Human Reproduction, Vol. 15, (Suppl. 2), pp. 18-27, 2000
Genetic control of oxidative phosphorylation and
experimental models of defects
Ian Trounce1
Mutation Research Centre, 7th Floor, Daly Wing St Vincent's Hospital,
Fitzroy, Melbourne, Victoria 3065, Australia
'To whom correspondence should be addressed at: Mutation Research Centre,
7th Floor, Daly Wing, St Vincent's Hospital, Fitzroy, Melbourne, Victoria 3065,
Australia. E-mail: [email protected]
Energy in the form of ATP is continually
produced by all cells for normal growth and
function. Anaerobic glycolysis can provide
enough ATP for some cells, but energetic
cells such as cardiomyocytes and neurons
require a more efficient ATP supply, which
can only be provided by mitochondrial
oxidative phosphorylation. Invented by bacteria that became symbiotically associated
with other bacteria to form eukaryotic cells
billions of years ago, oxidative phosphorylation carries with it a genetic legacy that is
unique. The mitochondrial oxidative
phosphorylation complexes are assembled
from protein subunits encoded by both the
mitochondrial genome (mtDNA) and the
nuclear genome (nDNA, located in the chromosomes). The mtDNA is a remnant
genome of the bacterial progenitor of mitochondria, and (unlike the biparental diploidy that characterizes the nuclear
genome) is present in thousands of copies
per cell, is replicated through life, and is
inherited (cytoplasmically) only from the
female parent. Oxidative phosphorylation
comprises five multimeric enzyme complexes that act as a redox pathway, passing
electrons from oxidizable intermediates
produced by the metabolism of food to
molecular oxygen in the mitochondrial mat18
rix, while producing an electrochemical gradient by pumping protons into the
intermembranal space. The proton (hydrogen ion) gradient across the inner mitochondrial membrane is used by the H + transporting ATP synthase to produce ATP
from ADP and inorganic phosphate, with
the protons released into the mitochondrial
matrix then combining with electronated
oxygen to form water. Many of the details
regarding the control of the synthesis of
oxidative phosphorylation enzyme complexes remain to be elucidated. Transmitochondrial cell culture systems have been
developed so that defective oxidative
phosphorylation can be studied in a controlled nuclear background. Such systems
may soon enable the development of
mtDNA 'knockout' mice in order to better
model mtDNA transmission and mitochondrial disease.
Key words: electron transport chain/mitochondrial DNA/oxidative phosphorylation/respiratory chain/transmitochondrial cells
Introduction
A glance at any biochemistry textbook will
reveal that mitochondria house a large number
© European Society of Human Reproduction & Embryology
Oxidative phosphorylation and defect models
ND6
NAD+
NADH
Figure 1. Genetic map of human mtDNA (upper) and scheme showing how the gene products contribute to the complexes of
oxidative phosphorylation (lower). The circular mtDNA genome is represented in the upper figure as a linear map, with tRNA
genes indicated by vertical lines, often separating reading frames. Heavy (H-) strand genes are above the line and are
transcribed from right to left; light (L-) strand genes are below the line and are read from left to right. As electrons pass from
complex I down the chain, oxygen is reduced to water and protons are pumped into the intermembranal space (lower right).
cyt b encodes apocytochrome b of complex III; ND genes encode NADH dehydrogenase subunits 1 to 6 of complex I;
CO genes encode cytochrome oxidase subunits I—III of complex IV; ATP6/8 encode ATP synthase subunits of complex V.
of metabolic pathways. Some of these, e.g.
part of the urea cycle in liver mitochondria
and thermogenesis in brown fat, are unique
to particular organs; others are common to
mitochondria in all cells, e.g. the citric acid
cycle and other catabolic pathways that generate reduced (i.e. readily oxidizable) forms of
nicotinamide- and flavin-adenine dinucleotide
intermediates (NADH and FADH2).
