The mitochondrial genome

HUMAN NUCLEAR GENOME24 chromosomes (haploid)3200 Mbp30,000 genes

Mitochondrial genome16569 bp37 genes

Human genome = nuclear genome + mitochondrial genome

• 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).

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

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-the point of no return-to live or to die

casp9Bcl2 Apaf1

ATP Caspase 3

Apoptosis

AIF

substratesAIF

Nuclear apoptosis

Smac/Diablo

XIAP

Pro-caspase 3

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• 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

2 - 10 copies/mitochondrion

Organization of the human genome

Limited autonomy of mt genomesmt encoded nuclear

NADH 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 ~80 Other mt proteins none mtDNA pol, RNA pol

etc.

The Human Mitochondrial Genomeexpression 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

9 genes

28 genes

L- strand

H- strand

24 genes specify a mature RNA product

2 mitochondrial rRNA molecules (23S and 16S)

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

Plastid genetic code: GUG, UUG, AUU, CUG can initiate translation

UGA encodes trp at low efficiency in E. coli

Mitochondrial inheritance pattern - uniparentalmaternal 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

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

Particular Genetical ProcessesHeteroplasmy

Relaxed replication

Mixed population of normal and mutant mitochondrial genomes in one cell

mtDNA is degradated and replicates continuously, even in non dividing cells

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

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

Mitochondrial-inherited diseasesExample 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)

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 %

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 40Present in increasing frequency in older heart muscle

-rats fed on restricted diets - live longer - fewer oxygen freeradicals 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

Bcl-2-follicular lymphoma

IgH Bcl-2

T(14; 18)

Bax-colon cancer

•>50% somatic frameshift mutations in the Bax gene

•colon cancers of the microsatellite mutator phenotype

•Accelerates tumorigenesis with reduced apoptosis in Bax-/- mice

Bcl-2 family & diseases

constitutive expression

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 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 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)