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.     

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 transfer of microtubule organizing centers in mouse tissue culture cells

A chloramphenicol-resistant, aminopterin-sensitive cell line (AMT) derived from a mouse mammary tumor MT-29240 was enucleated, and the cytoplasts were fused with nucleated chloramphenicol-sensitive, HAT-resistant SV40 3T3 mouse cells. The resulting cytoplasmic hybrids (cybrids) were selected for their resistance to chloramphenicol and the chromosome complement of the SV40 3T3 cells. In addition to transfer of chloramphenicol resistance, these cybrid clones, as studied in the electron microscope, contained the intracisternal A particle phenotype characteristic of only the AMT cells. The cytoplasmic microtubule complex (CMTC) in these cybrids was also studied and appears to resemble the elaborate CMTC of the AMT cells more closely than the more reduced CMTC of the SV40 3T3. When treated with a colcemid block and then a brief reverse, the microtubule organizing centers (MTOC) appear as bright fluorescent foci when tubulin antibody and indirect immunofluorescence techniques are used. When AMT or SV40 3T3 cells are treated in this manner, only one MTOC is present in interphase cells. One clone of these cybrids, however, contained two MTOCs in interphase cells. This CMTC and MTOC phenotype has persisted in this cybrid clone for over 3 months of continuous culture.     

Species specificity of interferon action: maintenance and establishment of the antiviral state in the presence of a heterospecific nucleus.

The expression of the interferon-induced antiviral state was studied in heterokaryons and cytoplasmic hybrids (cybrids). An autoradiographic assay for the antiviral state, in which the percentage of cells containing vaccinia viral DNA factories was determined, was used. The expression of the antiviral state was dominant in homokaryons and heterokaryons formed by fusion of interferon-treated cells with untreated cells. Cytoplasts derived from treated cells conferred resistance to virus growth on cybrids formed by fusing such cytoplasts with untreated cells. Treatment of L cell x HeLa cell heterokaryons with human interferon or mouse interferon was much less effective in inducing a detectable antiviral state than was similar treatment of parental cells with homospecific interferon. The antiviral state was fully induced when heterokaryons were treated simultaneously with both types of interferon. Cybrids formed by fusing L cell cytoplasts with HeLa cells or HeLa cytoplasts with L cells did not enter a detectable antiviral state after treatment with interferon specific for the cell type of the enucleated parent. However, treatment of cybrids with interferon specific for the cell type of the nucleated parent was effective in inducing a detectable antiviral state.     

Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells II: Fusions with human cell lines

Cytoplasmically inherited chloramphenicol (CAP) resistance in human cells has been used to study the interaction between sensitive and resistant mitochondria. Cybrids between two HeLa cells were stable for resistance, grew rapidly and cloned well in CAP, and were O2 tolerant. HeLa-HeLa hybrids were also stable up to 70 doublings in the absence of CAP. Cybrids between HeLa and WI-L2 cells were unstable for resistance for up to 40 doublings, grew slowly and cloned poorly in CAP, and were O2 sensitive (S phase). The growth rate then increased and the cells became stable for resistance, cloned well, and were not O2 sensitive (F phase). Doubling time for S but not F phase cells was proportional to CAP concentration, indicating that both kinds of mitochondria were present and functioning. The instability of CAP resistance in many interstrain but not in intrastrain mouse and human cybrids and hybrids is interpreted in relation to lower eukaryotes.     

