Jun-Ichi Hayashi

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.

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.

Identification of mitochondrial DNA species in interspecific cybrids and reconstituted cells using restriction endonuclease

1. Introduction Genes for mitochondrial proteins are known to be encoded in both nuclear DNA and mitochondrial DNAs (mtDNAs). So, it is important to elucidate whether the nucleus and mitochondria can function in harmony in cytoplasmic hybrids (cybrids) and reconstituted cells in which the nucleus and mito- chondria are of different species. Transfer of chloram- phenicol-resistant (CApr) cytoplasm to whole cells of different species was extremely difficult [ 11; this may be due to the incomplete cooperation of the products of nuclear DNA and mtDNAs of different species [ 11. However, viable interspecific reconstituted cells could be isolated by the fusion of human karyoplasts to CAP’ mouse cytoplasts [ 21. mtDNA species of the cells were identified by a difference in buoyant den- sities of the mtDNAs of the parent cells [2]. However, this identification technique is applicable only to mtDNA species having buoyant densities sufficiently different to be clearly distinguished. We developed another procedure for identification of mtDNA species in culture cells using restriction endonuclease. This procedure has many advantages: (i) It requires <O.l ml packed cells (~2 X 10’ cells) without labelling; (ii) The cleavage patterns of the mtDNAs are species- specific [3], and even intraspecifically different mtDNAs can be clearly distinguished [4-61; (iii) Sequence identification of mtDNAs is unambig- uous. Using this procedure we examined the mtDNA sequences of CAP’ interspecific cybrids, or reconsti- tuted cells, obtained by fusion of rat CAPr cytoplasts to hypoxanthine-aminopterin-thymidine-resistant (HAT? mouse whole cells or karyoplasts after culti-

Mitochondrial DNA analysis of mouse-rat hybrid cells: Effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAPR) and CAP-sensitive (CAPS) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAPR mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAPR parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAPR parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1–4 times that of rat mtDNAs in the mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Abstract Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al. , 1980, Biochem. Biophys. Res. Commun. 92 , 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg 2+ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg 2+ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg 2+ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg 2+ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg 2+ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg 2+ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg 2+ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.

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.

Polymorphisms of mitochondrial DNAs in Norway rats (Rattus norvegicus): Cleavage site variations and length polymorphism of restriction fragments

SummaryExtensive polymorphism was found in mitochondrial DNAs (mtDNAs) of Norway rats (Rattus norvegicus). The restriction endonuclease cleavage patterns of mtDNAs of laboratory rats, wild rats, tumor cells, and culture cells were compared The polymorphism is defined by two criteria; one is cleavage site variation and the other is length polymorphism of restriction fragments. The cleavage site variation may be caused by point mutation, and the length polymorphism by sequence deletions or insertions. At least five types, types A-E, were identified by cleavage site variations, and two groups, a and b, were identified by length polymorphism of one HpaII fragment, Hpa5. All types except type C belonged to either group-a or group-b, whereas both groups were found in type C. Differentiation of polymorphic Norway rat mtDNA types and the experimental use of the polymorphism are discussed.

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

Abstract 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).

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.

Identification of cytoplasmically transferred mitochondrial DNA in female germlines of Drosophila and its propagation in the progeny

SummaryThe composition of mitochondrial DNA (mtDNA) was analyzed in single female flies that developed from fertilized Drosophila melanogaster eggs, into which germ plasm of D. simulans had been introduced. HpaII cleavage patterns showed that all 12 individual female flies examined had developed from eggs in which 37%–71% of the total mtDNA was D. simulans mtDNA (Ds mtDNA) and the rest was D. melanogaster mtDNA (Dm mtDNA). The stability of this heteroplasmic state in these isofemale lines was monitored for seven generations at both individual and population levels. Results showed that the heteroplasmy of Dm and Ds mtDNAs was stably transmitted for at least three generations at the population level, but showed stochastic segregation at the individual level. After 4–6 generations, all individuals lost Ds mtDNA. The mechanisms of preferential loss of Ds mtDNA and of transmission of heteroplasmic mtDNA to descendants are discussed.