Two distinct types of mitochondrial DNA segregation in mouse-rat hybrid cells. Stochastic segregation and chromosome-dependent segregation

Two distinct patterns of mitochondrial DNA (mtDNA) segregation were found in different mouse-rat hybrid cell lines. On mouse-rat hybrid cell line, H2, retained complete sets of chromosomes and mtDNAs of both mouse and rat. Even after cultivation for about one year after cloning, the H2 cell population still retained both parental mtDNAs. However, when mtDNAs of H2 subclones were examined, it was found that some individual cells in the H2 cell population contained only mouse or only rat mtDNA, although they still retained complete sets of both kinds of parental chromosomes. This type of mtDNA segregation, named stochastic segregation, is bidirectional and may be caused by the repetition of random sharing of mouse and rat mtDNAs with daughter cells. This segregation occurred spontaneously during long-term cultivation. The second type of mtDNA segregation, named chromosome-dependent segregation, was found in the other mouse-rat hybrid cell lines that segregated either mouse or rat chromosomes. In these hybrid cells, chromosomes and mtDNA of the same species co-segregated. This second type of segregation is unidirectional. The types of mtDNA segregation appear to depend on the stability of the parental chromosomes in the hybrid cells. When both mouse and rat chromosomes retain stably, mtDNA shows stochastic segregation. On the contrary, when either species of chromosomes is segregated from the cells, mtDNA shows chromosome-dependent segregation.     

Stability of parental mitochondrial DNA species and expression of nuclear ribosomal RNA genes in mouse-rat hybrid cells

Mouse-rat hybrid somatic cells were isolated by fusion of chloramphenicol-sensitive (CAP^s) mouse fibroblast cells with hypoxanthine-guanine-phosphoribosyltransferase-deficient (HGPRT⁻) and CAP-resistant (CAP^r) rat myoblast cells and selected with hypoxanthine-aminopterin-thymidine (HAT) and CAP. Restriction endonuclease cleavage patterns showed that both mouse and rat mitochondrial DNAs (mtDNAs) were present in the hybrid cells and that the amount of rat mtDNA was one-quarter that of mouse mtDNA, even after cultivation for 3 months in the presence of CAP. Nuclear ribosomal RNA (rRNA) genes of mouse and rat were shown to be expressed stably in the hybrid cells by homochromatography fingerprinting of RNase T1 digests. The genetic compatibility between mouse and rat chromosomes in the mouse-rat hybrid cells assures retention of both parental chromosomes, and this may be responsible for the expression of both parental rRNA genes, and for the retention of both parental mtDNAs in the hybrid cells.     

Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells

Recombination of mammalian mitochondrial DNA (mtDNA) was examined using mouse X rat somatic cell hybrid clones and rat cybrid clones. The mouse X rat hybrids were isolated by fusion of chloramphenicol-sensitive (CAPs) mouse and CAP-resistant (CAPr) rat cells. The rat cybrids were isolated by fusion of rat cells with type B mtDNA and enucleated cells with type A mtDNA. Genetic and physical analyses showed that the mtDNAs of the hybrids and cybrids were simple mixtures of the two parental mtDNAs except in the following two cases: One was subclone H2-9 of mouse X rat hybrids, which was CAPr even though mtDNA from the CAPs mouse parent was predominantly retained. The other was rat cybrid subclones, Y12-24 and -61, which showed specific loss of one Hinf I fragment of type B mtDNA, B10. These observations suggest that, in contrast to the case with plant mtDNA, recombination of mammalian mtDNA occurs rarely, if at all.     

Effect of mitochondrial DNA composition on the cellular properties of interspecific hybrid cells

Four subclones with single species of mitochondria and three subclones with both parental mitochondria were isolated from a mouse-rat hybrid cell line H2. The effects of the coexistence of different species of mitochondria on cellular properties were examined in these clones. Growth properties were studied by comparing the plating efficiencies and doubling times. The numbers of growing colonies and the doubling times of all the subclones were found to be almost the same, indicating that these growth properties were not affected by the presence of both mouse and rat mitochondria within the cells. The correlation between the expression of chloramphenicol (CAP)-resistance and the relative contents of mtDNA of CAP-resistant (CAP^r) rat and CAP-sensitive (CAP^s) mouse parent cells in the subclones were also examined. The expression of CAP resistance was measured as the relative plating efficiency. Subclones with a high content of mtDNA from CAP^r rat parent cells showed high relative plating efficiency.     

Analysis of mitochondrial DNA species in interspecific hybrid somatic cells using restriction endonucleases. Identification of recombinant mtDNA molecules

Interspecific hybrid cells were isolated by fusion between thymidine kinase-deficient (TK⁻) mouse B82 cells and hypoxanthine-guanine-phosphoribosyl-transferase-deficient (HGPRT⁻) rat L6TG cells, and cultivating them in selective medium with hypoxanthine-aminopterin-thymidine (HAT). Karyo-type analysis revealed that they contained both mouse and rat chromosomes. Mitochondrial DNA (mtDNA) species of the hybrid cells were identified by digesting them with three kinds of restriction endonucleases, Hae II, EcoR I and Hpa II. Their restriction endonuclease cleavage patterns indicated that a portion of the mtDNAs was of mouse parent cell origin, while the remainings were recombinant molecules, i.e., part of the rat mtDNA sequence could be detected, but not whole rat mtDNA. The molecular weights of hybrid cell mtDNAs were calculated to be almost the same as that of the parent cells (˜10⁷ D).     

