Further this is more complicated by the facts that mitochondrial DNA segregates randomly and thus different proportions of mtDNA are found in the child.This creates the bottleneck effect shown in this figure.Thought is that different proportions of normal and mutant mtDNA has an influence on the phenotype.Mitochondrial DNA replicates continuously even in non dividing cells.
22gomori trichrome staining mtDNA Mutations (1)Affecting mitochondrial protein synthesisSingle deletions always one or more tRNA genesPoint mutations in rRNA or in tRNAAssociated with: multiple system disorders, lactic acidosis, “ragged red fibers” in muscle biopsyYou can find several mtDNA mutations.The once affecting mitochondrial protein synthesis, which are mainly single deletions (in 1 or > tRNA genes) or point mutations in rRNA or tRNA.These are associated with Ragged Red Fibers, which stain red with gomori trichrome staining.Cells which have a defective mitochondrial energy production start to proliferate more mitochondria sinds they need more energy, which leads to situations in which muscle fibers have mild to moderate mitochondrial proliferation (Red rim & speckled sarcoplasm)Or marked mitochondrial proliferation in which mitochondria have replaced most other structures in one of the muscle fibersgomori trichrome staining
23mtDNA Mutations (2) In mtDNA protein coding genes LHON Complex I (NADH dehydrogenase genes)ND 4 G11778AND 6 T14484CND 1 G3460ANARP/MILS ATPase 6 T8993G>100 mutations within 37 genesFurther mutations are found in mtDNA protein coding genes. Examples are Leber Hereditary Optic Neuropathy in which mutations are found in the NADH dehydrogenase genes orLeigh Disease or Maternally Inherited Leigh Syndrome which have an associated mutation on the ATPase 6 gene.(Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; alternate phenotype at this locus is reported as Leigh Disease)In total more that 100 mutations within the 37 mtDNA genes are associated with different mitochondrial diseases.
24Mitochondrial-inherited diseases Example Leigh’s Syndrome Cause - point mutation in either ATPase 6, mt tRNA (5 dif),NADH dehydrogenase 5, cytochrome oxidase IIIResult- ATP deficiencyPhenotype: Motor & Intellectual regression,Death often within 2 years of onset
25Mitochondrial DNA mutations in human genetic disease (Wallace Sci. Amer. 277:40)
27Pearson syndrome is currently recognized as a rare, multisystemic, mitochondrial cytopathy. Its features are refractory sideroblastic anemia, pancytopenia, defective oxidative phosphorylation, exocrine pancreatic insufficiency, and variable hepatic, renal, and endocrine failure. Death often occurs in infancy or early childhood due to infection or metabolic crisis. Patients may recover from the refractory anemia. Older survivors have Kearns-Sayre syndrome (KSS), which is a mitochondropathy characterized by progressive external ophthalmoplegia and weakness of skeletal muscle.
28Mitochondrial-inherited diseases Most decrease ATP-generating ability of the mitochondriaAffect function of nerve and muscle cellsSeverity of symptoms vary with amt of wt mtDNA presentIn ragged red fiber disease: % of mtDNA is wt (heteroplasmic)
29nDNA mutations affecting mtDNA stability/ expression A primary nuclear gene defect causes secondary mtDNA loss or deletion tissue dysfunctionMendelian inheritanceFactors for mtDNA maintenance and repair all encoded by nuclear genesOnly 1 nDNA mutation reported mtDNA (succinate dehydrogenase gene)There are many mitochondrial diseases which are thought to be due to nuclear DNA mutations which affect the mtDNA stability and expression.In these diseases a primary nuclear gene defect causes secondary mtDNA loss or deletion and thereby tissue disfunction.These diseases have a mendelian inheritance since nDNA is the first cause.Factors for mtDNA maintenance and repair all encoded by nuclear genes.Until now only 1 nDNA mutation reported to be associated with a mitochondrial disease.
