Transcript Why aren`t all the genes transferred?

Plant Energy Biology
New knowledge research…
What is an ARC Centre of Excellence?
•Large Funded Centres From Federal Government
•Aimed to Achieve Scale and Focus
•Funded for Period of 5 years
•Only 19 in total in Australia - in all Areas of Scientific Research
What is Plant Energy Biology?
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Four Chief Investigators
Jim Whelan
Harvey Millar
Steve Smith
Ian Small - will come from France to joint centre
What is Plant Energy Biology?
Funding $2.5 million a year for 5 years
Additional Funding From University and State Government =
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Total Funding = $22.5 Million
What is Plant Energy Biology?
Aim of Centre is to elucidate the mechanism(s) of control of energy
metabolism in cells by understanding the control switches and regulatory circuits
that control metabolism
Investigate master control switches controlling gene expression
for energy metabolism in cells
Achieve this using Functional Genomics, Genomics, Transcriptomics,
Proteomics, Metabolomics, Bio-informatics
Integrate all these approaches
Education Program
Honours
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Top up Scholarships ~ $18,000 + $7,000 = $25,000 (Tax free)
Annual training courses in various techniques and methods
Mitochondrial molecular biology 2
• evolution of mitochondria
• maternal inheritance of mtDNA
• mtDNA and human evolution
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)
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.
Yeast
mitochondrial
chromosome
yeast mtDNA
human 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
Mitochondrial replication
cell division: random distribution
of mitos between daughter cells
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.
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 aerobe’s
genome to the nucleus of the host allowed
the latter to control the former.
Endosymbiotic hypothesis of mitochondrial evolution
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.
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 aerobe’s genome to the nucleus of the host
allowed the latter to control the former.
endocytosis
host membrane
Chloroplasts of plants and algae are thought to have
arisen from endosymbiosis of a cyanobacterium
(blue-green alga)
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.
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.
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.
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.
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.
Despite having their own genome, most mitochondrial
proteins are encoded in the nucleus, made in the cytosol and
imported into the mitochondria
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.
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.
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.
Human Evolution and mtDNA
• Mitochondria divide by fission and are not made de novo
• they are inherited mainly from the mother:
>99% of our mitochondria are derived from those
(1000 or so) present in our mother’s ovum
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) 91:8739 (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
Human Evolution and mtDNA
D-loop:
origin of mtDNA
replication
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.
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)
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.
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.
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
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.
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 aren’t all the genes transferred?
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.
Yeast Mitochondrial Proteome
Classification Based on
