Copyright  2003 by the Genetics Society of America
Rapid, Selective Digestion of Mitochondrial DNA in Accordance With the matA
Hierarchy of Multiallelic Mating Types in the Mitochondrial Inheritance of
Physarum polycephalum
Y. Moriyama1 and S. Kawano
Laboratory of Plant Life System, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo,
Chiba 277-8562, Japan
Manuscript received December 16, 2002
Accepted for publication March 13, 2003
ABSTRACT
Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is
unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then
disappeared completely ⵑ1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of
the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the
loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated
by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all
possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a
hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted
to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10⫺4. Nevertheless, the matA
hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated
during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA
promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.
M
ITOCHONDRIA are inherited strictly maternally
in many species. The maternal inheritance of
mitochondria was first reported in 1974 in horse-donkey
hybrids (Hutchison et al. 1974). Related studies reached
the same conclusion in the rat (Hayashi et al. 1978;
Kroon et al. 1978), the pocket gopher Geomys pinetis
(Avise et al. 1979), the frog Xenopus laevis (Dawid and
Blackler 1972), the fruit fly Drosophila melanogaster
(Reilly and Thomas 1980), and humans (Giles et al.
1980). Particularly in oogamous species, uniparental inheritance of mitochondria has been attributed to the
small number of mitochondria in the male gamete. Although fertilized eggs are heteroplasmic (i.e., they contain mitochondria from both parents), a small population of mitochondria derived from the male gamete is
segregated rapidly after repeated cell division. Consequently, most cells are thought to contain mitochondria
from the female parent (Dawid and Blackler 1972;
Hutchison et al. 1974; Birky 1995; Ankel-Simons and
Cummins 1996). However, the idea of segregation of
parental mitochondrial DNA (mtDNA) has recently
been challenged in several reports. Backcrosses between
Mus musculus and M. spretus (an interspecific cross)
yielded offspring in which a very small proportion of
1
Corresponding author: Laboratory of Plant Life System, Department
of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-601, 5-1-5 Kashiwanoha, Kashiwa, Chiba
277-8562, Japan. E-mail: [email protected]
Genetics 164: 963–975 ( July 2003)
paternal mtDNA (0.01–0.1%) could be detected by sensitive PCR techniques (Gyllensten et al. 1991). This
promoted a reexamination, using PCR, of more common intraspecific crosses between mammals from which
Kaneda et al. (1995) concluded that, in intraspecific
crosses (M. musculus), the paternal mtDNA was eliminated by the two-cell stage.
Uniparental inheritance of mitochondria has also
been reported in the isogamous protist Physarum polycephalum (Kawano et al. 1987; Kawano and Kuroiwa
1989; Meland et al. 1991). The life cycle of Physarum
includes two distinct vegetative forms: the haploid amoeba
and the diploid plasmodium. The haploid myxamoebae
act as isogametes; individuals of different mating types
pair and fuse to form diploid zygotes that develop into
macroscopic, diploid plasmodia after repeated mitotic
cycles without cell division. Thus, the segregation of parental mtDNA is not involved in uniparental inheritance.
There are more than just two mating types of Physarum;
the mitochondria are transmitted uniparentally in accordance with the relative sexuality determined by the
mating-type locus matA, which has at least 13 alleles.
The matA alleles can be ranked in a linear hierarchy to
determine the loss of mtDNA (Kawano and Kuroiwa
1989; Meland et al. 1991): matA7 ⬎ matA2 ⬎ matA11 ⬎
matA12 ⬎ matA1//matA15 ⬎ matA6 (matA1 and matA15
have not been tested against each other). The mitochondrial donor is generally the amoeba that possesses the
dominant matA allele, and in each mating pair, one
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Y. Moriyama and S. Kawano
strain consistently acts as the mtDNA donor, although
this strain does not always act as the donor when combined in other mating pairs. Meland et al. (1991) suggested that the elimination of the mtDNA from one
parent is completed within 36 hr of mating. These facts
suggest that the universal phenomenon of uniparental
inheritance of mitochondria requires a species-specific
recognition system by which the zygote cytoplasm identifies and eliminates mitochondria or mtDNA from one
parent.
Recently, several studies reported selective destruction, rather than segregation, of sperm mitochondria
in the zygote, particularly in mammalian cells (Kaneda
et al. 1995; Sutovsky et al. 1999, 2000). The possible
involvement of ubiquitin in the destruction of sperm
mitochondria in fertilized cow and monkey eggs was
suggested. Conversely, in chloroplast inheritance, it has
been demonstrated that fluorescent chloroplast nucleoids derived from the male (mt⫺) parent disappear after
zygote formation in the isogamous green algae Chlamydomonas reinhardtii (Kuroiwa et al. 1982; Nishimura et al.
1999). Unfortunately, however, the behavior of mtDNA
before destruction is difficult to detect microscopically
because of the small copy number and molecular size
of mammalian mtDNA. In our work, to investigate the
mechanism for eliminating mitochondria from one parent, we observed the fate of mitochondria and mt-nucleoids (complexes of mtDNA and proteins) throughout
the mating of Physarum. The mitochondria and mtDNA
of Physarum are easily observed by phase-contrast and
epifluorescence microscopy. The mitochondria are well
developed and contain 20–40 ⵑ63-kb mtDNA molecules, which are highly organized by proteins into a
large rod-shaped mitochondrial nucleoid in each mitochondrion (Kuroiwa 1982; Takano et al. 2001). We
observed the rapid, selective digestion of the mtDNA
from one parent during early zygote development of
Physarum. The uniparental inheritance of mitochondria seems to be promoted by this rapid, selective digestion of mtDNA.
Some articles have reported that biparental inheritance of mtDNA does occur (Kondo et al. 1990; Gyllensten et al. 1991; Zouros et al. 1994; Kaneda et al. 1995;
Rawson et al. 1996). In particular, Gyllensten et al.
(1991) detected paternal mtDNA by PCR in interspecific mitochondrial congenic mice. Since the paternal
contribution was only 0.01–0.1%, these authors suggested that earlier failures to detect paternal mtDNA
were due to the low sensitivity of the assays used. The
situation remains ambiguous, however, because many
reported cases of paternal transmission involve interspecific rather than intraspecific hybrids. Since matings in
nature by definition occur mostly within species, it is
important to examine whether mtDNA is also biparentally transmitted in intraspecific hybrids. In this study,
we used all possible crosses between 16 strains with
matA1, matA2, matA11, matA12, matA15, or matA16 al-
leles to demonstrate that digestion of mtDNA from one
parent is highly selective and thorough, in accordance
with the matA hierarchy of multiallelic mating types.
In 21 of the 60 possible crosses, however, uniparental
mtDNA inheritance did not occur, and mtDNA from
both parents was transmitted to plasmodia at varying
frequencies. Since the rapid, selective digestion of
mtDNA in the recessive mitochondria was incomplete,
leakage of paternal mtDNA occurred.
