Mitochondrial disease model mice, mitomice, were created using zygotes of B6mtspr strain mice carrying mitochondrial DNA (mtDNA) from Mus spretus as recipients of exogenous mitochondria carrying wild-type and a deletion mutant mtDNA (mtDNA) of M. musculus domesticus. In these experiments, mtDNAs from different mouse species were used for identification of exo- and endogenous wild-type mtDNAs in the mitomice. Results showed transmission of exogenous mtDNA, but not exogenous wild-type mtDNA, of M. m. domesticus to following generations through the female germ line. Complete elimination of exogenous wild-type mtDNA would be due to stochastic segregation, whereas transmission of exogenous mtDNA would be due to its smaller size leading to a propagational advantage. Tissues in mitomice of the F₃ generation carrying exogenous mtDNA showed protection from respiration defects until mtDNA accumulated predominantly. This protection from expression of mitochondrial dysfunction was attained with the help of endogenous wild-type mtDNA of M. spretus, since mitomice did not possess exogenous wild-type mtDNA of M. m. domesticus. These observations provide unambiguous evidence for the presence of interaction between exogenous mitochondria carrying mtDNA and endogenous mitochondria carrying M. spretus wild-type mtDNA.

---

IN yeast and plant cells, the idea of mitochondrial interaction has received support from the evidence for recombination between two mtDNA molecules derived from both parental germ cells (DUJON et al. 1974; BELLIARD et al. 1979). In mammalian species, however, the opportunity for coexistence of mtDNAs from both parents is likely inhibited by their strictly maternal inheritance (KANEDA et al. 1995; SHITARA et al. 1998, 2000, 2001). Since the mammalian mtDNA population is homoplasmic throughout individuals due to maternal inheritance, recombination between maternal mtDNA molecules with the same sequences would not be productive. Although cell fusion techniques can mix mtDNA molecules from different mammalian individuals within single somatic cell hybrids, extensive mtDNA recombination as observed in yeast and plant cells (DUJON et al. 1974; BELLIARD et al. 1979) was not detectable even after their long-term cultivation (HAYASHI et al. 1985).

However, evidence for interactions between mammalian mitochondria is provided by translational complementation in cultured somatic cells (HAYASHI et al. 1994; TAKAI et al. 1999; ONO et al. 2001). For example, fusion of two different types of respiration-deficient somatic cells caused by different pathogenic mutant mtDNAs from patients with mitochondrial diseases resulted in overall restoration of respiration defects in their somatic cell hybrids (TAKAI et al. 1999; ONO et al. 2001). Moreover, evidence for rapid merging of normal mitochondria with wild-type mtDNA and respiration-deficient mitochondria without mtDNA was obtained by introduction of normal mitochondria into mtDNA-less HeLa cells using cell fusion techniques (HAYASHI et al. 1994).

Recently, the occurrence of interaction between mitochondria was extended from the in vitro to the in vivo level by the use of mitomice (INOUE et al. 2000; NAKADA et al. 2001). They were generated by introduction of respiration-deficient mitochondria carrying a predominant amount of mutated mtDNA with a large deletion (mtDNA) and a residual amount of wild-type mtDNA from cultured mouse cells into mouse zygotes (INOUE et al. 2000). In the mitomice, expression of mitochondrial dysfunction was not observed in any mitochondria in any cells carrying as much as 60% mtDNA, suggesting the presence of interaction between exogenous respiration-deficient mitochondria with mtDNA and endogenous mitochondria with wild-type mtDNA and the resultant restoration of respiratory function throughout the mitochondria (NAKADA et al. 2001; HAYASHI et al. 2002).