Teratoma cybrids: An analysis of the post-fusion effects of myoblast cytoplasms on embryonal carcinoma cells

The influence of rat myoblast cytoplasms in cybrids derived from fusions with mouse embryonal carcinoma cells (EC cells) has been considered. Cytoplasmic hybrids (cybrids) were identified by the use of nuclear and cytoplasmic markers. The presence of chromocenters was used as a marker for EC-cell nuclei. Phagocytosed polystyrene beads served as cytoplasmic markers. Shortly after fusion the cybrids had a drastically altered morphology. They lacked the cytoplasmic lipid granulum characteristic of EC cells and had gained demonstrable fibronectin deposits. These phenotypic changes disappeared during a 3-day period after fusion as the cybrids gradually regained normal EC-cell properties. It was considered that the lack of more stable phenotypic modifications in the cybrids was related to major abnormalities in the cytoplasm preparations. However, cytoplasms were found to be viable for up to 65 h post-enucleation and, as analysed by 2-D gel electrophoresis, continued to synthesize the same major polypeptides as did intact cells, for at least 10 h. Thus, the addition of a myoblast cytoplasm to an EC cell has significant short-term effects but has no detectable permanent or heritable effect on the EC phenotype.     

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.     

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.     

Molecular characterization of mtDNA depleted and repleted NT2 cell lines

Transmitochondrial cytoplasmic hybrids (cybrids) enable functional assessment of mitochondrial DNA (mtDNA)-encoded proteins. Cybrid production often utilizes cell lines depleted of endogenous mtDNA (rho0 cells), and a number of suitable rho0 cell lines exist for this purpose. We now provide molecular data characterizing an NT2 human teratocarcinoma rho0 cell line, as well as NT2 cybrid derivatives. NT2 rho0 cells contained no detectable mtDNA on a sensitive PCR assay. Eight weeks after exogenous mtDNA transfer cybrids showed no evidence of endogenous mtDNA reversion, and heteroplasmic ratios of a single nucleotide substitution roughly reflected that of the blood samples used to repopulate their mtDNA.     

CMS due to tapetal failure in cybrids between Nicotiana tabacum and Petunia hybrida

Cytoplasmic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Petunia hybrida were constructed. Three green plants were obtained after fusion of leaf protoplasts from a cytoplasmic chlorophyll deficient mutant of tobacco, with iodoacetamide inactivated protoplasts of P. hybrida. All regenerated plants were phenotypically similar to tobacco, but male and female sterile. Chromosome and isoenzyme analyses of the nuclear genome, and restriction and blot hybridization analyses of the organelle composition revealed that the regenerated cybrids possessed nuclear genome of N. tabacum, chloroplasts from P. hybrida and recombinant chondriomes. In vitro culture of ovules from one cybrid plant pollinated by N. tabacum resulted in the regeneration of cytoplasmic male sterile progeny plants. Cross-section of anthers from these CMS plants showed that male sterility was due to a failure of tapetum development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selection of proliferating cybrid cells by dual laser flow sorting : Isolation of teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids

An alternative method for the isolation of proliferating cybrid cells was developed, and was used to obtain teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids. Enucleated neuroblastoma (or endoderm) cells labelled with fluorescent microspheres were fused with (HPRT-deficient) unlabelled teratocarcinoma cells. The cells in the fusion mixture were stained with the vital DNA stain Hoechst 33342 and the cybrids, containing both a fluorescent nucleus and fluorescent beads, were isolated by dual laser flow sorting. The purity of the sorted fraction, as determined by the percentage of cells showing HPRT activity, was 86 and 57% for the neuroblastoma and endoderm cybrids, respectively. After single cell sorting in wells of Terasaki microtest plates, clones of proliferating cybrids were obtained with cloning efficiencies of 33% (neuroblastoma and endoderm cybrids respectively. After single cell sorting in wells of Terasaki microtest parental cell lines were analysed by two-dimensional gel electrophoresis. A number of differences were found between the parental cell lines but all isolated colonies (sixteen neuroblastoma cybrids and eight endoderm cybrids) resembled the teratocarcinoma parent. These results therefore give no evidence for the existence of cytoplasmic factors in neuroblastoma or endoderm cells capable of inducing permanent differentiation of teratocarcinoma cells.     

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.     

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

Summary Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.     

Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria.     