The respiratory chain (or electron transport
chain) together with the H+-transporting ATP
synthase comprise the final common fuel
oxidation pathway, known as oxidative
phosphorylation (or OXPHOS). Overall, oxidative phosphorylation involves oxidizable
intermediate metabolic substrates, which
include NADH and FADH2, generating ATP
from ADP and inorganic phosphate, and in
the process reducing molecular oxygen to
water. The details are explained below. Energetic cells require mitochondrially-produced
ATP, whereas quiescent cells can derive sufficient ATP from anaerobic, cytosolic glycolysis, so mitochondria are most abundant in
cells with the highest demand for energy. The
oxidative phosphorylation pathway comprises
five multisubunit enzyme complexes, in turn
composed of >80 different polypeptide units,
13 of which are encoded by a separate genome
to those contained in the (nuclear) chromosomes, the mitochondrial genome, composed
of mitochondrial DNA (mtDNA). The genetic
control of oxidative phosphorylation is therefore unusual, requiring the co-ordinated
expression of both nuclear and mitochondrial
genes. However, mitochondrial biogenesis and
self-replication, including mtDNA replication,
appear to be regulated entirely by nuclear
genes.
The mitochondrial genome
It is now accepted that mitochondria and
their genomes are vestiges of aerobic bacteria
(prokaryotes) that long ago became symbiotically incorporated into anaerobic prokaryotes
to give rise to eukaryotic cells (Margulis,
1981; Gray et al., 1999; Jansen, 2000). Most
19
LTrounce
of the endosymbiont's genome was then lost
or transferred to the nucleus, leaving a small,
circular, histone-free
'chromosome' of
mtDNA to remain within the cytoplasmic
organelle. In humans, mtDNA forms a 16.5 kb
circle, which (as in all multicellular animals
known) encodes 13 subunits of the OXPHOS
enzyme complexes, a complete set of 22
transfer RNAs (for 20 amino acids, two of
which have two distinct tRNAs), and two
rRNA components of the mitochondrial ribosomes (Figure 1). Replication and transcription
of mtDNA are controlled by nuclear-encoded
gene products and are discussed elsewhere is
this volume (Clayton, 2000).
The proteins encoded by mtDNA include
seven subunits of the 42-subunit NADHubiquinone oxidoreductase complex (complex
I), the apocytochrome b of the 11-subunit
ubiquinol-cytochrome c oxidoreductase complex (complex III), three core proteins of
the 13 subunits that comprise cytochrome c
oxidase (complex IV), and two of the 16
subunits of the H+ATPsynthase (complex V)
(Anderson et al., 1981; Saraste, 1999;
Figure 1).
Cytoplasmic inheritance, suspected since
the observations of Ephrussi (1950) with yeast,
was confirmed when mtDNA in the fungus
Neurospora was shown to be inherited from
the female parent (Reich and Luck, 1966).
Maternal inheritance of mtDNA was found to
be the rule for most of the (multicelled) animal
kingdom, including humans (Giles et al.,
1980). Plants also show uniparental inheritance of mtDNA (and plastid, or chloroplast,
DNA), although the contributing parent can
often be the male (Levings, 1983; Hoekstra,
2000). While tiny in comparison to the nuclear
genome, animal mtDNA exists in thousands
of copies per cell; in typical somatic cells
mtDNA comprises - 1 % of DNA mass, and
in the mature mammalian oocyte it can account
for as much as a half or more of total DNA
mass (Dawid, 1972). Mitochondria are
20
dependent on the nuclear genome for most of
the 80 or so OXPHOS proteins, and all of the
regulatory factors for mtDNA replication and
transcription. Enzymes of the many other
important metabolic functions housed in the
mitochondria of different cells are entirely
nuclear-encoded.
Plants have relatively large and complex
mitochondrial genomes compared with
animals (Levings, 1983; Gray et al., 1999).
An important class of mtDNA mutations in
plants result in cytoplasmic sterility, whereby
affected plants suffer an inability to make
viable pollen (Levings, 1983, 1996). A wide
variety of human diseases (individually rare,
but collectively important) are now also attributed to mtDNA mutations (Lightowlers et al.,
1997; DiMauro and Schon, 1998; Wallace,
1999; Christodoulou, 2000; Naviaux and
McGowan, 2000) and are gradually becoming
better understood. Controversy, however, still
engulfs the idea that it is an accumulation of
wider mtDNA mutations that contributes to
the manifestations of age and degenerative
disease (Beal, 1996; Lightowlers et al., 1999;
Wallace, 1999); discovery of mitochondrial
involvement in the signalling for cell death
(Newmayer et al., 1994; Kluck et al, 1997;
Yang et al., 1997), has heightened interest is
this area (Wallace, 1999), and the possibility
that altered mitochondrial function consequent
to mtDNA mutations could interfere with or
potentiate apoptotic signals warrants investigation. ATP is normally exported from the
mitochondrion by the adenine nucleotide
translocator, which forms part of the mitochondrial permeability transition pore, activated
during apoptosis. Generation of reactive
oxygen species (see below) and release of
cytochrome c through this pore are important
events in apoptotic cell death (Matsuyama
etal, 1998; Raff, 1998, Martinou, 1999).
