Cytoplasmically inherited mutations of a human cell line resulting in deficient mitochondrial protein synthesis.

A large number of mutants deficient in mitochondrial protein synthesis (mtPS-) have been isolated from the human cell line VA2-B by subjecting cells partially depleted of their mtDNA to mutagenic treatments thought to be specific for mtDNA. Each of these mtPS- mutants has less than 10% of the wild-type rate of mitochondrial protein synthesis, exhibits reduced cytochrome oxidase and rutamycin sensitive ATPase activities, requires high concentrations of glucose, and grows indefinitely in the presence of 100 micrograms/ml of chloramphenicol (CAP). Fusion of cytoplasts from seven mtPS- mutants to the nucleated thioguanine-resistant VA2-B derivative TG-6 has yielded numerous cybrid clones which grow in CAP plus thioguanine, whereas almost no clones have resulted from the fusion of nucleated mtPS- cells to TG-6 cells: these results suggest that the gene(s) coding for the phenotype of mtPS- cells is localized in the cytoplasm (mtDNA?).     

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.     

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.     

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.     

Biogenesis of mitochondria 47

Summary This paper consolidates and refines the physical map of genetic loci previously established in our laboratory, by molecular analysis of seven genetically characterized new petites (deletion mutants of mtDNA). A modified DNA-DNA hybridization procedure employing filters simultaneously bound with mtDNA from two different petites has been used to measure the overlaps in mtDNA sequences between the different petite mutants.Thus, by analysis of three new petites carrying the antibiotic-resistance loci, ery1, cap1 and par1 on their mitochondrial genomes, it has now been possible to improve our estimation of the maximum distance between the cap1 and ery1 loci. The cap1, ery1 loci, and the 21S ribosomal RNA gene have now been mapped within 5 units in the same region (map position 0 to 5 units). Similarly, by analysis of four new petites carrying the O _II and/or par1 loci on their mtDNAs, the map position of the O _II locus is also more accurately determined within 2 units in a region (map position 34 to 36 units) between the par1 and ana1 loci. The positions of other loci including par1, the 15S ribosomal RNA gene, and some mit ^- loci are also discussed.We have thus extended our library of genetically and molecularly defined petite mutants, resulting in a set of petites having overlapping regions distributed throughout the entire wild-type mitochondrial genome, consistent with the idea that yeast mtDNA is physically circular.     

Temperature-sensitive respiratory-deficient mitochondrial mutations: Isolation and genetic mapping

Summary In order to find new genetic loci and functions on the yeast mitochondrial DNA, especially mutations affecting the mitochondrial protein synthesis apparatus, temperature sensitive mutants have been isolated after MnCl₂ mutagenesis and mitochondrial and nuclear mutants classified according to their pattern of recombination with three rho^- tester strains.Eighteen cold- and heat-sensitive respiratory deficient mitochondrial mutants have been isolated and localized on the mitochondrial genome by deletion mapping using 113 rho^- strains. Eight of them appear to represent new loci, among which some are probably mutations of the tRNA and rRNA genes.     

Multiple forms of human dihydrofolate reductase messenger RNA: Cloning and Expression in Escherichia coli of their DNA coding sequence

The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA₂-B). A major fraction of this promoting activity is associated with a 3.8 × 10³ base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA₂-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 10³ bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 10³ base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 10³, 1.0 × 10³ and 0.8 × 10³ bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA₂B and HeLa BU25.     

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 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.     

Reversible tenfod reduction in mitochondrial DNA content of human cells treated with ethidium bromide

Summary Cells of the human line VA₂-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of ³H thymidine incorporation into mit-DNA decreased very rapidly to 60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70–80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3–4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of ³H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20–30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.     

Thymidine kinase-deficient mutants of Physarum polycephalum : Biochemical characterization

Thymidine kinase (TK) and deoxycytidine kinase (dCK) activity levels, [³H]thymidine (TdR) and 5-bromo-2′-deoxyuridine (BUdR) incorporation and 5-fluoro-2′-deoxyuridine (FUdR) sensitivity have been compared in TK-deficient (TU63 and TU84) and normal (TU291 and M₃b) strains of the myxomycete, Physarum polycephalum. The mutants had about 2% of the TK and 100% of the dCK activity of wild-type (wt) strains. They incorporated some TdR into both nuclear (nDNA) and mitochondrial DNA (mtDNA) but incorporated too little BUdR to give a buoyant density shift in nuclear DNA. They grew in the presence of levels of FUdR which completely blocked DNA synthesis in TU291. The FUdR sensitivity of strain M₃b could be increased by supplementing growth medium with folic acid.     

Complete mtDNA genome of Otus sunia (Aves,Strigidae) and the relaxation of selective constrains on Strigiformes mtDNA following evolution

Most of owls are nocturnal raptor and usually use their soft and fluffy feathers to flight silently to catch prey while other diurnal raptors prefer fierce attack and swift flight. For energy cost of these different hunting strategies can be greatly different, we speculate that mitochondrial gene of owls may undergo a different evolution pattern following raptors evolution. To test our hypothesis, we sequenced the mtDNA genome of Otus sunia and calculated the ratio of nonsynonymous to synonymous nucleotide substitutions (ω, Ka/Ks, dN/dS) of raptors. The mtDNA genome of O. sunia was 17,609 bp in length, containing 13 PCGs, 2 ribosomal RNAs, 22 transfer RNAs and a control region. Secondly structure of tRNAs and rRNAs were predicted and conserved sequence blocks (CSBs) on control region were identified. The Bayesian inference tree and maximum likelihood tree based on 13 PCGs and 2 rRNAs suggested the owls were related to other raptors. Finally, calculation of ω-values of each owls and other raptors mtDNA PCGs indicated that owls accumulated more nonsynonymous nucleotide substitutions relative to synonymous substitutions compared to other raptors. For mtDNA PCGs associated with energy metabolism, this finding may reveal the degeneration of flight abilities of owls.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Nuclear suppressors of the [poky] cytoplasmic mutant in Neurospora crassa. III. Effects on other cytoplasmic mutants and on mitochondrial ribosome assembly in [poky].

Summary We have previously isolated six non-allelic, nuclear mutations (su ^I loci) that partially suppress the growth, respiratory and cytochrome abnormalities of the extranuclear [poky] mutant.A comparison of the mitochondrial ribosome profiles of suppressed and unsuppressed [poky] strains revealed that five of the six suppressors alleviate at least partially the deficiency of mitochondrial small ribosomal subunits that is associated with the [poky] genotype.Six independently isolated Group I extranuclear mutants, namely [exn-1], [exn-2], [exn-4], [stp-B ₁], [SG-1] and [SG-3], which have growth and cytochrome phenotypes similar to [poky], also were found to be deficient in small subunits of mitochondrial ribosomes. Using cytochrome aa ₃ and b production as a criterion for mitochondrial protein synthesis, it could be shown that the nuclear su ^I suppressors of [poky] also suppress the other six Group I extranuclear mutants. However, differences in the efficiencies of suppression by su ^I suppressors suggest that at least some of Group I extrachromosomal mutants are not simply re-isolates of [poky], but represent distinct extranuclear mutations.     

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.     

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.).     

Effect of Mitochondrial Inhibitors On Metaphase-Telophase Progression and Nuclear Membrane Formation In Chinese Hamster Cells

Chinese hamster Don cells in log-phase were exposed to Colcemid during the G₂ period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. an inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca²⁺, stimulated the formation of micronuclei. This study indicates that Ca²⁺ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca²⁺ sequestration in the mitochondria and chelation of Ca²⁺ by EGTA as dominant factors.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.