Mitochondrial molecular biology 2

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Mitochondrial molecular biology 2 evolution of
mitochondria maternal inheritance of mtDNA
mtDNA and human evolution
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Summary of lecture 1 mitochondria are
essential for ATP synthesis in eukaryote cells
mitochondria have their own DNA small circular
chromosomes human mtDNA has no non-coding
regions and a unique organisation they
replicate by fission, separately from the rest of
the cell mtDNA encodes a few structural
proteins, ribosomal proteins and tRNAs most
mitochondrial proteins are encoded on nuclear
genes animal and fungal mitochondria have a
different genetic code (ie, non-universal)
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Organisation of the mitochondrial chromosome
Human mtDNA small, double stranded circular
chromosome 16,569 bp in total no non-coding
DNA no introns polycistronic replication
which is initiated from the D (displacement)-
loop region followed by splicing of
transcript to form messages.
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Yeast mitochondrial chromosome
yeast mtDNA
68-75 kb, similar in structure to bacterial
genome contains introns and non-regions between
genes. Same proteins made as in animals genes
transcribed separately
human mtDNA
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Mitochondrial replication
Mitochondria replicate much like bacterial cells.
When they get too large, they undergo fission.
This involves a furrowing of the inner and then
the outer membrane as if someone was pinching the
mitochondrion. Then the two daughter mitochondria
split. Of course, the mitochondria must first
replicate their DNA. An electron micrograph
depicting the furrowing process is shown in these
figures.
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Evolution of mitochondria
Mitochondria are generally thought to have
evolved endosymbiotically when an anaerobic
prokaryotic cell engulfed an aerobic bacterium
and formed a stable symbiosis. Loss of most of
the aerobes genome to the nucleus of the host
allowed the latter to control the former.
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Endosymbiotic hypothesis of mitochondrial
evolution
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Evolution of mitochondria
This hypothesis suggests that the animal mt
genome is most highly evolved as it has lost more
function than its yeast and plant counterparts.
MtDNA from some protozoa show the closest
homology to the ancestral mitochondrial genome.
Chloroplasts are thought to have arisen from
cyanobacteria in a similar fashion.
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Evolution of mitochondria
Mitochondria are generally thought to have
evolved endosymbiotically when an anaerobic
eukaryote cell engulfed an aerobic bacterium and
formed a stable symbiosis. Loss of most of the
aerobes genome to the nucleus of the host
allowed the latter to control the former.
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Chloroplasts of plants and algae are thought to
have arisen from endosymbiosis of a
cyanobacterium (blue-green alga)
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Clues to the endosymbiotic origin of organelles
come from studies of modern symbiotic
relationships - these can be either mutualistic
or parasitic - in symbioses where the
microsymbiont lives inside the host cell, the
asociation is referred to as endocytobiotic -
these associations have common structures around
the endosymbiont.
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The evidence for mitochondria and chloroplasts
Both mitochondria and chloroplasts have their own
protein-synthesizing machinery, and it resembles
that of prokaryotes not that found in the
cytoplasm of eukaryotes. Their ribosomal RNA
(rRNA) and the structure of their ribosomes
resemble those of prokaryotes, not eukaryotes.
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The evidence for mitochondria and chloroplasts
A number of antibiotics (e.g., streptomycin) that
act by blocking protein synthesis in bacteria
also block protein synthesis within mitochondria
and chloroplasts. They do not interfere with
protein synthesis in the cytoplasm of the
eukaryotes. Conversely, inhibitors (e.g.,
diphtheria toxin) of protein synthesis by
eukaryotic ribosomes do not have any effect on
bacterial protein synthesis nor on protein
synthesis within mitochondria and chloroplasts.
The antibiotic rifampicin, which inhibits
the RNA polymerase of bacteria, also inhibits the
RNA polymerase within mitochondria. It has no
such effect on the RNA polymerase within the
eukaryotic nucleus. Mitochondria and
chloroplast electron transport components show
great sequence homology with bacterial and
cyanobacterial components - these are not found
elsewhere in the eukaryote cell.
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Factors against the theory Mitochondria and
chloroplasts only code for a few proteins. Most
of the proteins found in the organelles are
actually coded for by the nuclear DNA. (Did the
organelle DNA jump to the nuclear DNA in
evolutionary history?) Mitochondrial and
chloroplast DNA have introns, a phenomenon never
seen in prokaryotes.(Did this characteristic jump
from the nuclear DNA to the organelle DNA?) If
the theory of endosymbiosis is true, then one
must ask what was the original eukaryotic cell
(without mitochondria or chloroplasts) and how
did it survive (glycolysis?). Why have not any
primitive eukaryotic cells ever be found that are
devoid of these organelles (is today's eukaryote
just too superior?) In modern symbioses, there
is no good evidence for gene transfer between
endosymbiont and the host.
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Most mitochondrial proteins are encoded in the
nucleus, synthesised in the cytosol and
transported to the mitochondrion. The