Oxidative phosphorylation
Organisms need to produce ATP continuously,
to do cellular work. Glycolysis, converting
Oxidative phosphorylation and defect models
glucose to pyruvate, produces two ATP molecules per glucose molecule, releasing around
238 kJ/mole. The citric acid cycle and oxidative phosphorylation, housed in the mitochondrion, catalyses the stepwise oxidation of
pyruvate to carbon dioxide and water, yielding
around 2870 kJ/mole and 38 ATP molecules
for each initial glucose molecule (Lehninger,
1964). Long before eukaryotes appeared, bacteria evolved many ways of producing ATP.
Oxidative phosphorylation was one of these
bacterial inventions and takes advantage of
the oxidizing power of molecular oxygen,
respiration being the controlled combustion
of carbohydrates, producing ATP instead of
excess heat.
Oxidative phosphorylation is achieved by
five multimeric enzyme complexes present
within the inner mitochondrial membrane.
Complexes I-IV (the respiratory, or electron
transport chain) pass electrons from NADH
and FADH2 (reduced intermediates derived
from the oxidation of carbohydrates and fatty
acids, ultimately to carbon dioxide) to molecular oxygen, so that electronated oxygen
products produced by the respiratory chain
enter the mitochondrial matrix. The energy
released is partly conserved by the pumping
of protons (hydrogen ions) into the intermembranal space (three protons for each electron
pair traversing the chain), which produces a
proton gradient with respect to both the cytosol
and the mitochondrial matrix. The concentration of protons is high enough to reverse an
inner
mitochondrial
membrane-located
enzyme H+-dependent ATPase (complex V;
the reversed ATPase is named H+-transporting
ATP synthase) and thus to produce ATP from
inorganic phosphate and ADP (when, by its
presence, the need for ATP-production is signalled). Meanwhile the protons discharged
into the mitochondrial matrix reduce the respiratory chain's electronated oxygen products
(which, potentially, constitute 'reactive oxygen
species') further to form water (Figure 1).
A wealth of exciting new structural information on the respiratory chain enzymes (for
review see Saraste, 1999) has paralleled continuing advances in mitochondrial genetics.
Complex I is the largest and least understood
OXPHOS complex, containing multiple ironsulphur centres that carry electrons from
NADH to ubiquinone. Complex II (succinate
dehydrogenase) contains only four subunits,
none encoded by mtDNA; this complex also
contains several iron-sulphur centres, as well
as FAD and a b-type cytochrome, to carry
electrons from succinate to ubiquinone.
Reduced ubiquinone (ubiquinol) is oxidized
in a two-step manner by complex III, as
electrons are passed via iron-sulphur centres
in the Rieske protein, and via cytochromes b
and c1? finally to reduce cytochrome c. The
last step in the respiratory chain sees complex
IV, or cytochrome c oxidase, pass electrons
through two copper atoms and two a-type
cytochromes, finally delivering four electrons
to molecular oxygen in the mitochondrial
matrix and, by employing four hydrogen ions
(protons) there for each molecule of oxygen,
producing two water molecules. Complexes I,
III and IV also pump a proton across the
mitochondrial membrane to the intermembranal space as they pass a pair of electrons,
producing the proton gradient and flow utilized
by complex V to condense ATP from ADP
and inorganic phosphate.
While it is tempting to view the electron
transport chain (and hence oxidative
phophorylation) as the linear passage of electrons, the molar ratios of complexes I, II,
III, IV and V in the inner membrane is
approximately 1:2:3:6:6 (Capaldi et al, 1988).
From inhibitor studies it is clear that complex
I and II, feeding electrons from NADH and
FADH2 to complex III, exert the greatest
control over respiratory chain flux; complexes
III, IV and V, however, exhibit specific activities more than an order of magnitude greater.
