Mitochondrial chloramphenicol-resistant mutants can have deficiencies in energy metabolism

Three pairs of mouse CAP-R PYR-IND OLI-R mitochondrial mutants, and the corresponding CAP-S parental lines, were assayed to determine if cellular expression of these phenotypes was accompanied by changes in cellular energy metabolism: glycolysis, cellular respiration, citric acid cycle activity, and mitochondrial electron transport. Relative to its parental CAPS line, the SVT2 CAP-R mutant had no significant deficiencies in any of the pathways analyzed. In contrast, the LA9 and SVA31 CAP-R mutants showed significant reductions in cellular respiration. At the biochemical level, respiration deficiency was accompanied by derangements in mitochondrial electron transport. It was also found that the CAP-R mutants had very high levels of glycolysis when the cells were maintained in the presence of chloramphenicol. The possibility is discussed that the sequence changes in the mitochondrial large rRNA gene which determine chloramphenicol resistance can also result, at least in some cases, in reduced levels of mitochondrial biogenesis, leading to respiration deficiency. The PYR-IND and OLI-R phenotypes, which also appear to be encoded by the CAP-R mutations, may result from a compensatory increase in glycolysis-generated ATP or metabolic intermediates.     

Expression of cytoplasmically inherited genes for chloramphenicol resistance in interspecific somatic cell hybrids and cybrids

Interspecies cybrids and hybrids were prepared in which one parent contributed the cytoplasmically inherited mutation for mitochondrial chloramphenicol (CAP) resistance. The transfer of CAP-resistant (R) mitochondria in human × mouse cybrids proved to be extremely difficult. Transfer from mouse to hamster cells occurred, but the cybrids had limited proliferative capacity. Hybrids between CAP-R mouse and CAP-sensitive (S) hamster cells could be selected in HAT + CAP at a very low frequency but comparable hybrids could not be isolated in CAP-R human × CAP-S mouse fusions unless ouabain was substituted for HAT. Hybrids selected directly in CAP retained the chromosomes of the resistant parent and segregated those of the sensitive. CAP-R hybrids (CAP-R human × CAP-S mouse) which segregate human chromosomes could be isolated only by sequential selection in HAT and then CAP. These results suggest that the mitochondrial genes of mammalian species are rapidly diverging from each other.     

Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants

The nucleotide sequences of the 3 halves of the mitochondrial 16S rRNA genes from four independent mouse chloramphenicol-resistant (CAP-R) mutants were determined. Each contained a different, single base change that encodes the mutational phenotype. The mitochondrial rRNAgene from the SVA31 CAP-R mutant contains a G-to-A transition at nucleotide 2161 of the noncoding strand; the SVIS CAP-R mutant, a G-to-A transition at position 2375; the LA9 CAP-R mutant, an A-to-T transversion at position 2379; and the SVT2 CAP-R mutant, a T-to-C transition at position 2433. Three of these CAP-R mutants appear to be heteroplasmic as the mtDNA populations contain both wild-type and mutant copies of the rRNAgene. The SVIS CAP-R mutation has not been observed in other mammalian CAP-R mutants, although it occurs at a site homologous to one of the yeast mitochondrial CAP-R mutations. Based upon the locations of the mutated sites within the 16S rRNA, and their proximity to previously analyzed sites of mutations conferring increased inhibitor resistance, all these mutations occur within the ribosomal RNA peptidyltransferase domain. These results provide an explanation for the pleiotropic nature of mitochondrial CAP-R mutations in mammalian cells, particularly the observations that some of the mutant lines are partially respiration deficient.     

Cytoplasmic genetics of mammalian cells: Conditional sensitivity to mitochondrial inhibitors and isolation of new mutant phenotypes

We report here that glucose, as a carbon source, and pyruvate are required for the phenotypic expression of cytoplasmically transmitted chlor-amphenicol-resistance (CAP-R) mutations, recovery of CAP-R mutants, and continuous growth in the presence of oligomycin or antimycin. We assume that glucose supplies additional energy when mitochondrial respiration is diminished and that pyruvate provides intermediates when the Krebs cycle is inhibited. Thus, the requirement for pyruvate is fully satisfied by an exogenous source of purines, and partially by -ketoglutarate or a pyrimidine source. Based upon these findings, we have obtained two types of mutations affecting mitochondrial function—oligomycin resistance and pyruvate-independent expression of chloramphenicol resistance. Both are cytoplasmically transmitted and provide new markers for a genetic analysis of mitochondrial biogenesis.This paper is dedicated to the memory of Boris Ephrussi, whose pioneering work in gene expression, mitochondrial biogenesis, and somatic cell genetics has been a continuing inspiration and guide to both of us. Ruth Sager further expresses her indebtedness for the intellectual companionship and moral support that Boris provided so generously in the dark dawn of cytoplasmic inheritance.     