However, Attardi and co-workers (ATTARDI et al. 2002) noted that the apparent rescue of mitomice from expression of mitochondrial defects could be explained by assuming intra-, but not inter-, mitochondrial interaction. In this case, a proportion of residual wild-type mtDNA preexisting in exogenous mitochondria has to increase preferentially for restoration of respiratory function, followed by elimination of endogenous mitochondria carrying wild-type mtDNA from mitomice. Although it is not obvious to assume such an exclusive increase of exogenous wild-type mtDNA to be predominant over endogenous wild-type mtDNA, this possibility could not be excluded completely. Therefore, experiments that could distinguish exogenous and endogenous wild-type mtDNAs had to be carried out for generation of mitomice before concluding the presence of an in vivo interaction of mammalian mitochondria.

In this study, we created mitomice using zygotes carrying mtDNA of a different mouse species, Mus spretus, so that both endogenous and exogenous wild-type mtDNAs in the mitomice could be distinguished. The results provided unambiguous evidence for the presence of the in vivo intermitochondrial interaction.

Cells and cell culture:

Cy4696 cybrids (INOUE et al. 2000) carrying 89.4 ± 2.3% mtDNA of M. m. domesticus were cultivated in RPMI1640 (Nissui Seiyaku, Tokyo) containing 10% fetal calf serum, 50 µg/ml uridine, and 0.1 mg/ml pyruvate. Uridine and pyruvate were supplemented, so that respiration-deficient Cy4696 cybrids caused by a predominant amount of mtDNA could grow (KING and ATTARDI 1989; INOUE et al. 1997).

Generation of mitomice carrying wild-type mtDNA of M. spretus and mtDNA of M. m. domesticus:

Zygotes of B6mtspr, which possess the nuclear genome of M. m. domesticus and the mitochondrial genome of M. spretus, were used as mtDNA recipients. Cytoplasts of the Cy4696 cybrids carrying 89.4 ± 2.3% mtDNA were used as mtDNA donors for generating mitomice. Introduction of mtDNA into B6mtspr zygotes was carried out as described previously (INOUE et al. 2000) with slight modifications. Briefly, pronuclear-stage zygotes were collected from oviducts of superovulated B6mtspr females at the age of 8–10 weeks after birth, and 10 Cy4696 cytoplasts were inserted into the perivitelline space of the zygotes with a piezo-driven micromanipulator. The cytoplasts were fused with the embryos by applying an electric pulse (3000 or 3750 V/cm, 10 msec) with a pre- and postpulse AC current (100 V/cm, 2 MHz, 30 sec each). After 24 hr cultivation, two-cell-stage embryos were transferred to the oviducts of pseudopregnant Jcl:ICR (Crea Japan, Tokyo) females.

Southern blot analysis:

Total DNA was extracted from cultured cells and tissues using a Pure Gene tissue kit (Gentra, Minneapolis). DNA (1 µg) was digested with the restriction enzyme BglII, and the resultant restriction fragments were separated in 0.6% agarose gel, transferred to a nylon membrane, and hybridized with alkaline phosphatase-labeled mouse mtDNA probes, which include nucleotide positions 1895–2762. This region was selected as a probe because of its high sequence similarity in M. m. domesticus and M. spretus mtDNAs. Probe labeling and signal detection were carried out as described in the protocols of the AlkPhos Direct (Amersham Pharmacia Biotech, Buckinghamshire, UK). For quantitation of wild-type mtDNA of M. spretus and mtDNA of M. m. domesticus, 10 replicate lanes were separately scanned and calculated by using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/) with control lanes with known amounts of mtDNAs.

PCR analysis:

Total DNA (300 ng) was used for PCR amplification. Three primer sets (11,927–11,946 and 12,388–12,365, 7587–7611 and 12,679–12,654, 7442–7469 and 7726–7696) were used for specific amplification of wild type and mtDNA of M. m. domesticus and wild-type mtDNA of M. spretus, respectively. Wild type and mtDNA of M. m. domesticus and wild-type mtDNA of M. spretus gave 462-, 397-, and 285-bp fragments, respectively. The cycle times were 30 sec of denaturation at 95°, 30 sec of annealing at 55°, and 60 sec of extension at 72° for 50 cycles.

