scholarly journals ON THE ORIGIN OF MITOCHONDRIAL MUTANTS: EVIDENCE FOR INTRACELLULAR SELECTION OF MITOCHONDRIA IN THE ORIGIN OF ANTIBIOTIC-RESISTANT CELLS IN YEAST

Genetics ◽  
1973 ◽  
Vol 74 (3) ◽  
pp. 421-432
Author(s):  
C W Birky
Keyword(s):  
Selective Medium ◽  
Wild Type ◽  
Respiratory Enzymes ◽  
Antibiotic Resistant ◽  
Homogeneous Population ◽  
Chloramphenicol Resistance ◽  
Mitochondrial Ribosomes ◽  
Constant Rate ◽  
A Cell ◽  
Mutant Cells

ABSTRACT In wild-type Sacchromyces cerevisiae, erythromycin and certain other antibacterial antibiotics inhibit the formation of respiratory enzymes in mitochondria by inhibiting translation on mitochondrial ribosomes. This paper is concerned with the origin of mutant cells, resistant to erythromycin by virtue of having a homogeneous population of mutant mitochondrial DNA molecules. Such mutant cells are obtained by plating wild-type (sensitive) cells on a nonfermentable substrate plus the antibiotic. Colonies of mutant cells appear first about four days after the time of appearance of established mutant cells; new colonies continue to appear, often at a constant rate, for many days. Application of the NEWCOMBrEes preading experiment demonstrates that most or all of the mutant cells which form the resistant colonies on selective medium arise only after exposure of the population to erythromycin. It is suggested that this result is most probably due to intracellular selection for mitochondrial genomes. Resistant mitochondria arising from spontaneous mutatLon are postulated to be at a selective disadvantage in the absence of erythromycin; reproducing more slowly than wild-type sensitive mitochondria, they cannot easily accumulate in sufficient numbers in a cell to render it resistant as a whole. In the presence of erythromycin, resistant mitochondria can continue to reproduce while sensitive mitochondria cannot, until there is a sufficient number to make the cell resistant, i.e. to permit normal cell growth. The same phenomenon is seen with respect to chloramphenicol resistance. Intracellular selection is considered more likely than direct induction of mutation by the antibiotic, since mutant cells do not accumulate in the presence of erythromycin if the mitochondrial genome is rendered nonessential by growth on glucose or nontranslatable by chloramphenicol. Intracellular selection provides a mechanism for direct adaptation at the cell level, compatible with currently acceptable ideas of spontaneous miitation and selection at the organelle level.

Growth in cell length in the fission yeast Schizosaccharomyces pombe

1985 ◽  
Vol 75 (1) ◽  
pp. 357-376 ◽  
Author(s):  
J.M. Mitchison ◽  
P. Nurse
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Size ◽  
Single Cells ◽  
Temperature Shift ◽  
Time Lapse ◽  
Cell Length ◽  
Wild Type ◽  
Cycle Growth ◽  
A Cell ◽  
Mutant Cells

The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the ‘old end’, which existed in the previous cycle. Later on, the ‘new end’, formed from the septum, starts to grow at a point in the cycle that we have called NETO (‘new end take-off’). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0.34 of the cycle with a cell length of 9.5 microns. With size mutants that are smaller at division, NETO takes place at the same size (9.0-9.5 microns) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0.32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9.0-9.5 microns; and (2) the cell has traversed the first 0.3-0.35 of the cycle and passed early G2. NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132. Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wild-type cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. cdc 11.123 is a mutant in which septation and division is blocked at 35 degrees C but nuclear division continues.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Disease-causing mutation in α-actinin-4 promotes podocyte detachment through maladaptation to periodic stretch

2018 ◽  
Vol 115 (7) ◽  
pp. 1517-1522 ◽  
Author(s):  
Di Feng ◽  
Jacob Notbohm ◽  
Ava Benjamin ◽  
Shijie He ◽  
Minxian Wang ◽  
...  
Keyword(s):  
Direct Consequence ◽  
Adhesion Assay ◽  
Actin Network ◽  
Wild Type ◽  
Spatially Correlated ◽  
Mechanical Responses ◽  
Podocyte Detachment ◽  
A Cell ◽  
Cross Links ◽  
Mutant Cells