Induction of supermelanin synthesis and morphological changes in interspecific reconstituted cells and its reversal by tumor promoter

Chloramphenicol-resistant (CAPr) reconstituted cells and cybrids were isolated by fusion of karyoplasts (or intact cells) of mouse amelanotic melanoma B16 cells with cytoplasts of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) -deficient, CAPr rat myoblastic cells, L6TG.CAPr, and double selection in HAT medium containing CAP. Reconstituted cells or cybrids exhibited unique cellular arrangement, and about one third of the isolated clones expressed high tyrosinase activity and marked melanin synthesis, although the parental mouse cells expressed low tyrosinase activity and the parental rat cells did not express tyrosinase activity. These phenotypic changes have been stable for more than a year. The phenotypic reversions of these clonal cells were induced by treatment with a tumor promoter. There were changes in the morphology of the treated cells to that of the mouse B16 cells and extinction of tyrosinase activity and melanin synthesis in pigmented clonal cells. These phenotypic changes and reversions induced by a promoter were repeatedly reversible.     

Cytoplasmic inheritance in mammalian tissue culture cells

Summary A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human × CAP-S mouse) occurred either at a moderate frequency and were stable or at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This work was supported by U.S.P.H.S. research grants GM-18186, GM-1948 and GM-21024 (to J. M. E.), and N.I.H. postdoctoral fellowship No. 1 F22 GM-02655 (to D. C. W.).     

Cytoplasmic inheritance in mammalian tissue culture cells.

A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human X CAP-S mouse) occurred either at a moderate frequency and were stable at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.     

Histone expressions in mouse-rat somatic reconstituted cells

A critical analysis of histone expression was performed on the four interspecific and the two intraspecific reconstituted cells formed between karyoplast from mouse B16 cells and the cytoplast from rat cells (L6TG.CAPr) or mouse cells (B82.CAPr). All the reconstituted cells had the same pattern of mouse histones and the same amount of mouse-specific H2B. 2 histone as that of mouse nuclear donor cells. A hybrid between B16 and L6TG.CAPr contained both mouse and rat-specific H1b subtypes, whereas no rat-specific H1b was detected in the interspecific reconstituted cells. In both intra- and interspecific reconstituted cells, the proportion of H1b content was lower than that of B16 cells but that of H1 degree was higher, indicating that the mouse H1 patterns from these cells slightly resembled the pattern of slower growing and differentiated cytoplast donor cells. As an effect of the tumor promoter, the H1 pattern tended to revert to that of the nuclear donor cells in agreement with the phenotypic reversion, without any significant change in cell growth.     

Fate of parental mitochondria in embryonic stem hybrid cells

When hybrid cells are created, not only nuclear genomes of parental cells unite but their cytoplasm as well. Mitochondrial DNA (mtDNA) is a convenient marker of cytoplasm allowing one to gain insight into the organization of hybrid cell cytoplasm. We analyzed the parental mtDNAs in hybrid cells resulting from fusion of Mus musculus embryonic stem (ES) cells with splenocytes and fetal fibroblasts of DD/c mice or with splenocytes of M. caroli. Identification of the parental mtDNAs in hybrid cells was based on polymorphism among the parental mtDNAs for certain restrictases. We found that intra- and inter-specific ES cell-splenocyte hybrid cells lost entirely or partially mtDNA derived from the somatic partner, whereas ES cell-fibroblast hybrids retained mtDNAs from both parents in similar ratios with a slight bias. The lost of the "somatic" mitochondria by Es-splenocyte hybrids implies non-random segregation of the parental mitochondria as supported by a computer simulation of genetic drift. In contrast, ES cell-fibroblast hybrids show bilateral random segregation of the parental mitochondria judging from analysis of mtDNA in single cells. Preferential segregation of "somatic" mitochondria does not depend on the differences in sequences of the parental mtDNAs but depends on replicative state of the parental cells.     

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (ρ°) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA^Arg genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA MboI polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient’s brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.     

Isolation and characterization of intraspecific cybrids : Effect of mitochondrial DNA on their cellular properties

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific beta-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.     