Figure 2 shows how oxidative phosphoryla21
I.Trounce
mito (0.5 mg)
I \
ADP 125 nmols
ADP 125 nmols
'. DNP
Figure 2. Polarographic (oxygen electrode) estimation of
oxidative phosphorylation in intact mouse cell mitochondria
showing coupling of active respiration (indicated by oxygen
consumption) with ADP stimulation, and the effects of
uncoupling oxidative phosphorylation with dinitrophenol
(DNP) (see text). Oxygen saturation (y axis) is measured as a
function of time (x axis), allowing comparison of specific
rates of respiration (O2 consumed/min/mg mitochondrial
protein). Mito = addition of mitochindria, 0.5 mg.
tion can be studied in vitro using freshly
isolated mitochondria in a polarograph chamber. Isolated from cultured mouse cells with
their membranes intact, the organelles maintain a proton gradient so that phosphorylation
as well as maximal respiratory flux can be
directly measured. This (real) polarograph
trace illustrates the basic principle of coupling
of respiration to ADP availability (Chance and
Williams, 1955); substrates are added to the
stirred chamber containing mitochondria in an
isosmotic buffer with phosphate present, and
a slow (state II) rate of respiration follows;
additions of ADP stimulate state III respiration
(the 'active' state in which phosphorylation
occurs); then, when added ADP has all been
phosphorylated to ATP, the slower state IV
respiration ensues. Figure 2 shows how the
extraordinary kinetics of oxygen utilization by
the respiratory chain ensures that respiration
can proceed maximally even at the lowest
oxygen levels measurable with the polaro22
graph, - 1 % saturated. Respiration can be
uncoupled, by adding dinitrophenol, which
causes the leakage of protons from the intermembranal compartment, dissipating the
proton gradient. The even faster respiration
observed with dinitrophenol-uncoupled mitochondria in Figure 2 is typical, and implies that
complex V shares some control of respiratory
chain electron flux in the phosphorylating state
(state III).
Mitochondria from cultured cells can also
be used to assay the individual respiratory
chain enzyme complexes to gain a full picture
of nuclear or mtDNA mutation effects on
oxidative phosphorylation (Trounce et al.,
1996).
During normal respiratory chain activity
some incompletely reduced reactive oxygen
species (ROS) are produced, with complexes I
and III being the sites of most ROS generation
(Boveris and Chance, 1973). An estimated 14% of oxygen consumed is directly converted
to such free radicals under normal conditions.
Inhibition of the respiratory chain greatly
increases ROS production (Kwong and Sohal,
1998). When the electron transport chain is
inhibited, electrons accumulate in the proximal
portion of the electron transport chain (complex I and ubiquinone) (Kwong and Sohal,
1998), and can be added to molecular oxygen,
forming superoxide anion (O2°~). Superoxide
is converted by mitochondrial manganese
superoxide dismutase (MnSOD) to give hydrogen peroxide. Hydrogen peroxide, which in
the presence of reduced transition metals can
itself be reduced to the highly reactive
hydroxyl radical (OH°) by the Fenton reaction,
is then converted to water by glutathione
peroxidase. Using a MnSOD knockout mouse
as a model of acute oxidative stress, Melov
et al. (1999) found the iron-sulphur centres of
complexes I, II, and III, as well as of aconitase
in the citric acid cycle, were inactivated,
severely disrupting mitochondrial energy production. Chronic exposure to free radicals
Oxidative phosphorylation and defect models
is thought to result in oxidative damage to
mitochondrial and cellular proteins, lipids and
nucleic acids, increasing the prevalence of
mtDNA mutations (Wallace, 1999).
Transmitochondrial cells
It can be useful to consider oxidatively-derived
ATP as an energy supply that is supplementary
(to anaerobic glycolysis) for many cells, but
essential for cells of highly energetic tissues,
such as myocardium, skeletal muscle, pancreatic islets, the central nervous system, retinal
cells, the kidneys and the liver. That human
cells can grow in vitro without OXPHOS has
been made clear by studies over the past
decade using chemically-derived rho-zero (p°)
cells, i.e. cells that have been completely
depleted of mtDNA. First described for avian
cells (Morais et al., 1988), King and Attardi
(1989) succeeded in producing a human p°
cell line by the same approach, namely treating
cells with the y-polymerase inhibitor, ethidium
bromide; as the cells divide in the presence
of the ethidium bromide the mtDNA copy
number is reduced until clones can be isolated
that have no mtDNA.