highlighted labels are drugs that can be used to
block the process and test the source of the
mitochondrial protein.
Mitochondrial ribosomes have a similar structure
to those of bacteria - ie, 70S (cf the cytosol
which are 80S). This enables mitochondrial
protein synthesis to be distinguished from that
in the cytosol using inhibitors such as
chloramphenicol and cycloheximide.
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Despite having their own genome, most
mitochondrial proteins are encoded in the
nucleus, made in the cytosol and imported into
the mitochondria
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Synthesis of mitochondrial proteins
In all organisms, only a few of the proteins of
the mitochondrion are encoded by mtDNA, but the
precise number varies between organisms
Subunits 1, 2, and 3 of cytochrome oxidase
Subunits 6, 8, 9 of the Fo ATPase Apocytochrome
b subunit of complexIII Seven NADH-CoQ
reductase subunits (except in yeast) The nucleus
encodes the remaining proteins which are made in
the cytosol and imported into the mitochondrion.
Most of the lipid is imported.
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Mitochondria are largely maternally inherited in
higher animals and plants In mammals, most of
the mitochondrial DNA (mtDNA) is inherited from
the mother.  This is because the sperm carries
most of its mitochondria its tail and has only
about 100 mitochondria compared to 100,000 in the
oocyte.  
Although sperm mitochondria penetrate the egg,
most are degraded after a few hours. As the cells
develop, more and more of the mtDNA from males is
diluted out.  Hence less than one part in 104 or
0.01 of the mtDNA is paternal.
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Mitochondria are largely maternally inherited in
higher animals and plants This means that
mutations of mtDNA are passed from mother to
child.  It also has implications for the cloning
of mammals with the use of  somatic cells.  The
nuclear DNA would be from the donor cell, but the
mtDNA would be from the host cell.  This is how
Dolly the sheep was cloned.  
In plants, the cytoplasm, including the
mitochondria and the plastids, are contributed
only by the female gamete and not by the pollen -
again, mutations in organelle DNA are inherited
maternally.
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Human Evolution and mtDNA
Mitochondria divide by fission and are not made
de novo
they are inherited mainly from the mother
gt99 of our mitochondria are derived from those
(1000 or so) present in our mothers ovum
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Extrapolating this in evolutionary terms, this
means that all mitochondria came from a single
ancestral female - the so-called
Mitochondrial Eve.
References Proceedings of National Academy Sci
(USA) 918739 (1994) Science 279 28 (1998)
However, this is based on the assumption that
mitochondrial inheritance is strictly clonal.
Recent evidence shows that mitos from sperm do
enter the egg and last for several hours. If
recombination occurs between mitos, then the Eve
hypothesis may be incorrect - or at least the
timing would be incorrect. Proc. R. Soc. Lond. B
(1999) 266, 477-483
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Human Evolution and mtDNA
Human evolution can be traced by analysis of the
base sequence in a small part of the
mitochondrial genome which does not encode a gene
and which is quite variable. - the so-called
D-loop.
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Human Evolution and mtDNA
The D-Loop of the mtDNA is the start of
replication/transcription site and contains
400-800 bp Unlike the rest of mtDNA in humans,
which is highly conserved, this region is very
variable between people It also has a very high
frequency of change during evolution (about 2
per million years)
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Human Evolution and mtDNA
This makes the D-loop a very powerful tool for
the study of evolutionary relationships between
organisms and for DNA typing of individuals. In
addition, because of the large number of mitos in
a cell, extracting mtDNA is easier from small
amounts of tissue - and it can be readily
separated form other DNA by centrifugation on
CsCl gradients.
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Human Evolution and mtDNA
By comparing different groups, we can get a
glimpse of human evolutionary lines. Eg, African
individuals have more variability between each
other than do Asians, indicating that the former
have had more time to accumulate changes - ie,
the Africans are a more ancient group.
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Human Evolution and mtDNA
Assuming that the rate of change in the D-loop is
constant and due only to mutation, the number of
difference s between Africans can be use to
calculate when their common ancestor lived. This
works out to be about 200,000 years ago.
This suggests that modern Homo sapiens came out
of Africa at about that time and migrated through
Europe and Asia, replacing other early humans
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Human Evolution and mtDNA
But we have to be careful the rate of change in
mtDNA may not be constant and heteroplasmy (due
to recombination of mtDNA) may cause
complications. Also, mtDNA represents a single
lineage and other genetic changes need to be
traced also.
However, when this was done with polymorphisms in
the Y chromosome, Adam was also traced back to
Africa, at about the same period.
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What are Mitochondria - Evolution Endosymbionts
- Bacterium engulfed by precursor to Eukaryotic
cells and formed a symbiotic relationship. Gene
Transfer - Accounts for the loss of mitochondrial
genes to the nucleus. Outstanding
Questions Are mitochondria simply
endosymbionts who have the majority of coding
capacity in Host? Why arent all the genes
transferred?