MATERIALS AND METHODS
Strains and culture: The amoebal strains of P. polycephalum
used in this study are listed in Table 1. Myxamoebae were
cultured on PGY plates (0.5% glucose, 0.05% yeast extract, 2
mm MgSO4, and 1.5% agar in 25 mm potassium phosphate
buffer, pH 6.6) at 23⬚ with live bacteria (Klebsiella aerogenes)
for food. Zygote formation was induced on SM-30 mating
plates (30 mm citrate buffer, pH 4.5, 10 mm MgSO4, and 1.5%
agar) at 23⬚. For efficient crossing, myxamoebae must carry
different matA alleles, and in each mating pair, one strain
consistently acts as the mitochondria donor. However, the
dominant strain does not necessarily act as the mitochondria
donor in other combinations; the donor in each pair is determined by the respective matA alleles.
Plasmodium formation: About 3 days after zygote formation, small agar blocks carrying young plasmodia were cut
from the mating plates and transferred to malt extract agar
(MEA) plates (Kawano et al. 1987) for further growth at 23⬚.
Microscopic observation and fluorometry: To observe the
mitochondria clearly, cells were fixed with 8% formaldehyde
in 10⫻ PBS (pH 11) containing 0.01% Tween 20 on SM-30
mating plates. DNA was stained with 4⬘,6-diamidino-2-phenylindole (DAPI), and a coverslip was placed over the stained
sample. Photographs were taken with a BX62 Olympus (Tokyo)
epifluorescence microscope equipped with a c4742 CCD camera (Hamamatsu Photonics, Shizuoka, Japan) and an Aquacosmos system. The length of mt-nucleoids and the relative
mtDNA fluorescence were determined using the same system.
Electron microscopy: Samples were fixed with 1% osmium
tetroxide in PBS, pH 7.6 for 6 hr at 4⬚. They were then dehydrated in a graded ethanol series and embedded in Spurr’s
resin (Spurr 1969). Ultrathin sections (0.06–0.09 ␮m) were
cut with a glass knife on an ultramicrotome (Leica Ultracut
UCT; Leica Mikrosysteme, Vienna) and mounted on Formvarcoated copper grids. The sections were stained with 3% uranyl
acetate for 10 min at room temperature and lead citrate (0.13
m lead nitrate, 0.2 m trisodium citrate dehydrate) for 5 min
at room temperature and then examined with an electron
microscope (H-7600; Hitachi, Tokyo).
Isolation of single cells: A single amoeba or zygote was
isolated from the SM-30 mating plate under a phase-contrast
microscope (IMT-2; Olympus) using a capillary system that is
typically used for microinjection (MO-202; Narishige, Tokyo).
Single cells were transferred to individual microtubes containing 10 ␮l 1⫻ PCR buffer, 0.5% Tween 20, and 2 ␮g/ml
proteinase K. The samples were incubated overnight at 37⬚
to digest proteins, and heated to 95⬚ for 5 min to inactivate
proteinase K. Each sample was divided into two tubes and
used directly as template DNA for PCR.
DNA isolation: Approximately 20 mg of amoeba cells grown
on the PGY plates or plasmodium harvested on the MEA
plates for 4 days was transferred to a microcentrifuge tube
and suspended in 500 ␮l of 10⫻ Tris/saline EDTA (100 mm
Tris-HCl, pH 8, 150 mm NaCl, 100 mm EDTA) containing 2%
Selective Digestion of mtDNA
965
TABLE 1
Strains used in this study
Class
Strain
Mating types
mtDNA
genotypes
Origin (Reference)
A
AI2
AI5
AI16
AI33
AI35
AI39
DP89
DP90
DP239
DP246
DP248
TU9
TU41
NG3
NG5
NG6
matA 1matB 4
matA 1matB 1
matA 1matB 4
matA 2matB 1
matA 2matB 1
matA 1matB 1
matA 15matB 12
matA 16matB 13
matA 15matB 13
matA 16matB 13
matA 15matB 12
matA 11matB 5
matA 12matB 5
matA 12matB 6
matA 11matB 6
matA 11matB 5
M
M
M
M
M
M
W
W
W
W
W
T
T
N
N
N
a ⫻ i (Dee 1960)
a ⫻ i (Dee 1960)
a ⫻ i (Dee 1960)
a ⫻ i (Dee 1960)
a ⫻ i (Dee 1960)
a ⫻ i (Dee 1960)
Wis-2 (Kirouac-Brunet et al. 1981)
Wis-2 (Kirouac-Brunet et al. 1981)
DP89 ⫻ DP90
DP89 ⫻ DP90
DP89 ⫻ DP90
ATCC38899/Tu9/Turtox (Collins 1975)
ATCC38899/Tu9/Turtox (Collins 1975)
Ng (Kawano et al. 1991a,b)
Ng (Kawano et al. 1991a,b)
Ng (Kawano et al. 1991a,b)
B
SDS and 0.5 mg/ml proteinase K. After incubation of the
suspension at 37⬚ for 2 hr, 1 ml of saturated NaI with distilled
water was added, and the lysate was incubated at 0⬚ for 30 min.
The lysate was then centrifuged at 20,000 ⫻ g for 10 min at
4⬚, the surface debris was removed, and 5 ␮l of Glassmilk from
a Gene Clean II kit (BIO 101, Vista, CA) was added. The Glassmilk was washed and the DNA eluted according to the manufacturer’s directions. The eluted DNA was used as the template
for PCR.
Detection of parental mtDNA types: According to the restriction fragment length polymorphism analyses, the mtDNA
genotypes of the amoebae used in this study are classified into
M-, W-, T-, and N-types (see Table 1). Unlike the M-type, the
mtDNA of the W-, T-, and N-types has a 2-kb deletion (Sakurai
et al. 2000). This difference was exploited to detect mtDNA
from single cells by semi-nested PCR (see Figure 3C). The
DNA from a single cell was separated into two subsamples and
each was amplified using the specific primers for either the
M-type or the other types (W, T, or N) of mtDNA. As one
complete round of PCR was insufficient to detect the mtDNA
from a single cell, a second round was performed with seminested primers. The primer sequences were as follows:
i. F1, 5⬘-TACCCTGTATATGGAACAG-3⬘;
ii. F2, 5⬘-GAATTGATAGAAGAACTCAGAAGAGG-3⬘;
iii. MR, 5⬘-GGTCCCCAAATATTTCTTATAGAATATGC-3⬘;
iv. TR, 5⬘-TGCTTCCATAATTGCATCGT-3⬘.
PCR reaction mixtures were prepared with ExTaq DNA
polymerase (Takara, Otsu, Japan) according to the manufacturer’s instructions in a final volume of 50 ␮l. The first and
second rounds of PCR included 35 cycles at 94⬚ for 0.5 min,
at 54⬚ for 0.5 min, and at 72⬚ for 1 min. M-type mtDNA was
amplified from the sample in one of the paired tubes with
primers i and iii for the first round of PCR and with primers
ii and iii for the second round. A 1-␮l sample of the mixture
from the first round of PCR was used as template for the
second round. T-type mtDNA was amplified from the remaining tube with primers i and iv for the first round of PCR
and with ii and iv for the second round. The lengths of the
fragments amplified with the second pairs of primers from
the M- and T-type mtDNA were expected to be 673 and 526
bp, respectively. The T-type-specific primer also amplified a
fragment of ⵑ3 kb from the mtDNA of M-type. However,
preliminary experiments showed that this 3-kb fragment was
not amplified from a mixture of mtDNA from two parents,
due to competition with the 526-bp fragment from the T-type.
