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.     

Cybrid formation in mouse L cells: the influence of cytoplast-to-cell ratio

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.     

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.     

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.     

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.     

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.).     

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

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.     

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 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.     

Leishmania tarentolae: streptomycin and chloramphenicol resistance of promastigotes.

Stable mutant strains of Leishmania tarentolae promastigotes resistant to chloramphenicol (CAP) were isolated by replica-plating techniques. In addition, cell lines stress-adapted to streptomycin and to high culture temperature (33 C) were obtained. Drug resistance was influenced by temperature, culture media, and plating technique. Inhibition of amino acid incorporation into protein occurs in CAP-resistant cells when exposed to 600 μg CAP/ml but this inhibition was 50–80% lower than that found in wild type sensitive cells. The primary site of CAP action appears to be inhibition of protein synthesis. CAP also adversely affected proline oxidation.     

Polyethylene-glycol-mediated cybrid formation: high-efficiency techniques and cybrid formation without enucleation.

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.     

Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli

Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing new drugs to inhibit the growth of the antibiotic-resistant bacteria. In the current study, comparative proteomic methodologies were used for identification of sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. Six differential OM proteins and another protein with unknown location were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC, and OmpW were critically altered proteins and potential targets for designing of the new drugs. Furthermore, a novel method of specific antibody combating bacterial growth was developed on these OM proteins. Only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT, and OmpW were separately utilized. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP. This observed result is basically the same to the finding that DeltatolC was survived in the same concentration of the antibiotic. Our study demonstrates that the enhancement of expression of antibody target with antibiotic could be very effective approach compared to using a drug alone, which highlights a potential way for treatment of infection by antibiotic-resistant bacteria.     

Genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of Escherichia coli

Complications of chemotherapy, such as appearance of multidrug resistance, have persuaded researchers to consider phage therapy as a new method to combat bacterial infections. In vitro experiments were performed to assess the therapeutic value of genetically modified phages for controlling gastrointestinal Escherichia coli O157:H7 cells in Luria–Bertani (LB) media and contaminated cow milk. We constructed a modified nonreplicating M13-derived phage expressing a lethal catabolite gene activator protein (CAP) that is a Glu181Gln mutant of CAP. The modified phagemid was propagated in the lethal CAP-resistant strain XA3DII. Time–kill assay experiments showed a considerable reduction in the number of surviving bacteria in both LB media and contaminated cow milk. Our further study using other test strains demonstrated that the host range of lethal phage is limited to E. coli strains that produce pili. This study provides a possible strategy for the exploitation of genetically engineered nonlytic phages as bactericidal agents by minimizing the risk of release of progeny phages and endotoxins into the environment. The phage was engineered to remain lethal to its bacterial target, but incapable of replicating therein. Furthermore, the addition of an inducer to express the lethal protein is not required.     

Effects of chloramphenicol on the mitochondrial respiratory chain in the wild strain and in a cytoplasmic chloramphenicol-resistant mutant of Tetrahymena pyriformis

The effects of chloramphenicol (CAP) on mitochondrial respiratory activity in the wild strain (ST) and in a cytoplasmic CAP-resistant mutant (STR1) of Tetrahymena pyriformis were studied by determining oxygen consumption, by spectrophotometry, and by cytochemistry. In the absence of CAP both strains had the same respiration capacity, and the low-temperature spectra of their isolated mitochondria were similar. Furthermore, the mitochondria of both strains showed a positive reaction with diaminobenzidine, denoting a similar cytochrome oxidase activity. However, when cells were grown in CAP for 24 or 48 h, the peaks of cytochrome oxidase and cytochromb b were almost absent in the wild type. In this type the oxygen consumption was greatly decreased, and the mitochondria were no longer stained by diaminobenzidine. In the mutant, the peaks of cytochrome oxidase and cytochrome b were decreased only; respiration was less affected than in the wild type, and cytochrome oxidase activity was still disclosed by the diaminobenzidine reaction. These results show that CAP inhibits the synthesis of two cytochromes (b and oxidase) which are partially translated into the mitochrondria of T. pyriformis. In the mutant, CAP reduces only the mitochondrial translation, resulting in reduced mitochondrial activity and reduced growth rate of the cell. These results are compared with the nucleo-mitochondrial regulation mechanisms discussed in our previous works.     