Analyses of cytochrome c oxidase activity:

Estimation of cytochrome c oxidase (COX) activity was carried out by examining the rate of cyanide-sensitive oxidation of reduced cytochrome c (SELIGMAN et al. 1968). In histochemical analyses, hearts were excised from mitomice, and their 10-µm cryosections were stained for COX activity. COX electron micrographs were carried out as described (NAKADA et al. 2001) with slight modifications. Briefly, 25-µm cryosections were fixed in 2% glutaraldehyde in PBS for 10 min at 0°. Ultrathin sections, which were not stained with uranyl acetate and lead nitrate, were viewed directly with an H-7000 electron microscope (Hitachi, Tokyo).

Single-cell PCR:

Three serial cryosections (10 µm) of heart from mitomice possessing 85.2 ± 3.0% mtDNA were used for single-cell PCR. The first and third sections were stained with dimethylaminoazobenzene for COX activity, and COX-positive and COX-negative fibers in both sections were used for quantitative PCR analysis. Each cell in the second sections was dissected with a PRO-300 laser scissors (Cell Robotics, Albuquerque, NM). Quantification of M. m. domesticus mtDNA and M. spretus wild-type mtDNA were carried out using a TaqMan PCR reagent kit and an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA) under the conditions recommended by the manufacturer. The primer set specific for M. m. domesticus mtDNA was nucleotide positions 7697–7725 and 12,528–12,508. The reporter dye 6-carboxyfluorescein (FAM)-labeled TaqMan MGB probe (Applied Biosystems) specific for M. m. domesticus mtDNA was nucleotide positions 7750–7758 and 12,455–12,464. The primer set specific for M. spretus wild-type mtDNA was nucleotide positions 7442–7469 and 7726–7697. The reporter dye FAM and the quencher dye TAMRA-labeled probe was nucleotide positions 7490–7513.

Generation of mitomice by introduction of mtDNA into zygotes with M. spretus mtDNA:

Cy4696 cybrids carrying 89.4 ± 2.3% mtDNA of M. m. domesticus were enucleated and the resultant cytoplasts were used as mtDNA donors for generating mitomice expressing mitochondrial defects. Zygotes of the mouse B6mtspr strain possessing only M. spretus mtDNA were used as recipients, so that endogenous and exogenous wild-type mtDNAs could be identified (Figure 1). The cytoplasts were fused with zygotes by electrofusion and resultant two-cell-stage embryos were transferred to the oviducts of pseudopregnant Jcl:ICR females for obtaining F₀ mice.

View larger version (70K): In this window In a new window Download PPT slide   FIGURE 1.— Identification of M. spretus wild-type mtDNA and of M. m. domesticus wild type and mtDNA. spretus, wild-type mtDNA of M. spretus; domesticus, wild-type mtDNA of M. m. domesticus; , mtDNA of M. m. domesticus. Lanes spr, dom, , and mix represent DNA samples prepared from tails of B6mtspr strain mice, C57BL/6J strain mice, Cy4696 cybrids with 89.4 ± 2.3% mtDNA, and their mixtures, respectively. Lanes F0, F1, F2, and F3 represent mtDNA prepared from tails of F0, F1, F2, and F3 progeny, respectively. (A) Southern blot analysis of BglII fragments. Wild-type mtDNA of M. spretus gave three BglII fragments (8.3, 5.1, and 2.9 kbp), and only one fragment with 5.1 kbp was detectable by the probe (nucleotide positions 1895–2762) used in this experiment. Wild-type mtDNA and mtDNA of M. m. domesticus gave 16.3 and 11.6 kbp fragments, respectively. (B) Specific detection of wild-type mtDNA of M. spretus and of wild type and mtDNA of M. m. domesticus by PCR analysis. Due to high sensitivity of PCR amplification, a small amount of wild-type mtDNA of M. m. domesticus observed by PCR analysis (B) was not detected by Southern blot analysis (A).

