The mitochondrial genome This presentation will be about mitochondria, their function, their genome and how errors in their and the nuclear genome influence their function and structure.

nuclear genome + mitochondrial genome Human genome = nuclear genome + mitochondrial genome Mitochondrial genome 16569 bp 37 genes HUMAN NUCLEAR GENOME 24 chromosomes (haploid) 3200 Mbp 30,000 genes

1-10 m small Mitochondria 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.

Endosymbiont 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.

Mitochondrial Genome Small circular genome >1000 copies/ cell 16569 bp 44% G+C H- Strand Guanines L- Strand Cytosines D- Loop 7S DNA The 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.

Mitochondrion plays a role in: Energy production  Oxidative phosphorilation (OXPHOS) Maintaining the intracellular homeostasis Protecting the rest of the cell from reactive oxygen species (ROS) Apoptosis  important development and disease Mitochondria 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.

Apoptosis ATP Caspase 3 casp9 Bcl2 Apaf1 substrates Mitochondria-the point of no return-to live or to die Pro-caspase 3 Smac/ Diablo XIAP ATP Caspase 3 casp9 AIF Bcl2 Apaf1 AIF substrates Apoptosis Nuclear apoptosis

Genome Structure The 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).

The Human Mitochondrial Genome 2 - 10 copies/mitochondrion Circular ~ 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 cytoplasm 100,000 copies of mitochondrial DNA in ovum

Organization of the human genome Limited autonomy of mt genomes mt encoded nuclear NADH dehydrog 7 subunits >41 subunits Succinate CoQ red 0 subunits 4 subunits Cytochrome b-c1 comp 1 subunit 10 subunits Cytochrome C oxidase 3 subunits 10 subunits ATP synthase complex 2 subunits 14 subunits tRNA components 22 tRNAs none rRNA components 2 components none Ribosomal proteins none ~80 Other mt proteins none mtDNA pol, RNA pol etc.

The 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 rRNAs Transcripts then procesed into individual genes prior to translation

Coding- Non-coding 37 genes 28 genes H- strand 9 genes L- strand 24 genes specify a mature RNA product 2 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 molecules 22 encode tRNA molecules 22 tRNA molecules 13 genes specify polypeptides

H strand enriched in G L strand enriched in C

Mitochondrial Genetic code is somewhat different… Human Mito Standard AGA Ter Arg AGG Ter Arg AUA, AUU Met Ile UGA Trp Ter UGA encodes trp at low efficiency in E. coli Plastid genetic code: GUG, UUG, AUU, CUG can initiate translation

Mitochondrial inheritance pattern - uniparental maternal in animals Paternal inheritance in gymnosperms, some angiosperms

Mitochondrial disease (1) Incidence from 1:10.000 to 1:4000 Affecting most energy demanding tissues Central nervous system Heart Skeletal muscle This is not always the case Mitochondrial diabetes Liver and kidney disease Pearson syndrome Specific, but highly variable clinical features with various gene defects Mitochondrial disease has an incidence from 1:10.000 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.

Clinical presentation of OXPHOS defects Unexplained combination of neuromuscular and/ or non-neuromuscular symptoms Progressive course Involvement of seemingly unrelated tissues or organs Clinical symptoms either isolated or in combination, may occur at any stage Frequent feature; increasing number of organs involved in the course of the disease While initial symptoms usually persist and gradually worsen, they may occasionally improve or even disappear, as other organs become involved Oxidative 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 symptoms Progressive course Involvement 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

Particular Genetical Processes Heteroplasmy Mixed population of normal and mutant mitochondrial genomes in one cell Relaxed replication mtDNA is degradated and replicates continuously, even in non dividing cells An 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.

gomori trichrome staining mtDNA Mutations (1) Affecting mitochondrial protein synthesis Single deletions  always one or more tRNA genes Point mutations  in rRNA or in tRNA  Associated with: multiple system disorders, lactic acidosis, “ragged red fibers” in muscle biopsy You 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 fibers   gomori trichrome staining

mtDNA Mutations (2) In mtDNA protein coding genes LHON Complex I (NADH dehydrogenase genes) ND 4 G11778A ND 6 T14484C ND 1 G3460A NARP/MILS ATPase 6 T8993G >100 mutations within 37 genes Further mutations are found in mtDNA protein coding genes. Examples are Leber Hereditary Optic Neuropathy in which mutations are found in the NADH dehydrogenase genes or Leigh 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.