Estimation of the ratio of parental mtDNA with a PCR
matrix: To estimate the ratio of mtDNA from each parent in
the plasmodium, a PCR matrix of different PCR cycles and
different template ratios was made using purified M- and
W-type mtDNA. The DNA was amplified by PCR with primer
sets i ⫹ iii or i ⫹ iv from AI35 (M-type) or DP246 (W-type)
and then purified with a GFX PCR DNA and gel band purification kit (Amersham Pharmacia Biotech) as recommended by
the manufacturer. The copy numbers of each product were
estimated from the DNA concentration, and the two products
were mixed at ratios of 1:109–109:1. Using such template mixtures, PCR was carried out for 30 sec at 94⬚, 30 sec at 55⬚, and
1 min at 72⬚, for 20, 25, 30, and 35 cycles with primer sets
ii ⫹ iii or ii ⫹ iv. The products amplified with the two primer
sets using the same template ratios were loaded in one lane
and electrophoresed together. The electrophoresis patterns
are shown in Figure 7. The parental mtDNA in the plasmodium was detected by PCR using plasmodial DNA isolated
with Glassmilk from a Gene Clean II kit as the template. For
PCR, 1 ␮l of 0.01⫻, 0.1⫻, and 1⫻ template solution was used.
PCR was performed with primer sets ii ⫹ iii or ii ⫹ iv for 20,
25, 30, and 35 cycles of 30 sec at 94⬚, 30 sec at 55⬚, and 1
min at 72⬚. The ratio of mtDNA from the parents in the
plasmodium was estimated by comparing this PCR pattern
with the PCR matrix.
RESULTS
Visualization of loss of mtDNA during zygote formation: Myxamoebae are uninucleate cells that act as isogametes in crosses. In our serial observation of mating,
syngamy occurred soon after mixing two myxamoebae
strains with different matA alleles. Cell nuclei fused ⵑ2
hr after syngamy, and the resultant diploid nucleus di-
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Y. Moriyama and S. Kawano
Figure 1.—Loss of mt-nucleoids during zygote
formation in P. polycephalum. (A–H) Merged images from phase-contrast and fluorescence microscopy. (A) Myxamoebae. (B) Zygote. (C–H)
Timing of mt-nucleoid loss. (C and F) Fused cell.
(D and G) Uninucleate zygote just after nuclear
fusion. (E and H) Uninucleate zygote ⵑ1.5 hr
after nuclear fusion. Bars: A–E, 5 ␮m; F–H, 1 ␮m.
vided repeatedly in the absence of cell division to form
a multinucleate, diploid plasmodium. Phase-contrast
observations of myxamoebae clearly revealed the elliptical mitochondria. After DAPI staining, cell nuclei and
mt-nucleoids emitted bright blue-white fluorescence.
Each amoeba contained ⵑ15 mitochondria before mating, and each mitochondrion contained a long rodshaped mt-nucleoid at its center (Figure 1A). Zygote
formation was induced on a mating plate by mixing two
strains of different mating types. Surprisingly, about half
of the total mitochondria in zygotes lacked an mt-nucle-
Figure 2.—Sequential images of mt-nucleoid loss. (A) Fluorescent image showing the simultaneous loss of half the mtnucleoids in the zygote. (B–E) Representative images of mtnucleoid loss. Phase-contrast and fluorescent images are
merged. (B) Mitochondrion and its nucleoid in a uninucleate
zygote just after nuclear fusion. (C and D) Beginning and end
of mt-nucleoid loss. (E) Mitochondrion completely lacking
an mt-nucleoid. Bars: A, 5 ␮m; B–E, 1 ␮m.
oid, although the shape of the mitochondria was consistent with the amoebal stage (Figure 1B). To investigate
the loss of mt-nucleoids, the behavior of mitochondria
during zygote formation was analyzed throughout the
course of mating. Soon after two myxamoebae fused, a
full complement of ⵑ30 parental mitochondria mixed
together. Each mitochondrion was characterized by the
presence of a fluorescent mt-nucleoid (Figure 1, C and
F). The fused cells formed a uninucleate zygote as a
result of nuclear fusion ⵑ2 hr after mating. The fluorescent mt-nucleoid persisted in every mitochondrion
until this stage (Figure 1, D and G). However, 1 hr after
nuclear fusion, mt-nucleoid fluorescence completely
disappeared in about half of the mitochondria in the
zygote (Figure 1, E and H).
The process of mtDNA loss: Rapid loss of mtnucleoids is expected to occur within an hour of nuclear
fusion. We investigated this stage of mating in detail
and found that mt-nucleoid fluorescence diminished
synchronously to small spots, regardless of the position
of the mitochondria in the zygote (Figure 2A). Within
another 30 min, the mt-nucleoid fluorescence in these
mitochondria disappeared completely. Mitochondria
were arranged according to the time course of mtnucleoid loss, as shown in Figure 2, B–E. In half of the
mitochondria, the long rod-shaped mt-nucleoid present
in each mitochondrion just after nuclear fusion (Figure
2B) disappeared, starting from both ends of the mtnucleoid ⵑ1 hr after nuclear fusion (Figure 2C). The
fluorescence of each mt-nucleoid grew fainter and was
rapidly reduced to a single small spot (Figure 2D). This
spot was consistently located at the margin of the mito-
Selective Digestion of mtDNA
967
Figure 3.—Detection of parental mtDNA from a single cell by PCR. (A and B)
Before and after isolation of a single zygote
from a mating plate with a capillary tube.
(C) Scheme for detecting parental mtDNA
by semi-nested PCR. The primer sets used
are marked by half arrows (see materials
and methods). (D) Inheritance of parental
mtDNA, as detected in single zygotes at several developmental stages of AI35 ⫻ U41.
Three representative samples are shown for
each stage of development. Bar, 30 ␮m;
arrows: (A) zygote; (B) after isolation of the
zygote.
chondrion, and it disappeared completely without any
other apparent major changes in the mitochondrion
(Figure 2E). These observations suggest the presence
of two types of mitochondria in the uninucleate zygote:
those in which the mt-nucleoid is lost and those in which
it persists. A mechanism for the uniparental inheritance
of mitochondria could be proposed if it is established
that lost mt-nucleoids originate from a single parent.