30Anu Suomalainen and Jyrki Kaukonen, Am J Med Genet 2001 adPEO (1)adPEO autosomal dominant progressive external opthalmoplegiaAutosomal dominantOnset yearsRRFMultiple mtDNA del in post-mitotic tissueBasal ganglia and cerebral cortex > 60 %Skeletal and ocular muscle + heart > 40 %Autosomal dominant progressive external opthalmoplegia is an autosomal dominant mitochondrial disease with an onset ofThe mendelian inheritance pattern of this disease suggests that a nuclear mutation should the primary cause of this disease.Typical are ragged red fibres in muscle biopsy and multiple mtDNA deletions in post mitotic tissue.In this disease you find more than 60% mutant mitochondrial DNA in basal ganglia and cerebral cortex andMore than 40% in skeletal and ocular muscle and in the heart.Anu Suomalainen and Jyrki Kaukonen, Am J Med Genet 2001
31Mitochondria and Aging The “mitochondrial theory of Aging”: as we live and produce ATP, our mitochondria generate oxygen free radicals (electrons “leak” from electron transport chain) that attack our mitochondria and mutate our mitochondrial DNA.Result: decrease in ATP needed for normal cell functionEvidence:-5000 bp deletion in mtDNA absent in heart muscle before age 40Present in increasing frequency in older heart muscle-rats fed on restricted diets - live longer - fewer oxygen freeradicals generated - fewer mitochondrial mutations accumulateElevated mtDNA defects in people with degenerative diseases(Parkinson’s, Huntingtons, Alzheimers, ALS...)
32mtDNA Mutations (3) Somatic mitochondrial DNA mutations with age in healthy individualsOld people typically harbour a wide range of different mtDNA deletions in post mitotic tissues; skeletal muscle, myocardium, brainOverall amount of mutant mtDNA very lowOne cell high percentage of one mutant mtDNADifferent cells different mutationsThreshold effect OXPHOSSomatic mitochondrial DNA mutations with age accumulate in healthy individuals.Old people typically harbor a wide range or different mt deletions in postmitotic tissues.The overall amount of mutant mtDNA is very low in tissue as a whole, but individual cells can contain high percentages of a single mutant species.Different cells usually contain different mutations.When the proportion of the mutant mtDNA exceeds a critical threshold concentration, a defect of mitochondrial oxidative phosphorylation results
33Bcl-2 family & diseases Bcl-2-follicular lymphoma Bcl-2 IgH T(14; 18)Bcl-2IgHconstitutive expressionBax-colon cancerAccelerates tumorigenesis with reduced apoptosis inBax-/- micecolon cancers of the microsatellite mutator phenotype>50% somatic frameshift mutations in the Bax gene
34In Search of Eve Mitochondrial DNA doesn’t undergo recombination It evolves faster than nuclear DNA (~1 change per 1,500-2,000 years)One theory estimates that all non-Africans descended from “Eve” who lived 150,000 years ago in Africa
35Mitochondrial DNA and Evolution The genetic diversity of African populations was confirmed by later studiesHowever, proponents of the out-of-Africa hypothesis assumed that genetic diversity reflected only the age of a population rather than population size.Africa has greater genetic diversity because its prehistoric population was probably larger than elsewhere.Recently John Relethford and Henry Harpending have argued that differences in ancient population size could mimic a recent African origin of modern humans. The data reflect population dynamics, they say, and do not support one model of modern human origins over another.
36Molecular analysis of Neanderthal DNA from the northern Caucasus Igor V. OvchinnikovAnders GötherströmGalina P. RomanovaVitaliy M. KharitonovKerstin LidénWilliam Goodwin
37Main informationsThe neandertal mtDNA placed outside the mtDNA pool of modern humans.The divergence between Neandertals and modern humans estimated to have occured between 317,000 and 741,000 years ago.
38September 11 and Mitochondria DNA Typing If cell is damaged, chromosomal DNA disintegratesHeavily damaged samples tested by “profiling” mitochondrial DNAEvery cell in the human body containsthousands of copies of maternally inherited mtDNA."We use mitochondrial DNA when there's almost nothing left. It's our last hope," - Phil Danielson, assistant professor of molecular biology at the University of DenverAs of July the medical examiner's office had identified 1,229 victims, or 44 percent of the total number of people listed as dead (500 using solely DNA technology)
39Mitochondrial disease (3) Many mitochondrial diseases have been associated with mutations on the mt genome.For example several mitochondrial syndromes in which hearing loss is one of the symptoms or diabetes.DEAF, Maternally inherited DEAFness or aminoglycoside-induced DEAFnessIn this picture is also shown a typical sporadic deletion for KSS, Kearns Sayre Syndrome and Pearson syndrome of 4977bpMERRF, Myoclonic Epilepsy and Ragged Red Muscle Fibers MELAS, Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes MILS, Maternally Inherited Leigh Syndrome