Clonal isolation and characterization of myoblast-like reconstituted cells formed by fusion of karyoplasts from mouse teratocarcinoma cells with rat myoblast cytoplasts

To examine the roles of the cytoplasms of differentiated somatic cells on nuclear gene expression, reconstituted cells (RC-cells) were isolated clonally by fusing karyoplasts (isolated nuclei) from neomycin-resistant mouse teratocarcinoma PCC4-neor cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L6TG.CAPr cells, and after double selection in the medium containing 400 micrograms/ml of neomycin and 100 micrograms/ml of CAP (G418 plus CAP medium). The RC-cells were characterized by the presence of two genetic markers, neomycin- and CAP-resistance, by the absence of latex beads which had incorporated into karyoplast donor PCC4-neor cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neor cells. In contrast to the teratocarcinoma cybrids previously isolated, all the isolated RC-clones expressed myoblast-like morphologies of three types. The phenotypic expression of these RC-cells was compared with that of PCD-1 cells, a teratocarcinoma-derived myoblast line. RC-cells and PCD-1 cells did not express alkaline phosphatase (ALPase) activity while parental PCC4-neor expressed it strongly. After induction of myogenic differentiation by treatments with excess thymidine and conditioned medium, two clones were capable of forming short multinucleated cells. The protein synthetic patterns of RC-cells analysed by two-dimensional polyacrylamide gel were different from PCC4-neor cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed alpha-tropomyosin, and contained 10 nm filaments, characteristic markers of early myogenic cells. These results suggest that the RC-cells are myoblast-like cells, that a few of them maturate to partially differentiated myogenic cells, that the rat myoblast cytoplasm contains regulatory factor(s) able to determine the myogenic cell lineage of the undifferentiated stem cells, and that this factor is continuously expressed in these myoblasts.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicol-resistant (CAPR) human x mouse hybrids and one CAPR cybrid derived from CAPR HeLa cells and CAPS mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with 32P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [32P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited "reverse" chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human-mouse hybrid is to retain human mitochondrial DNA.     

Recombination in yeast mitochondrial DNA

When haploid yeast strains containing mitochondrial DNAs (mtDNAs) of different buoyant densities are mated, the resulting zygotes contain a mixed population of mitochondria and mitochondrial DNAs. During vegetative growth of diploid cells formed from such a cross between a petite strain with mtDNA of density 1.677 g cm^?3 and a respiratory competent strain with mtDNA of density 1.684 g cm^?3, mtDNAs with intermediate buoyant densities are obtained. Virtually all newly synthesized mtDNA in diploid ρ^? progeny has the intermediate buoyant density. Therefore, within 2 generations of growth of the diploid cells, the intermediate buoyant density species predominate. In crosses between a respiratory competent strain and other petite strains with different values of genetic suppressiveness, it was found that the amount of recombination yielding mtDNAs of intermediate buoyant densities roughly parallels the degree of suppressiveness. Individual clones of respiratory deficient cells from such crosses were also isolated to confirm that stable mtDNAs with intermediate buoyant densities were obtained. Thus, it is apparent that some form of recombination takes place within the mtDNAs of yeast cells that results in stable mtDNA species.     

Segregation of mitochondrial DNA in human somatic cell hybrids

Summary The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and immortal). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to ³²P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

The Use of Hybrid Somatic Cells as an Approach to Mitochondrial Genetics in Animals

We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time.The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are most likely covalent bonds. This linkage may be described as "recombination" of mtDNA sequences from two different animals.     

Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells. I: Fusion with mouse cell lines

The segregation of cytoplasmically inherited chloramphenicol (CAP) resistance in mouse cells was investigated in fusions between CAP-resistant cells or cytoplasts (enucleated cells) and CAP-sensitive cells of varying tissue origin. All hybrids formed in cell-cell fusions were initially CAP-resistant, indicating that CAP resistance is dominant. Hybrids from fusions of cells of the same tissue origin (homologous) were stably CAP-resistant, whereas the hybrid population from fusions of different origins (heterologous) showed a rapid diminution of average CAP resistance. Individual hybrid clones from these heterologous fusions also showed an overall loss of CAP resistance, and a wide variation in CAP resistance which is consistent with a large number of genetic determinants (possibly mitochondrial DNA molecules) contributing to the CAP phenotype. Similar results were obtained from cytoplast-cell fusions, so the observed CAP segregation is not the result of nuclear-nuclear interactions. This segregation of CAP resistance constitutes a second criterion of cytoplasmic inheritance in mammalian cells.     

Mitochondrial genomes in intraspecies mammalian cell hybrids display codominant or dominant/recessive behavior

A unique type of nonstochastic mitochondrial DNA (mtDNA) segregation was found in mammalian cells. In human cell hybrids isolated from the fusion of HeLa cells with 23, GM639, A549, or 293 cells, HeLa mtDNA was always lost from the hybrids, whereas both parental mtDNAs were maintained in hybrids of HeLa X 143BTK-. Similar phenomena were observed in mouse cell hybrids isolated by the fusion of cells with different mtDNA types. Types 1, 2, and 3, can be distinguished from each other by restriction fragment-length polymorphisms. The mouse cell hybrids between cells with type 1 and type 2 mtDNA always lost type 2 mtDNA, whereas the hybrids between cells with type 2 and type 3 mtDNA retained both types stably. These observations suggest that either a codominant or a dominant/recessive relationship may be present in intraspecies mitochondrial genomes of human and mouse cells. When the mitochondrial genomes in cell hybrids are codominant, stochastic segregation occurs while nonstochastic segregation occurs when they are in the dominant/recessive relationship. These concepts may help elucidate organelle heredity in animals.