These p° cells, whether primary or transformed, can survive and grow with mitochondria that are without a functional respiratory
chain. The mitochondria show grossly abnormal morphology, being greatly enlarged and
lacking cristae (Figure 3). The cells produce
high levels of lactate and, unlike their p +
parents, require 'redox therapy' (additional
pyruvate and uridine in the culture medium)
for survival. Such uridine-dependence, or auxotrophy, among p° cells was found (Morais
et al., 1988) to be because dihydroorotate
dehydrogenase (an enzyme of the pyrimidine
biosynthesis pathway — thus essential for
nuclear DNA synthesis and mitosis - located
on the outer surface of the mitochondrion's
inner membrane), normally needs to pass
electrons released from the oxidation of dihy-
Figure 3. Electron microcrographs showing (A) a cybrid
made by fusion of enucleated mouse 3T3 cells with a mouse
p° cell line, showing normal mitochondrial morphology, and
(B) the p° cell line depleted of mtDNA, showing swollen and
hypodense mitochondria with few cristae. (A, B)
Magnification X15 000. The mouse p° cell line was produced
by the author in the laboratory of D.C.Wallace.
droorotate on to ubiquinone and thence to
complex III and the remainder of the electron
transport chain, all of which is missing in the
mitochondria of p° cells. The requirement for
exogenous pyruvate (King and Attardi, 1989)
probably provides oxidized NAD (NAD+)
necessary for ATP production from glycolysis
in the cytosol by further pushing lactate production via lactate dehydrogenase.
Omission of the nutritional requirements
for p° cells allows the selection of p + cybrids
made by fusing enucleated cells (cytoplasts)
from patients with mutant mtDNA with p°
cells, in turn permitting demonstration of the
association of different mtDNA disease
mutations with different OXPHOS phenotypes
in vitro (Chomyn et al., 1991; King et al.,
1992; Trounce et al, 1994).
Towards mtDNA 'knockout' mice
Successful mouse models of Mendelian
OXPHOS disease have been produced recently
23
I.TVounce
by knockout of the heart-muscle adenine nucleotide translocator isoform (Graham et al,
1997) and knockout of mitochondrial transcription factor A (mtTFA, or Tfam) (Wang
et al, 1999; Rantanen and Larsson, 2000).
Knockout mice with different mtDNA defects
will no doubt also be valuable tools. By
enabling direct study of segregation of deleterious heteroplasmic mutations through
development they will greatly aid the study
of the pathogenesis of, and possible treatment
modalities for, mitochondrial diseases, as well
as being useful to researchers investigating
nuclear genes that affect OXPHOS (including
mediators of apoptosis).
The unique features of mitochondrial
genetics present some technical barriers to
producing mtDNA knockout mice. Recombination has not been clearly demonstrated
in mtDNA (but the subject continues to be
controversial; Arctander, 1999), meaning that
the targeted knockout approach used for nuclear genes,utilizing homologous recombination of a mutated construct, cannot be used
for mtDNA. Transfection approaches using
artificial mtDNA constructs have not yet succeeded in transferring the construct into mitochondria within cells.
Transmitochondrial somatic cell techniques
provide another approach to producing
mtDNA knockout mice, but still require
mtDNA mutant cells as the starting point. The
inbred mouse strains exhibit little variation
in mtDNA sequence, although polymorphic
variants between two strains were used to
advantage in a pioneering study by
Shoubridge's group (Jenuth et al., 1996).
These authors constructed heteroplasmic
mouse embryos by fusion of a cytoplast from
one strain with a fertilized oocyte from another
strain. In other experiments, Meirelles and
Smith (1998) have used a similar approach of
either cytoplast or karyoplast fusion to further
show that oocyte mitochondria of perinuclear
(karyoplast) origin tend to show less stringent
24
segregation than peripheral (cytoplast origin)
mitochondria during preimplantation development.
A handful of drug-resistant mouse cell
mtDNA mutants have been identified in cell
culture, the best characterized being chloramphenicol resistance (CAP1) due to a 16S rRNA
point mutation. CAPr cells are not growthinhibited by chloramphenicol, providing a convenient selectable marker, and the cells have
defects in OXPHOS, probably due to disrupted
mitochondrial protein synthesis (Howell and
Nalty, 1988). Wallace et al. have recently
reported an attempt to make a transmitochondrial mouse with the CAPr mutation
(Levy et al., 1999). While succeeding in
transferring the mutant mtDNA into embryonic stem (ES) cells, they failed to get germline
transmission of the clone. However, a recent
update form the same group claimed to have
achieved germline transmission using a different XX ES cell line (Wallace et al, 1999).