 
Total DNA was prepared from tail biopsy samples of F₀ mice. Of 32 DNA samples, 20 showed PCR signals for the presence of both exogenous wild type and mtDNA of M. m. domesticus introduced from Cy4696, whereas the remaining 12 F₀ mice did not possess exogenous mtDNAs (Table 1). Then 8 F₀ females with PCR signals for the presence of exogenous mtDNAs in their tails were selected as mothers for obtaining the F₁ generation. We subsequently examined whether exogenous wild type and mtDNA of M. m. domesticus can be transmitted through the female germ line to the F₁ generation. Of 130 F₁ mice, 4 showed signals for mtDNA by both Southern blot and PCR analyses, whereas no mice gave signals for exogenous wild-type mtDNA of M. m. domesticus even by PCR analysis. These observations suggest that transmission of exogenous mtDNAs from the F₀ to the F₁ generation was limited to mtDNA (Table 1).

View this table: In this window In a new window   TABLE 1 Examination of exogenous wild type and mtDNA of M. m. domesticus in tails of F0–F3 mice by PCR analysis

 
Then we selected an F₂ female carrying 37.1 ± 3.0% mtDNA in its tail as a mother and obtained four F₃ mice, one female and three males. All DNA samples prepared from tails of F₃ mice gave PCR signals for the presence of mtDNA, while no signals for exogenous wild-type mtDNA were observed, suggesting exclusive transmission of exogenous mtDNA, but not exogenous wild-type mtDNA to following generations (Table 1). For quantitation of the amount of mtDNA, we carried out Southern blot analysis and found that the tails of four F₃ mice contained 10.5 ± 4.4, 45.1 ± 2.2, 45.2 ± 2.5, and 65.3 ± 2.7% mtDNA.

Examination of intermitochondrial interaction using mitomice with mtDNA:

Hearts excised from three F₃ male mice carrying 45.1 ± 2.2, 45.2 ± 2.5, and 65.3 ± 2.7% mtDNA in their tails were used for examination of their mtDNA composition by Southern blot analysis (Figure 2A). The results showed that they possessed 57.5 ± 2.4, 60.6 ± 3.4, and 85.2 ± 3.0% mtDNA, respectively. Moreover, complete absence of exogenous wild-type mtDNA from the F₃ hearts was observed in Southern blot (Figure 2A) and PCR analyses (Figure 2B).

View larger version (52K): In this window In a new window Download PPT slide   FIGURE 2.— Transmission of exogenous mtDNA, but not of wild-type mtDNA, of M. m. domesticus in hearts from F3 mice. Lane 1, mixtures of DNA samples including wild-type mtDNA of M. spretus, mtDNA, and wild-type mtDNA of M. m. domesticus. Lanes 2 and 3, DNA samples prepared from hearts of two F3 mitomice with 45.2 ± 2.5 and 65.3 ± 2.7% mtDNA, respectively, in their tails. (A) Southern blot analysis of BglII fragments. (B) Specific detection of wild-type mtDNA of M. spretus and of wild type and mtDNA of M. m. domesticus by PCR analysis.

 
We selected two F₃ hearts carrying 60.6 ± 3.4 and 85.2 ± 3.0% mtDNA for further examination of COX activity by COX histochemistry (Figure 3) and COX electron micrographs (Figure 4). Histochemical analysis of COX activity in hearts carrying 60.6 ± 3.4% mtDNA showed that all cardiac cells possessed COX activity: no COX-negative cells were observed (Figure 3). Moreover, COX electron micrographs, which can identify COX activity at the individual mitochondrial level, clearly showed that no mitochondria lost COX activity, even though the heart possessed 60.6 ± 3.4% mtDNA (Figure 4). Considering that exogenous wild-type mtDNA was not present in hearts carrying 60.6 ± 3.4% exogenous mtDNA, at least 60% mitochondria should be COX negative in the absence of interaction between COX-negative exogenous mitochondria and normal endogenous mitochondria. Therefore, the observations in Figure 4 could not be obtained in the absence of mitochondrial interaction.

