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1.
The frequency of cybrid colony formation was measured in fusions between enucleated chloramphenicol (CAP)-resistant mouse cells and CAP-sensitive mouse cells in varying ratios. By labeling the CAP-resistant cytoplasts with polystyrene beads and then performing the same fusions with CAP-sensitive cells, the frequency of cybrid fusions could be measured. Comparison of the frequency of viable cybrids (cybrid colonies) with the frequency of cybrid fusions showed that, with increasing fusion ratios of cytoplasts to cells, the proportion of cells fused to cytoplasts increased. Further, the viability of cybrid fusions increased from about 1 in 500 to nearly 1 in 60 over the range of cytoplast-to-cell ratios studied. 相似文献
2.
Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells. I: Fusion with mouse cell lines 总被引:3,自引:0,他引:3
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. 相似文献
3.
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. 相似文献
4.
Douglas C. Wallace Y. Pollack C. L. Bunn J. M. Eisenstadt 《In vitro cellular & developmental biology. Plant》1976,12(11):758-776
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.). 相似文献
5.
Jun-Ichi Hayashi Yusaku Tagashira Toshiyuki Watanabe Toyozo Sekiguchi 《Experimental cell research》1983,148(1):258-264
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 (CAPr) rat and CAP-sensitive (CAPs) 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 CAPr rat parent cells showed high relative plating efficiency. 相似文献
6.
Polyethylene glycol (PEG) can be used to promote the fusion of enucleated cytoplasts from chloramphenicol (CAP)-resistant mouse cells with intact cells, resulting in the formation of viable cybrids. The techniques are simple and highly efficient, yielding up to one viable cybrid per 20 intact cells fused. It also seems that PEG can be used to induce cybrid formation without the necessity of prior enucleation of the CAP-resistant cells. 相似文献
7.
M L Ziegler 《Somatic cell genetics》1978,4(4):477-489
This study has been directed toward the effect of cytoplasmic transfer on the expression of marker properties in hybrid cell systems. Conventional hybrids between two nucleated cells were constructed between tumorigenic and nontumorigenic cells. Cytoplasmic hybrids, or cybrids, were constructed between enucleated chloramphenicol resistant (CAP R) donor cells (cytoplasts) and nucleated recipient cells. Clear-cut evidence for the cytoplasmic transmission of CAP resistance was obtained. Although cytoplasmic transfer had no effect on tumorigenicity or growth in soft agar, preliminary evidence was found that saturation density of the recipient cells could be altered by cytoplasmic addition in cybrids. 相似文献
8.
Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells. 总被引:1,自引:1,他引:0
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HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis. 相似文献
9.
Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells 总被引:5,自引:0,他引:5
Jun-Ichi Hayashi Yusaku Tagashira Michihiro C. Yoshida 《Experimental cell research》1985,160(2):387-395
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. 相似文献
10.
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. 相似文献
11.
Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells II: Fusions with human cell lines 总被引:1,自引:0,他引:1
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. 相似文献
12.
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 inthe 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. 相似文献
13.
Segregation of mitochondrial DNA in human somatic cell hybrids 总被引:6,自引:0,他引:6
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. 相似文献
14.
Inheritance of organelle genomes in citrus somatic cybrids 总被引:4,自引:0,他引:4
Moreira Cristina D. Chase Christine D. Gmitter Fred G. Grosser Jude W. 《Molecular breeding : new strategies in plant improvement》2000,6(4):401-405
Restriction fragment length polymorphisms (RFLPs) were used for the characterization of citrus organelle inheritance in somatic cybrids produced during six different citrus protoplast fusions. All the cybrids in this work inherited their mitochondrial genome from the embryogenic fusion partner (callus or cell suspension). In some of the combinations, non-parental bands were observed among the mitochondrial configurations. In contrast, the cybrids inherited plastid DNA from either the embryogenic or the nonembryogenic (leaf) fusion partner. The relative abundance of organelle DNAs in the embryogenic and leaf cells was in accordance with these inheritance patterns. Stochastic processes may therefore influence the outcome of somatic cell fusions with respect to organelle genomes. 相似文献
15.
Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction. 总被引:10,自引:1,他引:9
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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. 相似文献
16.
G. H. Schaap A. Verkerk A. W. M. Van Der Kamp J. F. Jongkind 《Experimental cell research》1982,140(2):299-305
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. 相似文献
17.
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. 相似文献
18.
Intermediate filament expression and lifespan potential in human somatic cell hybrids 总被引:1,自引:0,他引:1
Clive L. Bunn Frank A. White W. Michael O’Guin Roger H. Sawyer Loren W. Knapp 《In vitro cellular & developmental biology. Plant》1985,21(12):716-720
Summary Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed
growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast
cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence
of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth
characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were
keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition,
low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated
with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative
potential in these hybrids.
This work was supported by grant no. AG 02664 from NIA (to C.L.B.) and by grant nos. 1R01 HD 18129-01 from NIH and PCM83-09068
from NSF (to R.H.S.).
Editor’s Statement The tight correlation between the expression of the intermediate filaments of the immortal parent in hybrids
of limited lifespan fibroblasts and HeLa cells with the transformed phenotype is of interest. It may offer important clues
to the mechanism involved in cellular senescence. Gordon H. Sato 相似文献
19.
The transformed phenotype is believed to be dominant in fusions between limited lifespan cells and transformed cells, based on heterokaryon experiments and on the isolation of transformed hybrids from mass cultures of fused cells. A series of fusions has been performed between limited lifespan Lesch-Nyhan fibroblast cells and a permanent HeLa cell line with a complementary genetic marker. The growth of independently isolated hybrid clones was followed in parallel with Lesch-Nyhan cells. In fusions involving Lesch-Nyhan cells which had completed about 50% of their lifespan, all hybrids initially show fibroblastic properties. Thirty-five hybrids had a limited lifespan slightly longer than Lesch-Nyhan controls. Three other hybrid clones, and all mass cultures of hybrids, showed the appearance of transformed colonies at a rate of approx. one transformant in 2 × 105 hybrid cells. These transformed cells showed anchorage independence, low serum requirement, chromosome loss, and have been maintained in culture for 50–100 population doublings with no signs of senescence. Fusions involving enucleated HeLa cells did not show transformation. Fusions with senescent Lesch-Nyhan cells yielded hybrids which grew beyond the normal Lesch-Nyhan cell lifespan, but which again showed limited lifespan and low frequency transformation. It is concluded that limited lifespan is expressed in a dominant manner in these fusions, and that transformation or “escape from senescence” is a low frequency event requiring the presence of the HeLa nucleus. 相似文献
20.
S. Linder 《Experimental cell research》1980,130(1):159-167
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. 相似文献