Creation of homoplasmic transmitochondrial cells without drug-resistant markers or
p° cells is possible with the use of the toxic
dye rhodamine 6-G (R6G) (Christodoulou,
2000). Pretreatment of cells including ES cells
(Levy et al, 1999) with low levels of R6G
for several days was shown to kill cells, but
the cells could be rescued by fusion with
enucleated untreated mitochondrial donor
cells. The R6G appears to irreversibly collapse
the mitochondrial membrane potential, and
the utility of the drug in creating transmitochondrial mouse cells has been demonstrated
(Trounce and Wallace, 1996).
Another approach is suggested by the findings of Moraes's group that mtDNA from
closely related primate species can be replicated in human p° cells, but that the 'xenomitochondrial' cybrids show OXPHOS defects
(Kenyon and Moraes, 1997). We have similarly produced a range a xenomitochondrial
cybrids by introducing mitochondria from several murinae species into a mouse p° cell
Oxidative phosphorylation and defect models
XX ES cell
Mouse cell with
mtDNA mutation
R-6G treatment
transfect mito. or rho-zero cells
rho-zero cell
with homoplasmic
mtDNA mutation
blastocyst injection
T
mice
Figure 4. Transmitochondrial cell approaches to producing mtDNA knockout mice (see text).
line, resulting in various OXPHOS defects
(McKenzie and I.Trounce, unpublished
results). Irwin et at. (1999) have also used
mitochondria from another species (Mus
spretus), directly injecting isolated mitochondria into oocytes. We are also attempting to
isolate mouse mtDNA mutants using classical
and novel mutagenesis approaches combined
with efficient mutant scanning using chemical
cleavage of mismatch, which identifies differences between heteroduplexed DNA molecules (see Forrest et ai, 1995). Different
mtDNA mutant cells with OXPHOS or ROS
phenotypes similar to human mtDNA disease
mutations may then be used to create mtDNA
knockout mice. Figure 4 shows a schematic
summary of these approaches.
Conclusions
Oxidative phosphorylation defects consequent
to mtDNA mutations cause an important group
of inherited diseases, and may be involved in
degenerative diseases of ageing and cancer. To
better understand pathogenesis and inheritance
of heteroplasmic mtDNA mutations, and to
develop new treatment modalities, mouse
models of mtDNA disease are required. Invitro manipulation of mitochondrial genotypes
in cultured somatic cells is now well established, as are techniques for karyoplast or
cytoplast transfer into fertilized oocytes or
early embryos. To mimic human mtDNA
mutations in mouse models we now need
techniques for creating custom mtDNA
knockouts and for isolating mtDNA mutants
in cell culture.
Acknowledgements
The author's research is supported by NHMRC grants
970543 and 98002, and grants-in-aid from the Ramaciotti
Foundation, ANZ Trustees and the Percy Baxter Trust.
References
Anderson, S., Bankier, A.T., Barrell, B.G. et al. (1981)
Sequence and organization of the human
mitochondrial genome. Nature, 290, 457^-65.
Arctander, P. (1999) Mitochondrial recombination?
Science, 284,. 2090-2091.
Beal, M.F. (1996) Mitochondria, free radicals and
neurodegeneration. Curr. Opin. Neurobiol., 6, 661666.
Boveris, A. and Chance, B. (1973) The mitochondrial
generation of hydrogen peroxide. Biochem. J., 134,
707-716.
Capaldi, R.A., Ganzalez-Halphen, D., Zhang, Y.-Z. and
Yanamura, W. (19&8) Complexity and tissue
specificity of the mitochondrial respiratory chain.
J. Bioenerg. Biomembr., 20, 291-311.
25
I.Trounce
Chance, B. and Williams, G.R. (1955) Respiratory
enzymes in oxidative phosphorylation I-IV. J. Biol.
Chem., 217, 383-438.