View larger version (149K): In this window In a new window Download PPT slide   FIGURE 3.— COX histochemistry of longitudinal sections of hearts carrying 0% (left), 60.6 ± 3.4% (middle), and 85.2 ± 3.0% (right) mtDNA. Hearts consist of mononuclear cardiac muscle fibers (cardiac cells) joined end to end by intercalated discs (arrowheads). All cardiac cells were COX positive in heart carrying 60.6 ± 3.4% mtDNA, whereas heart with 85.2 ± 3.0% mtDNA consisted of COX-positive and COX-negative fibers. Bar, 30 µm.

 

View larger version (123K): In this window In a new window Download PPT slide   FIGURE 4.— COX electron micrographs of longitudinal sections of hearts carrying 0% (left), 60.6 ± 3.4% (middle), and 85.2 ± 3.0% mtDNA (right). Arrowheads indicate intercalated discs. Hearts carrying 60.6 ± 3.4% mtDNA consist of all COX-positive cells, and all individual mitochondria in each cell were COX positive. On the other hand, hearts carrying 85.2 ± 3.0% mtDNA consist of cells with only COX-positive mitochondria or cells with only COX-negative mitochondria. No mosaic distribution of COX-positive and COX-negative mitochondria within single cardiac cells was observed, irrespective of whether hearts contained 60.6 ± 3.4% or 85.2 ± 3.0% mtDNA. Bar, 1 µm.

 
On the other hand, examination of a heart carrying 85.2 ± 3.0% mtDNA gave apparently different features of COX histochemistry and COX electronmicrographs, but the results again supported the presence of interaction between mitochondria. The heart consisted of COX-positive and COX-negative cardiac cells (Figure 3). Single-cell PCR analysis of the serial cross sections showed that COX-positive and COX-negative cells possessed 67.6 ± 12.9% (n = 22) and 92.2 ± 3.7% (n = 18) mtDNA, respectively. COX electron micrographs showed a uniform distribution of either COX-positive or COX-negative mitochondria within single cardiac cells (Figure 4). In the absence of mitochondrial interaction, 85% of the mitochondria would be COX negative in hearts carrying 85.2 ± 3.0% mtDNA. However, COX electron micrographs showed a homogeneous distribution of COX activity throughout mitochondria, and no mosaic distribution of COX-positive and COX-negative mitochondria within any single cardiac cell was observed (Figure 4).

We suggest that cardiac cells carrying <85% mtDNA retained normal mitochondrial translation and normal COX activity by complementing tRNAs transcribed from tRNA genes missing in mtDNA. On the other hand, cardiac cells carrying >85% mtDNA progressively lost COX activity due to an insufficient amount of the tRNAs required for normal mitochondrial translation. Therefore, the translation phase may be shifted from complementation to competition of the tRNAs in cells with >85% mtDNA, resulting in progressive inhibition of overall mitochondrial translation and resultant reduction of COX activity.

These observations consistently suggest the presence of extensive in vivo interaction between exogenous mitochondria carrying mtDNA and endogenous mitochondria carrying wild-type mtDNA in the mitomice.

Our recent study (NAKADA et al. 2001) provided evidence for the presence of intermitochondrial interaction using mitomice (INOUE et al. 2000), which were generated by the introduction of COX-negative mitochondria carrying 88% mtDNA and residual 12% wild-type mtDNA into zygotes carrying 100% wild-type mtDNA. All mitochondria in tissues with mtDNA showed normal COX activity until it accumulated to 80%. Moreover, no coexistence of COX-positive and COX-negative mitochondria within single cells was observed (NAKADA et al. 2001). These observations could be explained by the occurrence of in vivo intermitochondrial complementation by the extensive and continuous interchange of genetic materials between exogenous mitochondria carrying mtDNA and host mitochondria carrying wild-type mtDNA.