Quantitative effect of 4977 bp deletion of mitochondrial DNA on the susceptibility of human cells to UV-induced apoptosis

In this study, we used a series of human cytoplasmic hybrids (cybrids) harboring different proportions of 4977 bp-deleted mtDNA to investigate the quantitative effect of a pathogenic mutation of mtDNA on apoptosis. We found that the sensitivity of human cells to apoptosis triggered by UV irradiation increases with the proportion of 4977 bp-deleted mtDNA. Moreover, UV-induced activation of caspase 3 was preceded by the activation of caspases 8 and 9. Most importantly, we observed that UV-induced cytochrome c release from mitochondria occurred much earlier and was much more pronounced in the cybrids harboring higher proportions of 4977 bp-deleted mtDNA. We suggest that 4977 bp-deleted mtDNA increases the susceptibility of human cells to UV-induced apoptosis in a quantitative manner through cytochrome c release from mitochondria and caspase 3 activation.     

Carbomycin resistance in mouse L cells

A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.     

The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation

Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases.     

A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata

 Cytoplasmic somatic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Nicotiana alata were constructed. A total of 33 green regenerants were obtained after fusion of protoplasts from a tobacco cytoplasmic chlorophyll-deficient mutant and gamma irradiation-inactivated leaf protoplasts of N. alata. Twenty nine of them were male sterile and displayed an altered stamen morphology (formation of petaloid and stigmoid structures instead of stamens). Southern-blot analyses of eight CMS plants using N. alata-specific nuclear repetitive DNA and cpDNA probes revealed that they contained nuclear genetic material of N. tabacum and chloroplasts from N. alata. Restriction-enzyme analysis of mitochondrial DNAs of the cybrids in question showed different patterns consisting of an incomplete mix of mtDNA fragments from both parents, as well as new fragments. Southern-blot analysis of mtDNAs with a sunflower atpA probe gave the same recombinant hybridization pattern for all analyzed cybrids, indicating that high-frequency specific recombination occurs in the atpA region. Analysis of the progeny from three successive backcrosses of the studied cybrids with N. tabacum demonstrated a strict cytoplasmic inheritance of the male-sterile phenotype. Received: 10 May 1997 / Accepted: 31 March 1998     

Interaction of vinblastine with the secretagogue action of db-cAMP in the rat exocrine pancreas,.

Developmentally pluripotent embryonal carcinoma cells isolated from mouse teratocarcinomas were fused to whole cells, to cytoplasts, and to karyoplasts of 3T3 fibroblasts. The cybrids (cell X cytoplast fusion product) retained the developmental potency of the embryonal carcinoma cell parent. On the other hand, the karyobrids (cell X karyoplast fusion product) and the hybrids resembled the fibroblast parent cell and were incapable of differentiation. These experiments, therefore, failed to reveal the presence of cytoplasmic regulators of nuclear gene expression.     

Comparison of the organization of the mitochondrial genome in tomato somatic hybrids and cybrids

Summary The organization of the mitochondrial genome in somatic hybrids and cybrids regenerated following fusion of protoplasts from cultivated tomato, Lycopersicon esculentum, and the wild species, L. Pennellii, was compared to assess the role of the nuclear genotype on the inheritance of organellar genomes. No organellar-encoded traits were required for the recorvery of either somatic hybrids or cybrids. The organization of the mitochondrial genome was characterized using Southern hybridization of restriction digestions of total DNA isolated from ten cybrids and ten somatic hybrids. A bank of cosmid clones carrying tomato mitochondrial DNA was used as probes, as well as a putative repeated sequence from L. pennellii mitchondrial DNA. The seven cosmids used to characterize the mitochondrial genomes are predicted to encompass at least 60% of the genome. The frequency of nonparental organizations of the mitochondrial genome was highest with a probe derived from a putative repeat element from the L. pennellii mitochondrial DNA. There was no difference in the average frequency of rearranged mitochondrial sequences in somatic hybrids (12%) versus cybrids (10%), although there were individual cybrids with a very high frequency of novel fragments (30%). The frequency of tomato-specific mtDNA sequences was higher in cybrids (25%) versus somatic hybrids (12%), suggesting a nuclear-cytoplasmic interaction on the inheritance of tomato mitochondrial sequences.