Mitochondrial genetics of mammalian cells: A mouse antimycin-resistant mutant with a probable alteration of cytochromeb

Mouse LA9 antimycin-resistant mutants (ANT-R) were isolated and characterized. Genetic analyses established that this phenotype is encoded within the mtDNA: (1) the ANT-R phenotype showed frequent mitotic segregation and reassortment in hybrid clonal lines; (2) it was transmitted directly in cybrid crosses; and (3) it was cotransmitted in cybrid crosses with the mitochondrial CAP-R marker. Furthermore, the genetic studies suggested that the LA9 CAP-R ANT-R cells were heteroplasmic and contained at least two mtDNA genotypes, cap-r ant-s and cap-s ant-r. Cellular respiration of the ANT-R mutant was markedly more resistant to inhibition by antimycin than that of the parental ANT-S cells. The increased resistance of cellular respiration was entirely accounted for by an increase in the resistance of mitochondrial succinate-cytochrome coxidoreductase to antimycin inhibition. There was no detectable change in the specific activity of the oxidoreductase in mitochondria of resistant ANT-R cells nor in the sensitivity of the complex to three other specific inhibitors of the complex: TTFA, myxothiazol, and HQNO. Taken together, these studies indicate that the ANT-R phenotype is most likely encoded within the mitochondrial cytochrome bgene and, more specifically, within an antimycin binding domain.     

Mitotic segregation of mitochondrial dnas in human cell hybrids and expression of chloramphenicol resistance

The relationship between the chloramphenicol (CAP)-resistant phenotype and the mtDNA genotype was investigated in segregating human, HeLa × HT1080, somatic cell hybrids. The parental mtDNAs were quantitated in heteroplasmic cells by using restriction fragment length polymorphisms (RFLPs) detected in Southern blots. CAP-resistant (R) × CAP-sensitive (S) hybrids selected and grown in CAP for brief periods had as little as 25% CAP-R mtDNA. With prolonged selection, the CAP-R mtDNA increased to 90–95%. Hybrids selected and passaged without CAP either retained both mtDNAs or progressively lost one mtDNA (mitotic segregation). The CAP-resistance phenotype of these hybrids changed abruptly when the proportion of CAP-R mtDNAs fluctuated around approximately 10% (threshold effect). Hybrids with greater than 25% HT1080 mtDNA had an additional characteristic. They cloned better with CAP than without. The cloning efficiency in CAP of hybrids having 90% HT1080 mtDNA was more than fivefold greater than the control.     

Respiration-deficient Chinese hamster cell mutants: Genetic characterization

We present here genetic experiments with a series of Chinese hamster cell mutants defective in oxidative energy metabolism. The mutations were all shown to be recessive in intraspecies hybrids. Thirty-five mutants were sorted into eight complementation groups, but one of these mutants failed to complement representatives of two distinct complementation groups. The possibility was raised that this is a cell carrying two mutations or a deletion. Because of the greatly different frequencies with which such mutants could be isolated from two different Chinese hamster cell lines, CCL16 (DON) and V79, the stability of representatives from each cell line was examined, and it was found that revertants could be obtained after treatment with mutagens, while spontaneous revertants appeared at unmeasurable or extremely low frequencies, with one exception. The mutant with a very noticeable frequency of spontaneous reversion was defective in mitochondrial protein synthesis, and the question arose whether the mutation was on the mitochondrial genome. A detailed fluctuation analysis of reversion rate and comparison with rates for other mutations was consistent with a nuclear mutation. This conclusion was supported by experiments involving fusions with cytoplasts.     

Use of fluorescence-activated cell sorter to isolate mutant mammalian cells deficient in an internal protein,dihydrofolate reductase

Dihydrofolate reductase-deficient mutants of Chinese hamster ovary cells have been selected based on their inability to bind a fluorescent derivative of methotrexate, a substrate analog. Nonfluorescent mutant cells were isolated from mutagenized populations using a fluorescence-activated cell sorter. After multiple rounds of sorting plus regrowth, the mutant cell frequency was increased from an initial 10^–5 to greater than 0.9. This use of the cell sorter to isolate mutants deficient in an internal protein should be applicable to any gene product that is able to bind a fluorescent ligand tightly. The method has the advantage of allowing the screening of large numbers of cells and of selecting for partially expressed phenotypes.     

Lectin-resistant CHO cells: Selection of new mutant phenotypes

Cytotoxic plant lectins select for mutants which exhibit unique structural changes in surface carbohydrates reflecting specific defects in glycosylation reactions. However, lectins are not highly specific selective agents and, as a result, only the most frequently occurring mutants are obtained from single lectin selections. We have previously shown that the specificity of lectin selections may be improved by utilizing a combination of lectins added together or sequentially. This strategy has now been further exploited in the search for novel lectin-resistant mutants of Chinese hamster ovary cells. Five new Lee^R phenotypes have been uncovered. One belongs to a new, recessive complementation group, two behave dominantly in somatic cell hybrids, and the remaining two appear to represent new phenotypes which fall into previously described complementation groups.     