However, ATTARDI et al. (2002) noted that our observations (NAKADA et al. 2001) could be explained in the absence of interaction between mitochondria by assuming simultaneous occurrence of the following two events: first, clonal expansion of exogenous COX-negative mitochondria carrying mtDNA and the resultant elimination of most endogenous mitochondria, and second, an increase in the amount of wild-type mtDNA preexisting in exogenous mitochondria with the resultant recovery of COX activity in the overall mitochondria of mitomice. In these cases, protection of mitomice from mitochondrial defects was due to the interaction within exogenous mitochondria, but not to interaction between exogenous and endogenous mitochondria. Although the occurrence of these phenomena seems unlikely, experiments that can identify exogenous and endogenous wild-type mtDNA are required to exclude the possibility of clonal expansion of exogenous mitochondria and to draw a general conclusion with respect to the in vivo interaction between mitochondria.

In this study, we generated mitomice using zygotes of the mouse B6mtspr strain carrying mtDNA from a different mouse species, M. spretus, so that endogenous wild-type mtDNA could be distinguished from exogenous wild-type mtDNA by a restriction endonuclease. We obtained mitomice exclusively carrying endogenous M. spretus wild-type mtDNA and exogenous mtDNA, but not exogenous wild-type mtDNA of M. m. domesticus, suggesting the absence of clonal expansion of exogenous mitochondria with increased amount of exogenous wild-type mtDNA (Table 1). Moreover, none of the individual mitochondria in the mitomice expressed respiration defects until exogenous mtDNA accumulated predominantly, providing unequivocal evidence for the in vivo interaction between endogenous mitochondria with M. spretus mtDNA and exogenous COX-negative mitochondria with mtDNA.

In these experiments, transmission of exogenous mtDNA to following generations was observed in F₀–F₃ generations, whereas exogenous wild-type mtDNA was not. The rapid elimination of exogenous wild-type mtDNA of M. m. domesticus from female germ cells in F₀ mice could be caused by stochastic segregation, since a very small amount of exogenous mtDNAs could be introduced into zygotes by electrofusion techniques. Similar elimination of a small amount of exogenous mtDNA was observed when the transmission profile of paternal mtDNA in sperm introduced into zygotes on fertilization was examined (KANEDA et al. 1995; SHITARA et al. 1998). In intraspecies crossing, paternal mtDNA was completely eliminated from zygotes within 24 hr after fertilization. On the other hand, its leakage was observed exclusively in interspecies crossing (KANEDA et al. 1995). However, transmission of the leaked paternal mtDNA from interspecies hybrid mice to next generations through female germ cells was a rare phenomenon, if it occurred (SHITARA et al. 1998). Probably, the proportion of sperm-derived mtDNA was extremely small (<0.1%), so that the leaked paternal mtDNA disappeared very rapidly by stochastic segregation. Rapid and random mtDNA segregation was also reported in mice carrying heteroplasmic mtDNAs with neutral polymorphic mutations (JENUTH et al. 1996).

On the other hand, transmission of exogenous mtDNA through female germ cells to the following generations was observed (Figure 1). The escape of exogenous mtDNA from elimination may be due, at least in part, to its smaller size, which could give replication and propagational advantages over endogenous and exogenous wild-type mtDNAs. Therefore, even when the amount of mtDNA introduced into zygotes was extremely small, it could be transmitted and accumulate in tissues of F₃ progeny (Table 1).

The presence of intermitochondrial interaction was supported by our previous reports (HAYASHI et al. 1994; TAKAI et al. 1999; ONO et al. 2001), which provided evidence for the in vitro interaction between mitochondria by the fusion of cultured cells. Therefore, it can be generalized that extensive and continuous interchange of genetic materials occurs between mitochondria in cells both in vivo and in vitro, resulting in metabolic complementation of mitochondria to avoid direct expression of mtDNA mutations as respiration defects.