Chomyn, A., Meola, G., Bresolin, N. et al. (1991) In
vitro genetic transfer of protein sythesis and respiration
defects to mitochondrial DNA-less cells with
myopathy-patient mitochondria. Mol. Cell Biol., 11,
2236-2244.
Christodoulou, J. (2000) Genetic defects causing human
mitochondrial respiratory chain disorders and disease.
Hum. Reprod., 15 (Suppl. 2), 2 8 ^ 3 .
Clayton, D.A. (2000) Transcription and replication of
mitochondrial DNA. Hum. Reprod., 15 (Suppl. 2),
11-17.
Dawid, I.G. (1972) Cytoplasmic DNA. In Biggers, J.D.
and Schuetz, A.W. (eds) Oogenesis. University Park
Press, Baltimore, USA, pp. 215-226.
DiMauro, S. and Schon, E.A. (1998) Nuclear power and
mitochondrial disease. Nature Genet., 19, 214-215.
Ephrussi, B. (1950) The interplay of heredity and
environment in the synthesis of respiratory enzymes
in yeast. Harvey Lectures, 46, 45-67.
Forrest, S., Cotton, R.G.H., Landegren, U. and Southern,
E. (1995) How to find all those mutations. Nature
Genet., 10, 375-376.
Giles, R.E., Blanc, H., Cann, H.M. and Wallace, D.C.
(1980) Maternal inheritance of human mitochondrial
DNA. Proc. NatlAcad. Sci. USA, 77, 6715-6719.
Graham, B.H., Waymire, K.G., Cottrell, B. et al. (1997)
A mouse model for mitochondrial myopathy and
cardiomyopathy resulting from a deficiency in the
heart/muscle isoform of the adenine nucleotide
translocator. Nature Genet., 16, 226-234.
Gray, M.W, Burger, G. and Lang, B.F. (1999)
Mitochondrial evolution. Science, 283, 1476-1481.
Hoekstra, R. (2000) Evolutionary origin and
consequences
of
uniparental
mitochondrial
inheritance. Hum. Reprod., 15 (Suppl. 2), 102-111.
Howell, N. and Nalty, M.S. (1988) Mitochondrial
chloramphenicol-resistant
mutants
can
have
deficiencies in energy metabolism. Somat. Cell Mol.
Genet., 14, 185-193.
Irwin, M.H., Johnson, L.W. and Pinkert, C.A.. (1999)
Isolation and microinjection of somatic cell-derived
mitochondria and germline heteroplasmy in mice.
Transgenic Res., 8, 119-123
Jansen, R.P.S. (2000) Origin and persistence of the
mitochondrial genome. Hum. Reprod., 15 (Suppl. 2),
1-10.
Jenuth, J.P., Peterson, A.C., Fu, K. and Shoubridge, E.A.
(1996) Random genetic drift in the female germline
explains the rapid segregation of mammalian
mitochondrial DNA. Nature Genet., 14, 146-151.
Kenyon, L. and Moraes, C.T. (1997) Expanding the
functional human mitochondrial DNA database by the
establishment of primate xenomitochondrial cybrids.
Proc. NatlAcad. Sci. USA, 94, 9131-9135.
26
King, M.P. and Attardi, G. (1989) Human cells lacking
mtDNA: repopulation with exogenous mitochondria
by comlementation. Science, 246, 500-503.
King, M.P., Koga, Y., Davidson, M. and Schon, E.A.
(1992) Defects in mitochondrial protein synthesis and
respiratory chain activity segregate with the tRNA
Leu mutation associated with MELAS. Mol. Cell.
Biol., 12, 480-490.
Kluck, R.M., Bossy-Wetzel, E., Green, D.R. and
Newmaeyer, D.D. (1997) The release of cytochrome
c from mitochondria: a primary site for Bcl-2
regulation of apoptosis. Science, 275, 1132-1136.
Kwong, L.K. and Sohal, R.S. (1998) Substrate and site
specificity of hydrogen peroxide generation in mouse
mitochondria. Arch. Biochem. Biophys., 350,118-126.
Lehninger, A.L. (1964) The Mitochondrion: Molecular
Basis of Structure and Function. The Benjamin Co,
New York, USA.
Levings, C.S. (1983) The plant mitochondrial genome
and its mutants. Cell, 32, 659-661.
Levings, C.S. (1996) Infertility treatment: a nuclear
restorer gene in Maize. Science, 272, 1279-1280.