Mammalian mitochondrial mutants selected for resistance to the cytochromeb inhibitors HQNO or myxothiazol

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates hada common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochromec oxidoreductase, the respiratory complex containing cytochromeb. As with the previously described mitochondrial antimycinresistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochromeb gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochromeb gene. Finally, all four mitochondrial cytochromeb mutants have been analyzed in both cell plating studies and succinate-cytochromec oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochromeb other than the one used for selection.     

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination.     

Mitochondrial protein synthesis in interspecific somatic cell hybrids

The fusion of an oligomycin (OLI) -resistant mutant of mouse LM(TK^–) cells to a chloramphenicol (CAP) -resistant mutant of AK412 Chinese hamster cells resulted in a series of interspecific somatic cell hybrids. Hybrids selected in HAT medium retained only mouse mitochondrial genomes while hybrids selected in HA T plus CAP and OLI retained both hamster and mouse mitochondrial genomes in approximately equal amounts. Nuclear-coded mitochondrial proteins from both parental species were incorporated into mitochondria in all of the hybrids. However, the mitochondrially coded proteins of three individually isolated hybrid cell lines were predominantly mouse-specific, with only trace amounts of hamster protein detected.     

Analyses of the mitochondrial mutations in the Chinese patients with sporadic Creutzfeldt–Jakob disease

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause a variety of chronic diseases in central nervous system (CNS). However, the role of mtDNA mutations in sporadic Creutzfeldt–Jakob disease (sCJD) has still been unknown. In this study, we comparatively analyzed complete mtDNA sequences of 31 Chinese sCJD patients and 32 controls. Using MITOMASTER and PhyloTree, we characterized 520 variants in sCJD patients and 507 variants in control by haplogroup and allele frequencies. We classified the mtDNAs into 40 sub-haplogroups of 5 haplogroups, most of them being Asian-specific haplogroups. Haplogroup U, an European-specific haplogroups mtDNA, was found only in sCJD. The analysis to control region (CR) revealed a 31% increase in the frequency of mtDNA CR mutations in sCJD versus controls. In functional elements of the mtDNA CR, six CR mutations were in conserved sequence blocks I (CSBI) in sCJD, while only one in control (P<0.05). More mutants in transfer ribonucleic acid-Leu (tRNA-Leu) were detected in sCJD. The frequencies of two synonymous amino-acid changes, m.11467A>G, p.(=) in NADH dehydrogenase subunit 4 (ND4) and m.12372G>A, p.(=) in NADH dehydrogenase subunit 5 (ND5), in sCJD patients were higher than that of controls. Our study, for the first time, screened the variations of mtDNA of Chinese sCJD patients and identified some potential disease-related mutations for further investigations.     

High frequency of mutation to tubercidin resistance in CHO cells

The acquisition of high-level resistance to tubercidin (an adenosine analog) in CHO cells occurs in a single step at high frequency (10^–3 to 10^–4) without mutagenesis. Analysis of a large number of independent mutants by a fluctuation test (Luria and Delbruck, 1943) indicates that they arise independently of the selection medium and all fall into the same complementation group. All mutants tested lack detectable adenosine kinase activity. An analysis of hybrids between mutant and wild-type cells indicates that resistance to tubercidin is a recessive marker which segregates as would be expected if it were a haploid locus in the parental CHO cell. Resistance to tubercidin is not linked to the X chromosome in CHO cells and appears to occur at much lower frequency in primary Chinese hamster cells and other cultured cell lines.     

Different Chinese hamster cell lines express a G1 period for different reasons

Previous studies from our laboratory have shown that the absence of G1 (G1^– condition) in two lines of Chinese hamster cells is dominant over the presence of G1 (G1⁺ condition) in a variety of intraspecific cell hybrids. G1⁺ mutants or variants can be isolated from G1^– cells following mutagenesis and selection. These G1⁺ mutants fall into multiple complementation groups based on their abilities to form G1^– cell hybrids with one another. This is evidence that different mutants have G1 intervals for different reasons, possibly as the result of deficiencies in functions necessary for G1^– cell cycles. In this report we have used cell hybrid analysis to ask whether cells of different, naturally occurring G1⁺ lines of Chinese hamster are able to complement to produce G1^– hybrids. We have found three complementation groups among the four G1⁺ cell lines examined. Therefore, these lines define three different reasons or bases for the existence of a G1 interval. These results lead us to suggest that multiple requirements must be met for these cells to start the S period, but that failure to fulfill only a single and different requirement is responsible for the presence of a G1 interval in any given cell line.     