Cytoplasmically determined human cell mutants defective in mitochondrial ribosome assembly

Summary The mitochondrial macromolecular synthesis and assembly processes of three mutants of the human cell line VA₂-B, which are deficient to varying degrees in mitochondrial protein synthesis and resistant to chloramphenicol (CAP), have been analyzed. The mutant VA₂/CAP23 was selected directly for resistance in vivo to CAP, while the mutants VA₂/mtPS^- 1 and 4 were selected as respiration deficient, and subsequently found to be resistant to CAP. The phenotypes of the three mutants are inherited cytoplasmically and thus the mutations are probably localized in mitochonrial DNA (mtDNA). The gross mtDNA sequence organization of the three mutants, as analyzed by digestion with several restriction enzymes, was found to be indistinguishable from that of VA₂-B cells, and their relative mtDNA content comparable to or greater than that of the parental cell line. No difference was observed in the electrophoretic mobilities of mitochondrial rRNAs or mRNAs of the mutants analyzed on denaturing methylmercuric hydroxide-agarose gels as compared to the mobilities of the RNA species of the VA₂-B cells. Similarly, no significant change, or only a moderate decrease in the rate of synthesis of mitochondrial 12s or 16s rRNAs was found in the mutants. By contrast, the incorporation of³H-uridine into mitochondrial ribosomal subunits was significantly reduced in the three mutants, and in each of them, this decrease paralleled the decrease in the level of mitochondrial protein synthesis. The complex phenotypes of the mutants analyzed here could be accounted for by assuming that, as previously reported for other CAP-resistant mouse and human cell variants, the 16s rRNA gene is the site of the mutation. Such a mutation would produce a CAP-resistant phenotype and at the same time affect the assembly of the large ribosomal subunit, with a resulting reduction in mitochondrial protein synthesis and a pleiotropic respiratory deficiency. However, other possible sites for the primary defect in the mutants analyzed here cannot be excluded.     

New Djungarian hamster cell lines with selective cytoplasmic and nuclear genetic markers

Two new Djungarian hamster cell lines which are resistant to chloramphenicol (CAP) are described. The clonal DMCAP subline was obtained by incubation of HPRT-deficient DM-15 cells for 6 months in the medium containing 50 micron/ml of CAP. Resistance to CAP is determined in DMCAP cells by the cytoplasm: cytoplasts from these cells could transmit resistance to CAP into sensitive cells, such as L or DMCH-2/1 cells by hybridization. However, after transplantation of DMCAP nuclei into L cytoplasts, the resulting hybrid cells lost resistance to CAP to a great extent. Using the capacity of DMCAP cytoplasts to transfer CAP-resistance, we obtained a line of hybrids (cyt. DMCAP X DMCH-2/1) which was resistant to 8-azaguanine, CAP and colchicine. As in the original DMCH-2/1 cell line, colchicine-resistance in the cybrid line appeared to be associated with gene amplification. Thus, chromosomal analysis showed that the karyotype of the hybrids was identical to that of DMCH-2/1 cells. Both contained marker chromosomes with homogeneously staining regions (HSRs) and, during incubation in the colchicine-free medium, lost resistance to colchicine. The loss of resistance was accompanied by a decrease in the number of cells containing chromosomes with HSRs and an increase in the number with double minutes (DMs). Many cells containing small chromatin bodies in their cytoplasm also appeared. These chromatin bodies may be DMs lost from the nucleus during mitosis. These new sublines with cytoplasmic and nuclear genetic markers may be useful in the further study of cytoplasmic-nuclear interactions, particularly, in the analysis of possible activities of the DNA fragments which appear in the cytoplasm during reversion to colchicine sensitivity.     