Levy, S.E., Waymire, K.G., Kim, Y.L. et al. (1999)
Transfer of chloramphenicol-resistant mitochondrial
DNA into the chimeric mouse. Transgen. Res., 8,
137-145.
Lightowlers, R.N., Chinnery, P.F., Turnbull, D.M. and
Howell, N. (1997) Mammalian mitochondrial
genetics: heredity, heteroplasmy and disease. Trends
Genet., 13, 450-^55.
Lightowlers, R.N., Jacobs, H.T. and Kajander, O.A.
(1999) Mitochondrial DNA: all things bad? Trends
Genet., 15, 91-93.
Margulis, L. (1981) Symbiosis in Cell Evolution. W.H.
Freeman and Co, San Francisco, USA.
Martinou, J.-C. (1999) Key to the mitochondrial gate.
Nature, 399, 411^12.
Matsuyama, S., Xu, Q., Velours, J. and Reed, J.C. (1998)
The mitochondrial FOFl-ATPase proton pump is
required for function of the proapoptotic protein Bax
in yeast and mammalian cells. Mol. Cell, 1, 327-336.
Melov, S., Coskun, P., Patel, M. et al. (1999)
Mitochondrial disease in superoxide dismutase 2
mutant mice. Proc. NatlAcad. Sci. USA, 96, 846-851.
Meirelles, F.V. and Smith, L.C. (1998) Mitochondrial
genotype segregation during pre-implantation
development in mouse heteroplasmic embryos.
Genetics, 148, 877-883.
Morais, R., Desjardins, P., Turmel, C. and ZinkewichPeotti, K. (1988) Development and characterization of
continuous avian cell lines depleted of mitochondrial
DNA. In Vitro Cell Dev. Biol., 24, 649-658.
Naviaux, R.K. and McGowan, K.A. (2000) Organismal
effects of mitochondrial dysfunction. Hum. Reprod.,
15 (Suppl. 2), 44-56.
Oxidative phosphorylation and defect models
Newmeyer, D.D., Arschon, D.M. and Reed, J.C. (1994)
Cell-free apoptosis in Xenopus egg extracts: inhibition
by Bcl-2 and requirement for an organelle fraction
enriched in mitochondria. Cell, 79, 353-364.
Raff, M. (1998) Cell suicide for beginners. Nature, 396,
119-122.
Rantanen, A. and Larsson, N.-G. (2000) Regulation of
mitochondrial
DNA
copy
number
during
spermatogenesis. Hum. Reprod., 15 (Suppl. 2), 86-91.
Riech, E. and Luck, D.J.L. (1966) Replication and
inheritance of mitochondrial DNA. Proc. Natl Acad.
Sci. USA, 55, 1600-1608.
Saraste, M. (1999) Oxidative phosphorylation at the fin
de siecle. Science, 283, 1488-1493.
Trounce, I. and Wallace, D.C. (1996) Production of
transmitochondrial mouse cell lines by cybrid rescue
of rhodamine-6G pre-treated L cells. Somat. Cell.
Mol. Genet., 22, 81-85.
Trounce, I., Neill, S. and Wallace, D.C. (1994)
Cytoplasmic transfer of the mtDNA nt8993 T-G
(ATP6) point mutation associated with Leigh
syndrome into mtDNA-less cells demonstrates
cosegregation with a decrease in state III respiration
and ADP/O ratio. Proc. Natl Acad. Sci. USA, 91,
8334-8338.
Trounce, I., Jun, B., Kim, Y. and Wallace, D.C. (1996)
Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts,
and transmitochondrial cell lines. Methods Enzymol,
264, 484-509.
Wallace, D.C. (1999) Mitochondrial diseases in man and
mouse. Science, 283, 1482-1488.
Wallace, D.C, Levy, S.E. and Sligh, J.E. (1999) The
creation of mouse models for mitochondrial DNA
(mtDNA) disease. Am. J. Hum. Genet., 65, 34.
Wang, J., Wilhelmsson, H., Graff, C. et al. (1999)
Dilated
cardiomyopathy
and
atrioventricular
conduction blocks induced by heart-specific
inactivation of mitochondrial DNA gene expression.
Nature Genet., 21, 133-137.
Yang, J., Liu, X., Bhalla, K. et al. (1997) Prevention of
apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science, 275, 1129-1132.
27