Detection of parental mtDNA from a single cell by
PCR: To determine the parental origin of the lost mtnucleoids, parental mtDNA in single cells was analyzed
during zygote formation using semi-nested PCR. A single gamete or zygote was isolated under a phase-contrast
microscope using a microinjection capillary (Figure 3,
A and B), and its mtDNA was amplified by PCR. Two
amoebal strains of different mating types were used,
AI35 (matA2; mtDNA, M-type) and TU41 (matA12; mtDNA,
T-type). Unlike M-type, the mtDNA of the T-type has a
2-kb mtDNA deletion, so that mtDNA specific for the
M- and T-types can be distinguished with PCR primers
(Figure 3C). The results from three representative samples at each developmental stage are shown in Figure
3D. When AI35 and TU41 were crossed, the parental
mtDNA coexisted in the fused cell and was detectable
in the uninucleate zygote just after nuclear fusion. However, ⵑ1.5 hr after nuclear fusion, no parental mtDNA
from TU41 was detected, and this was correlated with
the loss of mt-nucleoids. Thus, mt-nucleoid loss appears
to be the result of the selective digestion of mtDNA
from one parent. Such selective digestion may account
for uniparental inheritance of mitochondria.
Fate of mitochondria lacking mtDNA during plasmodial development: To investigate the fate of mitochondria that lost mtDNA, mitochondria in the developing
zygote were observed from 0 to 60 hr after mating. As
the zygote of myxomycetes undergoes repeated nuclear
division without cell division after mating, all of the mitochondria derived from the parents are kept in a single
cell during zygote development. The total number of mitochondria in a single cell was counted at 8, 12, 24, 36,
48, and 60 hr after crossing AI35 and TU41 (Figure 4).
AI35 and TU41 had ⵑ14 and 16 mitochondria per cell,
respectively. There were ⵑ32 mitochondria in the zygote,
16 of which lost mtDNA by 8 hr after mating. As expected, the number of mitochondria with mtDNA increased ⬎25-fold to 430 by 60 hr after mating as a result
of repeated mitochondrial fission (Figure 4A). By contrast, the number of mitochondria without mtDNA remained at 16 until 36 hr after mating (Figure 4B). Then,
the number decreased to ⵑ3 in a single cell by 48 hr,
and all were lost by 60 hr after mating (Figure 4B).
Since the decrease in mitochondria lacking mtDNA
might have been due to mitochondrial sheath destruction, including destruction of the outer and inner membranes and cristae, mitochondrial morphology was examined during early zygote development. Morphological
changes were examined by light and electron microscopy during the early stages of zygote development to
plasmodium. The fused, diploid nucleus divided to form
a binucleate zygote (a small plasmodium). At ⵑ24 hr
after mating, no morphological changes were observed
by phase-contrast microscopy in mitochondria lacking
mtDNA (Figure 5A). The plasmodium became ⵑ50 ␮m
in diameter and had many nuclei ⵑ36 hr after mating
(Figure 5B). The vacant mitochondrial sheaths remained
visible at this time, and no degraded mitochondrial
sheaths were observed. By 48 hr after mating, almost
all of the vacant mitochondria had been eliminated
(Figure 5C).
Although phase-contrast microscopy suggested that
those that remained were unchanged morphologically
(Figure 5C), electron microscopy clearly revealed ultra-
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Y. Moriyama and S. Kawano
Figure 4.—Changes in the number of mitochondria derived from each parent in a single cell during early plasmodial
development. The number of mitochondria containing
mtDNA in a cell (䊊) and the number of mitochondria that
lost mtDNA (䊉) are shown in A and B in different ranges of
the y-axis. All measurements are shown as mean ⫾SD. A t-test
was used to estimate the significance of differences in the
numbers of mitochondria between one stage and a previous
stage (P ⫽ 0.01).
structural changes in the inner membrane by ⵑ36 hr
after mating. Figure 5D represents two typical mitochondria (left and right) at this stage. The left mitochondrion
preserves an electron-dense mt-nucleoid at the center
of the matrix and has well-developed tubular cristae.
Almost all of the mitochondria at this stage were of this
type. In contrast, the mitochondrion on the right has
no mt-nucleoid and has collapsed cristae. Almost the
entire mitochondrion was an electron-transparent region, although some parts retained double membranes
and tubular cristae, enabling their detection using
phase-contrast microscopy (Figure 5, B and D). Mitochondrial degradation was observed by ⵑ36 hr after
mating, and the elimination of the mitochondrial sheath
was completed by 60 hr after mating. Compared with
the rapid and selective digestion of mtDNA from one
parent within 0.5 hr at 3 hr of mating, elimination of
the vacant mitochondria derived from one parent was
a lengthy procedure.
Mitochondrial inheritance according to the matA hierarchy: In Physarum, there are multiple mating types,
and the mitochondrial inheritance mode is determined
by alleles matA1–matA16, which are ranked in a linear
hierarchy with respect to mitochondrial inheritance
(Kawano et al. 1987; Kawano and Kuroiwa 1989;
Meland et al. 1991). We investigated whether selective
digestion of mtDNA occurred in accordance with the
relative sexuality of matA. To determine whether the
mtDNA from TU41 could survive when crossed with a
second strain with a lower matA rank, AI16 (matA1;
mtDNA, M-type) was crossed with TU41. Since AI16 and
AI35 have the same parent (Dee 1960), they have the
same mitochondrial genotype (Table 1). Consequently,
crosses of these strains with TU41 also permitted examination of the relationship between digestion selectivity
and mtDNA molecules. In crosses of AI16 with TU41,
mt-nucleoid loss occurred in the same manner as in
the original AI35 ⫻ TU41 cross. In the plasmodium of
AI35 ⫻ TU41, mtDNA of TU41 was lost, as previously
described (Figures 3D and 6A). In the plasmodium of
AI35 ⫻ TU41, however, the TU41 mtDNA survived, and
the lost mt-nucleoids were of AI16 origin (Figure 6B).
The results show that the digestion of parental mtDNA
from one strain is highly selective and is in accord with
the matA hierarchy.
Figure 5.—Elimination of mitochondria that
lost mtDNA during zygote development. (A–C)
Merged images from phase-contrast and fluorescence microscopy. (A) Zygote 24 hr after mating.
(B) Zygote 36 hr after mating. (C) Zygote 48 hr
after mating. (D) Ultrastructural change in the
mitochondria. Arrow, disordered, degraded, destroyed mitochondrion. Arrowhead, mitochondrion that contains mt-nucleoid (MN). Bars: A–C,
5 ␮m; D, 500 nm.
Selective Digestion of mtDNA
Figure 6.—Transmission pattern of mtDNA in four representative crosses. Parental mtDNA was detected from five plasmodia in each cross of (A) AI35 ⫻ TU41, (B) AI16 ⫻ TU41,
(C) AI35 ⫻ DP246, and (D) AI16 ⫻ DP246.
To confirm the strictly uniparental inheritance at the
PCR level in any combination of mating type, the transmission patterns of mtDNA were examined in all possible crosses between the strains listed in classes A and B
in Table 1. The six strains ranked in class A are progeny
of a ⫻ i and have either matA1 or matA2. Their mtDNA
is M-type. The strains ranked in class B have different
origins and have matA11, matA12, matA15, or matA16,
depending on their origin. Since they have W-, T-, or
N-type mtDNA, the mtDNA transmission pattern can
be detected when they are crossed with class A strains.
