Transcript Diapositiva 1

The mitochondrial genome
Human genome =
nuclear genome + mitochondrial genome
Mitochondrial genome
HUMAN
NUCLEAR GENOME
24 chromosomes (haploid)
3200 Mbp
30,000 genes
16569 bp
37 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).
Endosymbiont Hypothesis
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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.
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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
Pro-caspase 3
Smac/
Diablo
XIAP
AIF
ATP
Bcl2 Apaf1casp9
AIF
Nuclear apoptosis
Caspase 3
substrates
Apoptosis
Genome Structure
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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
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Circular
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~ 16 kb (some plants ~100
kb!)
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Crowded (~40 genes)
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13 genes involved in
oxidative phosphorylation +
other genes (DNA pol, rDNAs,
tRNAs)
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Most proteins in
mitochondria are imported
from cytoplasm
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100,000 copies of
mitochondrial DNA in ovum
2 - 10 copies/mitochondrion
Organization of the human genome
Limited autonomy of mt genomes
NADH dehydrog
Succinate CoQ red
Cytochrome b-c1 comp
Cytochrome C oxidase
ATP synthase complex
tRNA components
rRNA components
Ribosomal proteins
Other mt proteins
mt encoded
nuclear
7 subunits
0 subunits
1 subunit
3 subunits
2 subunits
22 tRNAs
2 components
none
none
>41 subunits
4 subunits
10 subunits
10 subunits
14 subunits
none
none
~80
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)
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
AGA
Ter
AGG
Ter
AUA, AUU Met
UGA
Trp
Standard
Arg
Arg
Ile
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
Clinical presentation of
OXPHOS defects
• Unexplained combination of neuromuscular and/ or nonneuromuscular 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 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
mtDNA Mutations (1)
• Affecting mitochondrial protein synthesis
• Single deletions  always one or more tRNA
genes
• Point mutations  in rRNA or in tRNA
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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
– ND 6
– ND 1
G11778A
T14484C
G3460A
• NARP/MILS
ATPase 6
• >100 mutations within 37 genes
T8993G
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)
adPEO (1)
• adPEO autosomal dominant progressive external
opthalmoplegia
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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 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
Bcl-2 family & diseases
Bcl-2-follicular lymphoma
T(14; 18)
IgH
Bcl-2
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
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The genetic diversity of African populations was
confirmed by later studies
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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.
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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
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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.
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"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)