Chromosomal Transmission Bias in Laboratory Hybrids between Wild Strains of the Two European Subspecies of House Mice

Laboratory crosses between wild strains of the two European house mouse subspecies Mus musculus domesticus (2n = 34) and M. m. musculus (2n = 40) were performed to analyze the selective processes involved in the non-introgression of centromeric regions of Robertsonian (Rb) fusions in the Danish hybrid zone. The chromosomal analysis of 226 backcross progeny from 22 reciprocal crosses showed that the segregation of the three Rb fusions present did not significantly differ from Mendelian expectations. However, a significant negative correlation was found between Rb transmission rates and the average litter sizes of the F(1) pairs. Among the different models of selection discussed, the most likely one supported the existence of two opposing selective factors resulting in an overall compensation of chromosomal types in the backcross progeny. A two-phase selective process involving embryo competition was postulated with non-Rb carriers being favored during pre-implantation but disadvantaged after implantation. Such balanced selective pressures acting on musculus non-Rb centromeres are compatible with the steep slope and off-centered position of the chromosomal cline observed in the Danish hybrid zone. These results suggested that these selective factors may be more related to centromere origin (musculus or domesticus) than to centromere structure (Rb or non-Rb).     

Human lysosomal genes: Arylsulfatase A and β-Galactosidase

The segregation of human lysosomal arylsulfatase A (ARS-A) has been evaluated in 50 primary hybrid clones derived from four separate fusions involving WBCs from two unrelated individuals and three hamster cell lines. ARS-A was expressed in the hybrids as a dimeric molecule of very similar or identical subunits. The expression of this enzyme was concordant with that of mitochondrial aconitase (ACON-M), an isozyme assigned to chromosome 22, in all 50 clones and with chromosome 22 segregation in all but one of the 29 karyotyped hybrids. No other human chromosome cosegregated with 22 in these clones, suggesting that this enzyme is specified in hybrid cells by a locus (or loci) on a single chromosome. beta-Galactosidase (B-GAL) expression was analyzed with two different electrophoresis systems and with a number of cell extract preparation methods in 39 of the primary hybrid clones. The B-GAL isozyme expressed in these hybrid cells was concordant with the expression of glutathione peroxidase-1 (GPX-1), an isozyme assigned to chromosome 3, in all 39 clones and with the segregation of this chromosome in 97% of the 29 karyotyped hybrids. These observations substantiate the prior tentative assignments of an ARS-A locus to chromosome 22 and a B-GAL locus to chromosome 3 (Bruns et al., 1978a, b). The implications of the chromosome assignments of loci for 12 human lysosomal enzymes for the cellular assembly of these organelles are discussed.     

Hybrids between irradiated and unirradiated mammalian cells: Survival and chromosome segregation

We have studied the effect of X or γ irradiation, of one parent of a cell hybrid, on hybrid viability and chromosome segregation. The hybrid types studied were mouse-Chinese hamster (which spontaneously lose a few hamster chromosomes) and Chinese hamster-human (which spontaneously lose most of the human complement). Preirradiation of the segregated and retained cell parent resulted in highly asymmetric hybrid survival curves; survival was greatly reduced when the retained parent was irradiated, especially for hamster-human fusions. Preirradiation of the parents of mouse-hamster hybrids modified both the direction and the extent of chromosome segregation, but no consistent effect on elimination was observed for hamster-human hybrids, and reversal of the direction of loss was never observed. These results are more consistent with the hypothesis that chromosome segregation from hybrids results from an intracellular chromosome selection, than with the hypothesis that cellular selection acts on randomly generated chromosome variants.     

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.     

Assignment of the structural gene for human beta glucuronidase to chromosome 7 and tetrameric association of subunits in the enzyme molecule.

The structural locus for human beta glucuronidase is assigned to chromosome 7, a localization based upon concordant segregation of the expression of the human enzyme and the presence of human chromosome 7 in somatic cell hybrid clones derived independently from fusions of different human and mouse cells. Hybrid clones containing only human chromosome 7 are included in this study. Electrophoresis of beta glucuronidase also has revealed that human beta glucuronidase has a tetrametric structure.