The mtDNA of parents was detected in plasmodia 10
days after mating by using PCR for 35 cycles. In 39 of
60 possible crosses, strict uniparental inheritance was
confirmed (Table 2). For example, the mtDNA of AI35
(M-type) was transmitted in AI35 ⫻ TU41, while the
mtDNA of TU41 (T-type) was transmitted in AI16 ⫻
TU41, as shown in Figure 6, A and B. These results are
arranged in Table 2. Strict uniparental inheritance of
mtDNA occurred in all of the crosses of matA1 and -2
strains with matA11 and -12 strains. The mtDNA of
matA11 and -12 strains (N-type) was transmitted in the
crosses with matA1 strains, but was not transmitted in
the crosses with matA2 strains. These results are in accord with the matA hierarchy: matA2 ⬎ matA11 ⬎
matA12 ⬎ matA1. The matA2 strains were mitochondrial
donors when they were crossed with matA16 strains. In
21 of 60 crosses, however, mtDNA from both parents
was detected (Table 2). For example, when AI35 was
crossed with DP246, the mtDNA of AI35 (M-type) was
transmitted to the plasmodium (Figure 6C). However,
when AI16 (M-type) was crossed with DP246 (W-type),
uniparental transmission of mtDNA did not occur: Instead, mtDNA from both parents was transmitted to
969
plasmodia (Figure 6D). Such biparental inheritance of
mtDNA occurred only in the crosses matA1 ⫻ matA15,
matA1 ⫻ matA16, and matA2 ⫻ matA15.
Ratio of parental mtDNA in biparental inheritance of
mtDNA: We examined the mtDNA type of every plasmodium at 10 days after mating by PCR with 35 cycles, as
shown (Figure 6). With this number of PCR cycles, it
is possible to detect the presence/absence of either
parental mtDNA, but it is not possible to estimate the
ratio of parental mtDNA, because the PCR product is
saturated under this condition. Therefore, to estimate
the ratio of parental mtDNA in the 21 crosses that inherited mtDNA biparentally, the PCR efficiency of mixtures
of the two different kinds of mtDNA (M- and W-types)
in copy-number ratios of 1:109–109:1 was examined at
20, 25, 30, and 35 PCR cycles using different primer
sets for the M- and W-types. The two PCR products using
the different primer sets were loaded in one lane. The
results were arranged in a matrix, as shown in Figure
7, to show the PCR efficiency with different numbers
of cycles and different ratios of the two templates. This
efficiency matrix can detect at least 1 ⫻ 105 molecules
of mtDNA and can detect mtDNA from one genotype
in a 105–100 times excess of mtDNA from the other.
Therefore, using this PCR matrix, we estimated the ratios of parental mtDNA in 1 ␮l of 0.01⫻, 0.1⫻, and 1⫻
template DNA solution that was isolated from plasmodium 10 days after mating for 20, 25, 30, and 35 cycles of
PCR (Table 2). There were biased ratios in the parental
mtDNA for the 21 crosses that inherited mtDNA biparentally. For example, the AI5 ⫻ DP246 plasmodium,
which had inherited mtDNA biparentally, contained
10⫺3 as much mtDNA from AI5 as from DP246 (Table
2). Conversely, AI16 ⫻ DP246 contained mtDNA from
both parents in equal amounts, according to the PCR
matrix. Equal biparental inheritance occurred in 5 of
the 21 crosses, including AI16 ⫻ DP246. The mtDNA
genotypes of each plasmodium were abbreviated using
Ⰶ, ⬍, ⫽, or ⬎ according to the ratio of mtDNA from
the parents (Table 2), and these results are arranged in
Table 3 according to mating types. The bias of parental
mtDNA in the crosses matA1 ⫻ matA15, matA1 ⫻
matA16, and matA2 ⫻ matA15 seems to obey the matA
hierarchy. Furthermore, in matA1 ⫻ matA15, 5 of 12
crosses (AI5 ⫻ DP89, AI39 ⫻ DP89, AI2 ⫻ DP248, AI5 ⫻
DP248, and AI39 ⫻ DP248) showed exceptional uniparental inheritance of mtDNA, resulting in matA1 ⬎
matA15.
Biased biparental inheritance caused by partial mtDNA loss: To investigate the mechanism of the biased
biparental inheritance in particular crosses, we observed
the behavior of mitochondria during plasmodium formation with DAPI-fluorescence and phase-contrast microscopy. In the crosses that showed biased biparental
inheritance, e.g., AI5 ⫻ DP246, the disappearance of
fluorescent mt-nucleoids was observed after mating in
about half the mitochondria. However, the digestion of
b
a
UI
NG3
NG3
NG3
NG3
NG5
NG5
NG5
NG5
NG6
NG6
NG6
NG6
TU9
TU9
TU9
TU9
TU41
TU41
TU41
TU41
DP89
DP89
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
AI2
AI5
AI16
AI39
AI2
AI5
AI16
AI39
AI2
AI5
AI16
AI39
AI2
AI5
AI16
AI39
AI2
AI5
AI16
AI39
AI5
AI39
Strains
Ratio
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
N
N
N
N
N
N
N
N
N
N
N
N
T
T
T
T
T
T
T
T
W
W
B Ib
UI
Inheritance
M W, T, N Genotype
mode
U I: uniparental inheritance.
B I: biparental inheritance.
a
Inheritance
mode
DP246
DP90
DP246
DP248
DP239
⫻
⫻
⫻
⫻
⫻
AI2
AI16
AI16
AI33
AI39
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10⫺2
10⫺2
10⫺2
10⫺1
10⫺1
DP90
DP239
DP239
DP90
DP246
⫻
⫻
⫻
⫻
⫻
AI2
AI2
AI16
AI39
AI39
1
1
AI5 ⫻ DP90 10⫺3
AI5 ⫻ DP246 10⫺3
1
1
1
1
1
1
0
0
0
M
AI2 ⫻ DP89 10⫺4
AI16 ⫻ DP89 10⫺4
AI16 ⫻ DP248 10⫺4
DP248 ⫻ AI2
DP248 ⫻ AI16
DP248 ⫻ AI39
Strains
M
M
M
M
M
m
m
m
m
m
⫽
⫽
⫽
⫽
⫽
⬍
⬍
⬍
⬍
⬍
W
W
W
W
W
W
W
W
W
W
mⰆW
mⰆW
mⰆW
mⰆW
mⰆW
W
W
W
UI
BI
Inheritance
W, T, N Genotype
mode
Ratio
⫻
⫻
⫻
⫻
DP239
DP239
DP239
DP248
NG3
NG3
NG3
NG5
NG6
NG6
TU9
TU9
TU41
TU41
DP90
DP90
DP246
DP246
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
⫻
AI33
AI35
AI33
AI35
AI33
AI35
AI33
AI35
AI33
AI35
AI33
AI35
AI33
AI35
AI33 ⫻ DP89
AI35 ⫻ DP89
AI5
AI33
AI35
AI35
Strains
Ratio
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
10⫺2
10⫺2
10⫺1
10⫺1
10⫺1
10⫺1
⬎
⬎
⬎
⬎
w
w
w
w
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M⬎w
M⬎w
M
M
M
M
M W, T, N Genotype
Estimated ratio of mitochondrial DNA from the two parents in plasmodia derived from the 60 possible crosses of 16 strains
TABLE 2
970
Y. Moriyama and S. Kawano
Selective Digestion of mtDNA
971
Figure 7.—PCR matrix of different PCR cycles from template solution containing different copy numbers of the two mitochondrial DNA types. PCR was performed with strain-specific primers from template solution containing different copy numbers of
the two mtDNA types for 20, 25, 30, and 35 cycles. Each mtDNA sample was amplified separately using different primer sets and
applied to one lane. The upper fragment (673 kb) is the M-type product, and the lower fragment (526 kb) is the W-type.
had well-developed mt-nucleoids (0.5–1.8 ␮m, 0.4–2.2
rfiu), but some mitochondria contained small mt-nucleoids that emitted very faint fluorescence (0.1–0.25 ␮m,
0–0.5 rfiu). These very small mt-nucleoids were generated by incomplete mtDNA digestion. Such small mtnucleoids were rarely observed 36 hr after mating (Figure 8, D–F), because they appear to become larger
(0.25⫹ ␮m, 0.5⫹ rfiu). During plasmodial development,
mtDNA is replicated, and the size of these surviving mtnucleoids increases, as shown in Figure 9.
At 36 hr after mating, the vacant mitochondria that
had lost mt-nucleoids completely remained at the points
of origin. In the plasmodia, the vacant mitochondria
were completely eliminated, and the surviving mt-nucleoids and mitochondria were indistinguishable from each
other (0.4–1.8 ␮m, 0.3–1.9 rfiu). However, the biased
mtDNA in the recessive mitochondria from AI16 was
not complete 24 hr after mating; very faint, small spots,
representing fluorescent mt-nucleoids, persisted in some
mitochondria (Figure 8, A–C). Surprisingly, 24–36 hr
after mating, the persistent mt-nucleoids seemed to increase gradually in size due to mtDNA replication (Figure 8, D–F).
We measured the length and fluorescence intensity
of mt-nucleoids stained with DAPI in cells 24 and 36
hr after mating and in mature plasmodium, using an
epifluorescence microscope equipped with a CCD camera and fluorometric software. The values of their major
axes (in micrometers) and fluorescence intensities (rfiu;
relative fluorescent intensity unit) are arranged in a
scatter plot in Figure 9. At 24 hr after mating, 18% of
the mitochondria were vacant (0 ␮m, 0 rfiu), and 69%
TABLE 3
The relation between mating type and the inheritance mode of mtDNA
matA 11
matA 12
matA 15
matA 16
Mating
type
mtDNA
genotype
Strain
N
NG5
N
NG6
T
TU9
N
NG3
T
TU41
W
DP89
W
DP239
W
DP248
matA 1
M
M
M
M
M
M
AI2
AI5
AI16
AI39
AI33
AI35
N
N
N
N
M
M
N
N
N
N
M
M
T
T
T
T
M
M
N
N
N
N
M
M
T
T
T
T
M
M
mⰆW
W
mⰆW
W
M⬎w
M⬎w
m⬍W
m⬍W
m⬍W
m⬍W
M⬎w
M⬎w
W
W
mⰆW
W
M⫽W
M⬎w
matA 2
W
DP90
m
m
M
m
⬍W
ⰆW
⫽W
⬍W
M
M
W
DP246
M
m
M
m
⫽W
ⰆW
⫽W
⬍W
M
M
972
Y. Moriyama and S. Kawano
Figure 8.—Incomplete digestion of mtDNA in
a plasmodium showing biparental inheritance of
mtDNA. (A–F) Merged images from phase-contrast and DAPI fluorescence microscopy of whole
cells. (A–C) Binucleate zygote ⵑ24 hr after mating. (D–F) Multinucleate zygote ⵑ36 hr after mating. B and E are enlargements of areas in A and
D, respectively. Bars: A and D, 10 ␮m; B and E,
5 ␮m.
biparental inheritance of mtDNA suggests that the incompletely digested mtDNA was of uniparental origin
and that the normal copy number in mitochondria was
restored during plasmodial development. These results
suggest that complete digestion of each mt-nucleoid in
mitochondria is needed to destroy and eliminate the
mitochondrial sheath. Incomplete digestion of mtDNA
enables biased biparental inheritance.
DISCUSSION
Figure 9.—Scatter plots of fluorescence intensity vs. mtnucleoid length at 24 and 36 hr after mating and in plasmodia
in AI5 ⫻ DP246. The relative fluorescence intensity of mtnucleoids stained with DAPI was plotted against the length of
the major axis. Vacant mitochondria that had lost their mtnucleoid were plotted at the origin of the coordinate axes.
Rapid, selective digestion of mtDNA causes uniparental inheritance of mitochondria: Mitochondrial inheritance is thought to be predominantly uniparental in
nearly all eukaryotes. The combination of mainly uniparental inheritance and frequent mutation invites great
interest in mtDNA as an indicator of evolutionary relationships (Ingman et al. 2000). However, the mechanism
behind uniparental inheritance has been unclear. In
this study, we observed the rapid and simultaneous loss
of mt-nucleoids in about half of the mitochondria during an early stage of zygote maturation (Figures 1 and
2). Molecular analysis of a single cell showed that mtnucleoid loss coincided with the uniparental inheritance of mitochondria (Figure 3); after mt-nucleoid loss,
mtDNA from only one of the two parents was detected
by PCR. These results indicate that the lost mt-nucleoids
were of uniparental origin. The loss of mtDNA has also
been shown in higher plants and algae (Kuroiwa and
Hori 1986; Corriveau and Coleman 1991; Nagata et
al. 1999). This loss of mtDNA organized in mt-nucleoids
occurs before fertilization in the mature generative cell
inside a pollen grain or in the male gamete before
fertilization. In Physarum, however, mt-nucleoid loss
occurs in the zygote after mating. Although mitochondria from both parents were well mixed in the zygote,
mtDNA from the mitochondrial recipient strain was
digested synchronously and completely, while that from
the mitochondrial donor strain was completely pro-
Selective Digestion of mtDNA
tected from digestion. The mt-nucleoid loss progressed
synchronously after nuclear fusion (Figure 2). This suggests that a nuclease, or nuclease activation signal, is
synchronously transported from the cytoplasm to targeted mitochondria within a very limited period. The
digestion of mtDNA seemed to be independent of the
mtDNA sequence, since mtDNA with the same sequence
were digested in the AI35 ⫻ TU41 cross, but not in the
AI16 ⫻ TU41 cross (Figure 6). The results of reciprocal
crosses in which one strain (TU41) played a dual role
in uniparental inheritance, acting as a recipient in one
cross (AI35 ⫻ TU41) and a donor in another (AI16 ⫻
TU41), confirmed that mtDNA of uniparental origin
are not destined to be digested before mating. The
uniparental inheritance of mitochondria seems to involve mechanisms that recognize the origin of mtDNA
and promote the selective digestion of mtDNA from
one parent in the zygote.