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Rickettsia - 834 open reading frames (obligate
intracellular parasite) E. coli - 4, 288
ORF Human mit genome - 13 ORF Yeast mit genome -
7 ORF Arabidopsis mit genome - 57
ORF Reclinomonas americana - 67 ORF These
figures would suggest that mitochondria
are Endosymbionts that have transferred most of
their coding capacity to the host. However the
process of gene transfer was (or is) not as
straightforward as it may appear.


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Yeast Mitochondrial Proteome Classification
Based on Phylogenetic Origin
Gray et al. 2001
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Why arent all the genes transferred?

Rickettsia - 834 open reading frames E. coli - 4,
288 ORF Human mit genome - 13 ORF Yeast mit
genome - 7 ORF Arabidopsis mit genome - 57
ORF Reclinomonas americana - 67 ORF


Hydrogenosomes are likely to be organelles that
were mitochondria but have lost all DNA
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Mitochondrial DNA of animals and fungi uses a
different genetic code than the universal code
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• Gene Transfer
• Multi-step process
• Several potential
• barriers

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Screening for Gene Transfer
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Multiple transfers and activation mechanisms for
a ribosomal protein
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Genes Encoded in All Mitochondrial Genomes
COX 1
Apocytochrome b
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COX Subunit Composition
Mitochondria
Poyton and McEwen 1996
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Gene Transfer of cox 2 in legumes
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Topology of Cox2 in Inner Mitochondrial Membrane
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In vitro protein import into mitochondria
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Nuclear-Mitochondrial Cox2 Chimerics
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Organelle Encoded cox2 Cannot Be Imported
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Transmembrane Region I of Mit Encoded cox2
Inhibits Import
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Hydrophobicity Changes in Transmembrane
Regions Associated with cox2 Gene Transfer in
legumes
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Hydrophobicity Verse Coding Location of Cox2 from
a Variety of Legume Species
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Hydrophobicity Verse Coding Location of Cox2 from
a Variety of Species
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Amino Acid Changes that Reduce Hydrophobicity
in Mature Protein Required for Import
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_
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Why arent all the genes transferred?

• Hydrophobicity is a barrier to import and thus
  Gene Transfer - but only for some genes.
• In non-plant systems Hydrophobicity should not be
  a problem - non-universal genetic code is the
  barrier.
• This implies in plants some other mechanism(s)
  operating to maintain mitochondrial genome.

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Missing Ribosomal Proteins Genes for rps 8 and 13
of the mitochondrial ribosome missing
fromArabidopsis nuclear and mitochondrial
genomes. These genes are considered essential
No EST Hard to envisage how ribosome can
function without these proteins - experimental
evidence indicates impossible


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Phylogenetic Analysis of Mitochondrial and
Chloroplast rps13 Genes
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In vitro Mitochondrial and Chloroplast Import
Assays
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Duplicated rps13 is Targeted to Mitochondria
But not Chloroplasts
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Phylogenetic Analysis of rps15a Genes
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Replaced
Missing Ribosomal Proteins Current mitochondrial
proteome has a complex genetic history. In this
case the Arabidopsis mitochondrial ribosome is
derived from at least three different ancestors.

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Why arent all the genes transferred?

All Plant mitpchondrial Genomes encode some
ribosomal Proteins - not hydrophobic Assembly -
All organelle encoded proteins function
in Multisubunit complexes. Defined and sequential
assembly Pathways may dictate some proteins
encoded. Ribosomes are very complex and have
very specific Assembly pathways


Hydrogenosomes are likely to be organelles that
were mitochondria but have lost all DNA