Therefore, these observations do not support the conventional "mitochondrial theory of aging" (LINNANE et al. 1989; WALLACE 1992; SHIGENAGA et al. 1994; NAGLEY and WEI 1998), which proposes that age-associated mitochondrial dysfunction appears as the consequence of age-associated accumulation of somatic mutations in the mtDNA population. This hypothesis was supported by the evidence that various pathogenic mtDNA mutations accumulated with age in mitotic (MICHIKAWA et al. 1999) and postmitotic tissues (CORRAL-DEBRINSKI et al. 1992; SOONG et al. 1992). However, our previous reports provided direct evidence for the functional integrity of mtDNAs from mitotic (ISOBE et al. 1998) and postmitotic tissues (ITO et al. 1999) of aged subjects using mtDNA transfer techniques. For example, nuclear transfer experiments from mtDNA-less HeLa cells to human skin fibroblasts showed that nuclear-recessive mutations, but not mtDNA mutations, are responsible for the age-associated mitochondrial dysfunction observed in the fibroblasts (ISOBE et al. 1998). Moreover, introduction of mtDNA of autopsied brain tissues from aged human subjects into mtDNA-less HeLa cells resulted in complete restoration of mitochondrial respiratory function in mtDNA repopulated HeLa cells (cybrids), although brain donors possessed mtDNAs with various pathogenic mutations, which were transferred to the cybrids (ITO et al. 1999). These observations could be explained by intermitochondrial complementation, which rescues aged tissues from direct expression of mtDNA mutations as age-associated mitochondrial dysfunction.

This work was supported in part by a grant for a Research Fellowship from the Japan Society for Promotion of Science for Young Scientists to A.S.; by a grant for the Hayashi project of Tsukuba Advanced Research Alliance, University of Tsukuba; and by Grants-in-Aid for Scientific Reseach from the Ministry of Education, Science, Sports and Culture of Japan to K.N. and J.-I.H.

ATTARDI, G., J. A. ENRIQUEZ and J. CABEZAS-HERRERA, 2002 Inter-mitochondrial complementation of mtDNA mutations and nuclear context. Nat. Genet. 30: 360.[CrossRef][Medline]

BELLIARD, G., F. VADEL and G. PELLETIER, 1979 Mitochondrial recombination in cytoplasmic hybrids of Nicotiana tabacum by protoplast fusion. Nature 281: 401–403.[CrossRef]

CORRAL-DEBRINSKI, M., T. HORTON, M. T. LOTT, J. M. SHOFFNER, M. F. BEAL et al., 1992 Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat. Genet. 2: 324–329.[CrossRef][Medline]

DUJON, B., P. P. SLONIMSKI and L. WEILL, 1974 Mitochondrial genetics IX: a model for recombination and segregation of mitochondrial genomes in Saccharomyces cerevisiae. Genetics 78: 415–437.[Free Full Text]

HAYASHI, J.-I., Y. TAGASHIRA and M. C. YOSHIDA, 1985 Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells. Exp. Cell Res. 160: 387–395.[CrossRef][Medline]

HAYASHI, J.-I., M. TAKAMITSU, Y. GOTO and I. NONAKA, 1994 Human mitochondria and mitochondrial genome function as a single dynamic cellular unit. J. Cell Biol. 125: 43–50.[Abstract/Free Full Text]

HAYASHI, J.-I., K. NAKADA and T. ONO, 2002 Reply to ‘Inter-mitochondrial complementation of mtDNA mutations and nuclear context’. Nat. Genet. 30: 361.[CrossRef]

INOUE, K., S. ITO, D. TAKAI, A. SOEJIMA, H. SHISA et al., 1997 Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations. J. Biol. Chem. 272: 15510–15515.[Abstract/Free Full Text]

INOUE, K., K. NAKADA, A. OGURA, K. ISOBE, Y. GOTO et al., 2000 Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes. Nat. Genet. 26: 176–181.[CrossRef][Medline]

ISOBE, K., S. ITO, H. HOSAKA, Y. IWAMURA, H. KONDO et al., 1998 Nuclear-recessive mutations of factors involved in mitochondrial translation are responsible for age-related respiration deficiency of human skin fibroblasts. J. Biol. Chem. 273: 4601–4606.[Abstract/Free Full Text]