After the complete digestion of mtDNA, the number
of mitochondria that contained mtDNA increased
greatly, whereas the number of mitochondria that lost
mtDNA remained unchanged until ⵑ36 hr after mating.
After another 24 hr, they were lost completely (Figure
4). Degraded or disintegrated mitochondria were not
observed directly at these stages by phase-contrast microscopy (Figure 5, A–C). In Saccharomyces cerevisiae and
C. reinhardtii, it is well known that organelles derived
from both parents fuse. However, the decrease in the
number of mitochondria that lost mtDNA was not due
to fusion with mitochondria that contained mtDNA,
since no fused mitochondria were observed at these
stages in Physarum. Moreover, the strains used here do
not have an mF plasmid, which is known to promote
mitochondrial fusion in P. polycephalum (Kawano et al.
1993).
More detailed observation by electron microscopy revealed that degradation of the mitochondrial inner
membrane occurred without any changes in size of mitochondria ⵑ36 hr after mating (Figure 5D). Morphological changes that do not result in changes in the overall
size of mitochondria have also been noted during apoptosis and necrosis (Lemasters et al. 1998; Scorrano et
al. 2002). In Physarum, it is likely that the inner membrane of mitochondria that lose mtDNA is disrupted,
and then the empty, nonfunctional mitochondria are
removed, probably by lysosomes. In hamsters, rats, and
bovines, degradation of sperm mitochondria has been
observed during the early stage of embryonic development (Szollosi 1965; Hiraoka and Hirao 1988;
Kaneda et al. 1995; Sutovsky et al. 1999, 2000). Sutovsky et al. (1999, 2000) reported that sperm mitochondria were ubiquitinated before fertilization and subsequently destroyed in the mammalian egg. They insisted
that the destruction of mitochondria invites uniparental
inheritance of mitochondria. The candidate ubiquitin
substrate was proposed to be prohibitin, an integral
protein of the inner mitochondrial membrane. It is
973
reasonable to assume that modification and destruction
of the mitochondrial membrane plays an important role
in mitochondrial inheritance. In Physarum, however,
such ubiquitination before gamete fusion is unlikely,
since the gamete has only relative sexuality, which is
determined by the matA allele of the mating partner.
As the choice of a fusion partner is random, the mitochondria or mtDNA cannot be primed before mating.
At least the integrity of the mitochondrial membrane
is conserved just before the digestion of mtDNA, as the
digestion of mtDNA in dividing mitochondria occurs
(Figure 1B). We believe that prohibitin ubiquitination
may perform an important role in the destruction of the
inner mitochondrial membrane after mtDNA digestion.
Incomplete digestion of mtDNA causes biased biparental inheritance of mitochondria: In Physarum, the
inheritance mode of mtDNA is determined by the mating-type locus matA, which has at least 13 alleles. We
examined the recognition and digestion of mtDNA among
multiple mating types, using PCR for 35 cycles of DNA
from plasmodia 10 days after mating. Of the eight possible combinations of six mating types, five (39 of possible
60 crosses) showed strict uniparental inheritance of mitochondria in accordance with the relative sexuality of
matA (Figure 6, Table 3). Conversely, mtDNA from both
parents was transmitted in three combinations of matA
(21 crosses).
To estimate the ratio of parental mtDNA readily, we
made PCR matrices that showed the efficiency of the
PCR reaction depending on the ratio of parental mtDNA. Our PCR matrix method is very useful for treating
many samples to estimate the ratio of parental mtDNA.
The ratio ranged from 1:10⫺4 to equal amounts (Tables
2 and 3), and one of the parental mtDNA genotypes
always dominated in accordance with the matA hierarchy. In the zygotes of these crosses, the digestion of
mtDNA was incomplete (Figure 8, A–C). The ratios of
mtDNA from parents, listed in Table 2, may depend on
the effectiveness of the digestion of mtDNA. The aberrant
mitochondria with small mt-nucleoids (Figures 8, C and
F) become normal sized by replicating their mtDNA
(Figure 9). The temporary reduction and subsequent
replication of mtDNA can explain the biased biparental
inheritance of mtDNA from parents in Physarum.
The presence of leaked paternal mtDNA, particularly
in interspecific hybrids between closely related species,
occurs in several genera, such as Mytilus (Zouros et al.
1994; Rawson et al. 1996), Drosophila (Kondo et al.
1990), and Mus (Gyllensten et al. 1991; Kaneda et
al. 1995), and in humans (Schwartz and Vissing
2002). In such cases, the selection or digestion of mtDNA from one parent in the zygote might fail. The
biased biparental inheritance of mtDNA in Physarum
suggests that the destruction of mitochondria from a
strain lower in the hierarchy never occurs unless the
mtDNA is digested completely. If the destruction of
mitochondria rather than the digestion of mtDNA were
974
Y. Moriyama and S. Kawano
the critical mechanism of mitochondrial inheritance,
strict uniparental inheritance of mitochondria would
occur even though complete digestion of mtDNA failed.
Our results suggest that complete digestion of mtDNA
is needed for the destruction of mitochondria and that
uniparental inheritance of mitochondria is directly
caused by the rapid and selective digestion of the mtDNA from one parent. The mechanisms for recognizing
the parental origin of mtDNA and for promoting the
selective digestion of mtDNA are unknown. We are now
investigating mitochondrial nuclease(s) that are nuclear
DNA coded and involved in the recognition and digestion of mtDNA from one parent. The isolation and
characterization of nuclease(s) should explain the leakage of paternal mtDNA to subsequent generations.
We thank T. Kuroiwa (Graduate School of Science, University of
Tokyo) for helpful discussions and A. Hirata (Graduate School of
Frontier Science, University of Tokyo) for advice on electron microscopy. We also thank S. Matsunaga (Graduate School of Engineering,
University of Osaka) for helpful technical advice. This study was supported by grants for Scientific Research in Priority Areas (no.
13440246 to S.K.) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
LITERATURE CITED
Ankel-Simons, F., and J. M. Cummins, 1996 Misconceptions about
mitochondria and mammalian fertilization: implications for theories on human evolution. Proc. Natl. Acad. Sci. USA 93: 13859–
13863.
Avise, J. C., C. Giblin-Davidson, J. Laerm, J. C. Patton and R. A.
Lansman, 1979 Mitochondrial DNA clones and matriarchal
phylogeny within and among geographic populations of the
pocket gopher, Geomys pinetis. Proc. Natl. Acad. Sci. USA 76:
6694–6698.
Birky, C. W., Jr., 1995 Uniparental inheritance of mitochondrial
and chloroplast genes: mechanisms and evolution. Proc. Natl.
Acad. Sci. USA 92: 11331–11338.
Collins, O.R., 1975 Mating types in five isolates of Physarum polycehalum. Mycologia 67: 98–107.
Corriveau, J. L., and A. W. Coleman, 1991 Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species. Dev. Biol. 147: 271–280.