Mitochondrial-inherited diseases Example Leigh’s Syndrome Cause - point mutation in either ATPase 6, mt tRNA (5 dif), NADH dehydrogenase 5, cytochrome oxidase III Result- ATP deficiency Phenotype: Motor & Intellectual regression, Death often within 2 years of onset

Mitochondrial DNA mutations in human genetic disease (Wallace Sci. Amer. 277:40)

Mitochondrial disease (3): deletions

Pearson 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.

Mitochondrial-inherited diseases Most decrease ATP-generating ability of the mitochondria Affect function of nerve and muscle cells Severity of symptoms vary with amt of wt mtDNA present In ragged red fiber disease: 2 - 27% of mtDNA is wt (heteroplasmic)

nDNA mutations affecting mtDNA stability/ expression A primary nuclear gene defect causes secondary mtDNA loss or deletion  tissue dysfunction Mendelian inheritance Factors for mtDNA maintenance and repair all encoded by nuclear genes Only 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.

Anu Suomalainen and Jyrki Kaukonen, Am J Med Genet 2001 adPEO (1) adPEO autosomal dominant progressive external opthalmoplegia Autosomal dominant Onset 18-40 years RRF Multiple mtDNA del in post-mitotic tissue Basal 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 of 18- 40. The 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 and More than 40% in skeletal and ocular muscle and in the heart. Anu Suomalainen and Jyrki Kaukonen, Am J Med Genet 2001

Mitochondria 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 function Evidence: -5000 bp deletion in mtDNA absent in heart muscle before age 40 Present in increasing frequency in older heart muscle -rats fed on restricted diets - live longer - fewer oxygen free radicals generated - fewer mitochondrial mutations accumulate Elevated mtDNA defects in people with degenerative diseases (Parkinson’s, Huntingtons, Alzheimers, ALS...)

mtDNA Mutations (3) Somatic mitochondrial DNA mutations  with age in healthy individuals Old people typically harbour a wide range of different mtDNA deletions in post mitotic tissues; skeletal muscle, myocardium, brain Overall amount of mutant mtDNA very low One cell  high percentage of one mutant mtDNA Different cells  different mutations Threshold effect  OXPHOS Somatic 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

Bcl-2 family & diseases Bcl-2-follicular lymphoma Bcl-2 IgH T(14; 18) Bcl-2 IgH constitutive expression Bax-colon cancer Accelerates tumorigenesis with reduced apoptosis in Bax-/- mice colon cancers of the microsatellite mutator phenotype >50% somatic frameshift mutations in the Bax gene

In 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

Mitochondrial DNA and Evolution The genetic diversity of African populations was confirmed by later studies However, 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.

Molecular analysis of Neanderthal DNA from the northern Caucasus Igor V. Ovchinnikov Anders Götherström Galina P. Romanova Vitaliy M. Kharitonov Kerstin Lidén William Goodwin

Main informations The 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.

September 11 and Mitochondria DNA Typing If cell is damaged, chromosomal DNA disintegrates Heavily damaged samples tested by “profiling” mitochondrial DNA Every cell in the human body contains thousands 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 Denver As of July 2002 - 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)

Mitochondrial 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 DEAFness In this picture is also shown a typical sporadic deletion for KSS, Kearns Sayre Syndrome and Pearson syndrome of 4977bp MERRF, Myoclonic Epilepsy and Ragged Red Muscle Fibers MELAS, Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes MILS, Maternally Inherited Leigh Syndrome