ITO, S., S. OHTA, K. NISHIMAKI, Y. KAGAWA, R. SOMA et al., 1999 Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer's disease. Proc. Natl. Acad. Sci. USA 96: 2099–2103.[Abstract/Free Full Text]

JENUTH, J. P., A. C. PETERSON, K. FU and E. A. SHOUBRIDGE, 1996 Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nat. Genet. 14: 146–151.[CrossRef][Medline]

KANEDA, H., J.-I. HAYASHI, S. TAKAHAMA, C. TAYA, K. F. LINDAHL et al., 1995 Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc. Natl. Acad. Sci. USA 92: 4542–4546.[Abstract/Free Full Text]

KING, M. P., and G. ATTARDI, 1989 Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246: 500–503.[Abstract/Free Full Text]

LINNANE, A. W., S. MAZUKI, T. OZAWA and M. TANAKA, 1989 Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1: 642–645.[CrossRef][Medline]

MICHIKAWA, Y., F. MAZZUCCHELLI, N. BRESOLIN, G. SCARLATO and G. ATTARDI, 1999 Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication. Science 286: 774–779.[Abstract/Free Full Text]

NAGLEY, P., and Y.-H. WEI, 1998 Ageing and mammalian mitochondrial genetics. Trends Genet. 14: 513–517.[CrossRef][Medline]

NAKADA, K., K. INOUE, T. ONO, K. ISOBE, A. OGURA et al., 2001 Inter-mitochondrial complementation: mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA. Nat. Med. 7: 934–940.[CrossRef][Medline]

ONO, T., K. ISOBE, K. NAKADA and J.-I. HAYASHI, 2001 Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria. Nat. Genet. 28: 272–275.[CrossRef][Medline]

SELIGMAN, A. M., M. J. KARNOVSKY, H. L. WASSERKRUG and J. S. HANKER, 1968 Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J. Cell Biol. 38: 1–14.[Abstract/Free Full Text]

SHIGENAGA, M. K., T. M. HAGAN and B. N. AMES, 1994 Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. USA 91: 10771–10778.[Abstract/Free Full Text]

SHITARA, H., J.-I. HAYASHI, S. TAKAHAMA, H. KANEDA and H. YONEKAWA, 1998 Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage. Genetics 148: 851–857.[Abstract/Free Full Text]

SHITARA, H., H. KANEDA, A. SATO, K. INOUE, A. OGURA et al., 2000 Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis. Genetics 156: 1277–1284.[Abstract/Free Full Text]

SHITARA, H., H. KANEDA, A. SATO, K. IWASAKI, J.-I. HAYASHI et al., 2001 Non-invasive visualization of sperm mitochondria behavior in transgenic mice with introduced green fluorescent protein (GFP). FEBS Lett. 500: 7–11.[CrossRef][Medline]

SOONG, N. W., D. R. HINTON, G. CORTOPASSI and N. ARNHEIM, 1992 Mosaicism for a specific somatic mitochondrial DNA mutation in adult human brain. Nat. Genet. 2: 318–323.[CrossRef][Medline]

TAKAI, D., K. ISOBE and J.-I. HAYASHI, 1999 Transcomplementation between different types of respiration-deficient mitochondria with different pathogenic mutant mitochondrial DNAs. J. Biol. Chem. 274: 11199–11202.[Abstract/Free Full Text]

WALLACE, D. C., 1992 Mitochondrial genetics: A paradigm for aging and degenerative diseases? Science 256: 628–632.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Sato, T. Kono, K. Nakada, K. Ishikawa, S.-I. Inoue, H. Yonekawa, and J.-I. Hayashi Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation PNAS, November 15, 2005; 102(46): 16765 - 16770. [Abstract] [Full Text] [PDF]

International Access Link

Online ISS 1091-6490
Copyright 2008 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu