Mitochondrial structure

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Mitochondrial structure
Respiring mitochondrion
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Membrane protein density in the inner and outer
membranes is different
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Membrane heterogeneity permits separation of
different membranes by equilibrium or velocity
sedimentation
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Mitochondrial electron transport chain
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Reduction potentials in the electron transport
chain
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Mitochondria can respire if fed succinate alone
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Succinate dehydrogenase (SDH) structure
Redox centers in SDH
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Polyacrylamide gel electrophoresis analysis of
protein composition
Each band represents one or more polypeptide
chains of a certain size
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Mitochondrial genomes have only a few
protein-coding genes
human
yeast
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Plant mitochondrial genomes vary in size, even in
a single species
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All mitochondrial genomes encode subunits of
cytochrome oxidase, the terminal complex of the
chain
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Sticky ends
Blunt ends
Restriction/modification systems are widespread
in bacteria and constitute a defense mechanism.
Useful restriction systems caused double
stranded breaks at specific sequences.
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Southern blot analysis (Northern blot is similar)
For us these are restriction fragments of the
mitochondrial DNA
At this stage, the bands are not visible
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Cloning DNA into a plasmid vector
Of course, the probe is also DNA here
This is generally how plasmids like ours are made
in our case the blue DNA is a cox sequence
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This is the plasmid we use to generate the probe
We use random priming short pieces of DNA are
added as primers. Since they are short, we
expect them to find complementary sequences
scattered randomly through the plasmid. Since
the plasmid is mostly cox sequence, most of the
DNA pieces generated will be cox sequences as well
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This derivative of dUTP is incorporated into the
DNA synthesized from the short primers we add
We have antibodies that will bind to this part of
the structure the digoxigenin.
Alkaline phosphatase is covalently attached to
these antibodies, and catalyzes the color-forming
reactions shown here