Phylogenetic Origin
Gray et al. 2001
Why aren’t 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
Mitochondrial DNA of animals and fungi uses a different genetic code
than the “universal” code
•Several potential
barriers
RT
RNA DNA
•Multi-step process
DNA
Gene Transfer
Mitochondrial gene
Integration and acquisition
Of Nuclear Signals
Expression
Import and assembly
Dual Expression
Nuclear gene
1. Gene i nteg rates into nuclear genom e
Nucleu s
2. Gene aqui res: (a) expression , and (b) mitochond rial ta rgeting signal s
(a) (b)
Mitochond ria l
Targetin g
3(a)
3(b)
Mitochond ria
Inte rme mb ran e Spa ce
Matrix
Imp ort o f prepro tein an d
p resequen ce cleavag e
p rotein assembl y
5
4
Screening for Gene Transfer
Multiple transfers
and activation
mechanisms for a
ribosomal protein
Genes Encoded in All Mitochondrial Genomes
COX 1
Apocytochrome b
H+
H+
H+
H+
c
UQ
I
IV
II I
Fo
V
Intermembrane
Space
Inner
Membrane
II
Matrix
N AD H
1/2
O2
Succinate
N AD +
H2 O
F1
Fumarate
ADP + Pi
ATP
COX Subunit Composition
Mitochondria
Poyton and McEwen 1996
Gene Transfer of cox 2 in legumes
Topology of Cox2 in Inner Mitochondrial Membrane
A)
N -loop
C -loop
TM2
Intermembrane
space
Mitochondrial
inner membrane
TM1
Matrix
Mat rix- loop
B)
N -loop
MTD
1
TD
60 61
MSS
124 125
Presequence
136 137
Mat rix- loop
TM1
161 162
C -loop
TM2
181 182
202 203
228 229
Mat ur e protein
376
In vitro protein import into mitochondria
Nuclear-Mitochondrial Cox2 Chimerics
Organelle Encoded cox2 Cannot Be Imported
Transmembrane Region I of Mit Encoded cox2 Inhibits Impor
Hydrophobicity Changes in Transmembrane Regions
Associated with cox2 Gene Transfer in legumes
<H> 17
Kyte and D oolittle
<H>19
aWW
Species
Gene
TM1
TM2
TM1
TM2
Soybean ( Gm)
mt
n
3.253
2.559
2.306
2.288
5.83
3.82
0.99
1.78
Amphicarpea
bracteata (Ab)
mt
n
3.524
2.024
2.306
2.265
6.52
3.03
0.98
2.15
Dumasia
villos a
mt
n
3.524
2.729
2.306
2.306
6.52
2.96
0.98
1.77
Lespedeza
formosa
mt
n
3.524
1.888
2.306
2.265
6.52
1.87
0.98
0.90
Neonotonia
wightii
mt
n
3.465
2.712
2.306
2.306
7.09
2.92
0.98
1.49
Pseudeminia
comos a
mt
n
3.524
2.753
2.306
2.247
6.52
2.34
0.98
1.31
Hydrophobicity Verse Coding Location
of Cox2 from a Variety of Legume Species
Hydrophobicity Verse Coding Location
of Cox2 from a Variety of Species
Amino Acid Changes that Reduce Hydrophobicity
in Mature Protein Required for Import
Construct
<H> 17
Kyt e and Doolitt le
<H> 19
aWW
Import
TM1
TM2
TM1
TM2
nGm1-123/mGm124-383
3.253
2.306
5.17
0.99
nGm1-123/mGm124-383 L169Q
2.824
2.306
4.03
0.99
_
_
8
nGm1-123/mGm124-383 L169Q /L171G
2.576
2.306
3.46
0.99
4
nGmCox2
2.559
2.288
3.16
1.78
nGmCox2
2.559
2.288
3.16
1.78
nGmCox2 Q 169L
2.988
2.288
4.30
1.78
nGmCox2 Q 169L/G 171L
3.235
2.288
4.87
1.78
nGm1-123/mGm124-383
3.253
2.306
5.17
0.99
8
+
4+
4+
4+
_
8
8_
Why aren’t 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.
Rib os ome
RuBisCO
Cyto chrome b 6/ f
Pho tos ystem I
Stroma
Chloroplas t
Pho tos ystem I I
ATP s yn thas e
Lume n
Ribos ome
Matrix
Mitochond ria
Compl ex I
Compl ex II
Inte rme mb rane Space
Compl ex III
Compl ex IV
Compl ex V
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
Phylogenetic Analysis of
Mitochondrial and Chloroplast
rps13 Genes
In vitro Mitochondrial and Chloroplast Import Assays
cDNA
cell-free lysate
plant
transcription &
translation
[ 35 S] labelled
precursor protein
incubation
room temp 20 min
25ÞC 25 min
mitochondria
chloroplasts
+ proteinase K
+ thermolysin
Divide into 2
equal aliquots
Phosphor imaging of
polyacrylamide gel
precursor
alone
precursor
+ mito
+ chloro
precursor
+ mito
+ chloro
+ protease
Precursor
Mature form
protease
Precursor
Mature
Duplicated rps13 is Targeted to Mitochondria
But not Chloroplasts
Phylogenetic Analysis
of
rps15a Genes
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.
Why aren’t 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