Dawid, I. B., and A. W. Blackler, 1972 Maternal and cytoplasmic
inheritance of mitochondrial DNA in Xenopus. Dev. Biol. 29:
152–162.
Dee, J., 1960 A mating type system in an acellular slime-mould.
Nature 185: 780–781.
Giles, R. E., H. Blanc, H. M. Cann and D. C. Wallace, 1980 Maternal inheritance of human mitochondrial DNA. Proc. Natl. Acad.
Sci. USA 77: 6715–6719.
Gyllensten, U., D. Wharton, A. Josefsson and A. C. Wilson, 1991
Maternal inheritance of mitochondrial DNA during backcrossing
of two species of mice. Nature 352: 255–257.
Hayashi, J., H. Yonekawa, O. Gotoh, J. Motohashi and Y. Tagashira, 1978 Two different molecular types of rat mitochondrial
DNAs. Biochem. Biophys. Res. Commun. 81: 871–877.
Hiraoka, J., and Y. Hirao, 1988 Fate of sperm tail components after
incorporation into the hamster egg. Gamete Res. 19: 369–380.
Hutchison, C. A., III, J. E. Newbold, S. S. Potter and M. H. Edgell,
1974 Maternal inheritance of mammalian mitochondrial DNA.
Nature 251: 536–538.
Ingman, M., H. Kaessmann, S. Paabo and U. Gyllensten, 2000
Mitochondrial genome variation and the origin of modern humans. Nature 408: 708–713.
Kaneda, H., J. Hayashi, S. Takahama, C. Taya, K. F. Lindahl et al.,
1995 Elimination of paternal mitochondrial DNA in intraspe-
cific crosses during early mouse embryogenesis. Proc. Natl. Acad.
Sci. USA 92: 4542–4546.
Kawano, S., and T. Kuroiwa, 1989 Transmission pattern of mitochondrial DNA during plasmodium formation in Physarum polycephalum. J. Gen. Microbiol. 135: 1559–1566.
Kawano, S., R. W. Anderson, T. Nanba and T. Kuroiwa, 1987 Polymorphism and uniparental inheritance of mitochondrial DNA
in Physarum polycephalum. J. Gen. Microbiol. 133: 3175–3182.
Kawano, S., H. Takano, K. Mori and T. Kuroiwa, 1991a A mitochondrial plasmid that promotes mitochondrial fusion in Physarum polycephalum. Protoplasma 160: 167–169.
Kawano, S., H. Takano, K. Mori and T. Kuroiwa, 1991b The oldest
laboratory strain of Physarum polycephalum. Physarum Newslett.
22: 70–75.
Kawano, S., H. Takano, J. Imai, K. Mori and T. Kuroiwa, 1993
A genetic system controlling mitochondrial fusion in the slime
mould, Physarum polycephalum. Genetics 133: 213–224.
Kirouac-Brumet, J., S. Mansson and D. Pallota, 1981 Multiple
allelism at the matB locus in Physarum polycephalum. Can. J. Genet.
Cytol. 23: 9–16.
Kondo, R., Y. Satta, E. T. Matsuura, H. Ishiwa, N. Takahata et
al., 1990 Incomplete maternal transmission of mitochondrial
DNA in Drosophila. Genetics 126: 657–663.
Kroon, A. M., W. M. de Vos and H. Bakker, 1978 The heterogeneity
of rat-liver mitochondrial DNA. Biochim. Biophys. Acta 519: 269–
273.
Kuroiwa, T., 1982 Mitochondrial nuclei. Int. Rev. Cytol. 75: 1–59.
Kuroiwa, T., and T. Hori, 1986 Preferential digestion of male chloroplast nuclei and mitochondrial nuclei during gametogenesis
of Bryopsis maxima Okamura. Protoplasma 133: 85–87.
Kuroiwa, T., S. Kawano, S. Nishibayashi and C. Sato,
1982 Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature 298: 481–483.
Lemasters, J. J., A. L. Nieminen, T. Qian, L. C. Trost, S. P. Elmore
et al., 1998 The mitochondrial permeability transition in cell
death: a common mechanism in necrosis, apoptosis and autophagy. Biochim. Biophys. Acta 1366: 177–196.
Meland, S., S. Johansen, T. Johansen, K. Haugli and F. Haugli,
1991 Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum. Curr. Genet. 19: 55–59.
Nagata, N., C. Saito, A. Sakai, H. Kuroiwa and T. Kuroiwa, 1999
The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance. Planta 209: 53–65.
Nishimura, Y., O. Misumi, S. Matsunaga, T. Higashiyama, A.
Yokota et al., 1999 The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezers. Proc. Natl. Acad. Sci. USA
96: 12577–12582.
Rawson, P. D., C. L. Secor and T. J. Hilbish, 1996 The effects of
natural hybridization on the regulation of doubly uniparental
mtDNA inheritance in blue mussels (Mytilus spp.). Genetics 144:
241–248.
Reilly, J. G., and C. A. Thomas, Jr., 1980 Length polymorphisms,
restriction site variation, and maternal inheritance of mitochondrial DNA of Drosophila melanogaster. Plasmid 3: 109–115.
Sakurai, R., N. Sasaki, H. Takano, T. Abe and S. Kawano, 2000 In
vivo conformation of mitochondrial DNA revealed by pulsed-field
gel electrophoresis in the true slime mold, Physarum polycephalum.
DNA Res. 7: 83–91.
Schwartz, M., and J. Vissing, 2002 Paternal inheritance of mitochondrial DNA. N. Engl. J. Med. 22: 576–580.
Scorrano, L., M. Ashiya, K. Buttle, S. Weiler, S. A. Oakes et al.,
2002 A distinct pathway remodels mitochondrial cristae and
mobilizes cytochrome c during apoptosis. J. Dev. Cell 2: 55–67.
Spurr, A. R., 1969 A low-viscosity epoxy resin embedding medium
for electron microscopy. J. Ultrastruct. Res. 26: 31–43.
Sutovsky, P., R. D. Moreno, J. Romalho-Santos, T. Dominko, C.
Simerly et al., 1999 Ubiquitin tag for sperm mitochondria. Nature 402: 371–372.
Sutovsky, P., R. D. Moreno, J. Romalho-Santos, T. Dominko, C.
Simerly et al., 2000 Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance
in mammalian embryos. Biol. Reprod. 63: 582–590.
Selective Digestion of mtDNA
Szollosi, D. J., 1965 The fate of sperm middle-piece mitochondria
in the rat egg. Exp. Zool. 159: 366–377.
Takano, H., T. Abe, R. Sakurai, Y. Moriyama, Y. Miyazawa et al.,
2001 The complete DNA sequence of the mitochondrial genome of Physarum polycephalum. Mol. Gen. Genet. 264: 539–545.
975
Zouros, E., B. A. Oberhauser, C. Saavedra and K. R. Freeman,
1994 An unusual type of mitochondrial DNA inheritance in the
blue mussel Mytilus. Proc. Natl. Acad. Sci. USA 91: 7463–7467.
Communicating editor: N. Takahata