Selection of mammalian thymidine auxotrophic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate

Thymidine auxotrophic mutants were selectively isolated from mutagenized mouse FM3A cells by resistance to methotrexate in the presence of thymidine and 5-methyl-tetrahydrofolate with a frequency of 10^–5–10^–6. In most of the thymidine auxotrophs the activity of thymidylate synthase was very low or undetectable, but dihydrofolate reductase activity was normal. Upon starvation of thymidine, the mutant cells immediately stopped growing and started to lyse within one day. In the presence of thymidine, the mutant cells grew quite normally. This phenotype behaved recessively in cell—cell hybrids, and the segregation profile of its marker indicated that the lesions in the mutants are not linked to the X chromosome. Prototrophic revertants could be isolated from these mutants, and they showed almost the normal level of thymidylate synthase activity. The selection method described here should be useful for isolating large numbers of thymidylate synthase-negative mutants from various mammalian cell lines.     

Isolation and characterization of temperature-sensitive mutants of Chinese hamster ovary cells after treatment with UV and X-irradiation

The isolation of ten conditionally lethal temperature-sensitive mutants of the Chinese hamster ovary cell (CHO-Kl, pro^–) by the BUdR-visible light selection procedure is described. Treatment with radiation at doses known to cause single gene mutation in mammalian cells increases the mutation frequency by a factor of at least 14. These mutants will grow with normal plating efficiency at 34.5° but will not grow at 39.5°. Complementation analysis by two independent methods indicates that all mutants are recessive and allows the assignment of the mutants to six genetically independent complementation groups. Reversion analysis indicates that the TS-mutants are stable, spontaneous revertants arising at a frequency of <10^–6. Preliminary chromosome analysis revealed no systematic chromosomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5° and 39.5°. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.     

Lactic acidemia and mitochondrial disease

Lactic acidemia is present in the majority of patients with mitochondrial oxidative defects as well as in disorders of gluconeogenesis. An understanding of the dynamics of lactic acid metabolism in the human body and the influences on lactate/pyruvate ratios exerted by changes in cellular redox state allows for the development of diagnostic algorithms based on clinical and biochemical phenotypes. Mitochondrial disorders can be due to defects in nuclear genes directly affecting the respiratory chain assembly or function, mtDNA genes affecting the respiratory chain or nuclear genes influencing mtDNA structure and viability. In this review, we look at the classification of mitochondrial disease from the perspective of not just the genetic and biochemical etiology but also from the perspective of the clinical phenotypic expression.     

A selection system specific for the thy mutator phenotype

Thy^– mutants, in addition to being resistant to arabinosyl cytosine (arcC), show cross-resistance to 5-fluorouracil (5FU). When Chinese hamster ovary (CHO) cells were exposed to a selection system using both araC and 5FU, the resistant clones isolated were identical to thy^– mutants by the following criteria: (1) all were auxotrophic for thymidine with a high reversion frequency to thymidine prototrophy; (2) those tested had a high level of dCTP relative to wild-type cells, while dTTP and dATP levels were unaffected; and (3) all tested had a 7- to 50-fold higher rate of spontaneous mutation than the wild-type strain for at least one independent genetic marker. Although spontaneous thy^– mutants were rare, the frequencies of thy^– mutants in untreated and mutagenized cultures are consistent with the conclusion that the thy^– phenotype is the consequence of a single mutation in CHO cells.     

Effect of selection cell density on the recovery of mutagen-induced 6-thioguanine-resistant cells (CHO/HGPRT system)

The relationship between the cell density utilized in the selection for 6-thioguaninine-resistant mutants induced by ethyl methanesulfonate and the recovery of mutant colonies was determined in Chinese hamster ovary cells. After an adequate phenotypic expression time of 9 days post mutagen treatment, cultures were plated in selection medium at cell densities ranging from 5 × 10⁴ to 2 × 10⁶ cells/100 mm dish (0.9 to 36.0 × 10³ cells/cm²). A decline in the frequency of mutant colonies was observed with increasing cell density, and analysis of the data showed that the loss was independent of the frequency of mutants in the cell population. This loss occurred in an exponential fashion, consistent with a random-target effect. Regression analysis of the data points yielded a best-fit line defined by the equation log(y) = 2.01 - 0.26(10^?6) x, where y = percentage recovery and × = selection cell density (cells per 100 mm dish). This study defines the cell densitydependent loss of mutants under selection conditions, and provides a basis for further study of the influence of agents on cell contact-mediated crossfeeding and possible effects on mutation induction determinations. These results also demonstrate the importance of the use of appropriate cell densities in mutant selections.