α-Actinin-4 (ACTN4) bundles and cross-links actin filaments to confer mechanical resilience to the reconstituted actin network. How this resilience is built and dynamically regulated in the podocyte, and the cause of its failure in ACTN4 mutation-associated focal segmental glomerulosclerosis (FSGS), remains poorly defined. Using primary podocytes isolated from wild-type (WT) and FSGS-causing point mutant Actn4 knockin mice, we report responses to periodic stretch. While WT cells largely maintained their F-actin cytoskeleton and contraction, mutant cells developed extensive and irrecoverable reductions in these same properties. This difference was attributable to both actin material changes and a more spatially correlated intracellular stress in mutant cells. When stretched cells were further challenged using a cell adhesion assay, mutant cells were more likely to detach. Together, these data suggest a mechanism for mutant podocyte dysfunction and loss in FSGS—it is a direct consequence of mechanical responses of a cytoskeleton that is brittle.

scholarly journals A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sonal Nagarkar-Jaiswal ◽  
Sathiya N Manivannan ◽  
Zhongyuan Zuo ◽  
Hugo J Bellen
Keyword(s):  
Cell Cycle ◽  
Gene Inactivation ◽  
Inversion Method ◽  
Wild Type ◽  
Flip Flop ◽  
Temporal Cues ◽  
A Cell ◽  
Novel Method ◽  
Mutant Cells

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila. Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

scholarly journals Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles

1994 ◽  
Vol 14 (4) ◽  
pp. 2699-2712
Author(s):  
M Yoneda ◽  
T Miyatake ◽  
G Attardi
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Wild Type ◽  
Mitochondrial Dna Mutation ◽  
Experimental Conditions ◽  
Chloramphenicol Resistance ◽  
Dna Mutation ◽  
Mitochondrial Dnas ◽  
A Cell ◽  
Type Gene

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

scholarly journals Characterization of bcsA Mutations That Bypass Two Distinct Signaling Requirements for Myxococcus xanthus Development

2002 ◽  
Vol 184 (18) ◽  
pp. 5141-5150 ◽  
Author(s):  
John K. Cusick ◽  
Elizabeth Hager ◽  
Ronald E. Gill
Keyword(s):  
Cell Density ◽  
Myxococcus Xanthus ◽  
Wild Type ◽  
Amino Acid Similarity ◽  
A Cell ◽  
Protease Deficient ◽  
Mutant Cells ◽  
Low Cell Density ◽  
Significant Amino Acid

ABSTRACT The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Ω951 insertion was localized to a gene, bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism. Mutations in bcsA bypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling. Bypass of the B-signaling requirement by the bcsA mutation was accompanied by restored expression of a subset of developmentally induced lacZ fusions to the BsgA protease-deficient strain. bcsA mutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (Ω4427), suggesting that the bcsA gene product may normally act to inhibit development in a cell-density-regulated fashion. Bypass of the requirements for both B and C signaling by bcsA mutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements.

scholarly journals A bacterial tragedy of the commons that masks the actual frequency of mutants

2020 ◽  
Author(s):  
Henrique Iglesias Neves ◽  
Gabriella Trombini Machado ◽  
Taíssa Cristina dos Santos Ramos ◽  
Hyun Mo Yang ◽  
Ezra Yagil ◽  
...  
Keyword(s):  
Carbon Source ◽  
Bacterial Culture ◽  
Tragedy Of The Commons ◽  
Selective Medium ◽  
High Rate ◽  
Wild Type ◽  
Selective Plate ◽  
The Commons ◽  
Mutant Cells ◽  
Mutant Frequency

AbstractThe frequency of mutants in a population is central to the understanding of evolution. Mutant frequency is usually assessed by plating a bacterial culture on selective medium in which only specific rare mutants can grow, assuming that all mutant cells present on the plate are able to form colonies. Here we show an exception to this rule. Wild-type Escherichia coli cells are unable to grow with glycerol-2-phosphate (G2P) as a carbon source. In contrast, PHO-constitutive mutants can hydrolyse G2P to glycerol and form colonies on plates having G2P as their sole carbon source. However, the frequency of PHO-constitutive colonies on the selective plate is exceptionally low. Here we show that such mutations occur at a relatively high rate, but the growth of the existing mutants is inhibited due to a competition with the surrounding wild-type cells for the limited amounts of glycerol produced by the mutants. This scenario in which neither the wild-type nor the majority of the mutants are able to grow constitutes an unavoidable case of the ‘tragedy of the commons’. Evidence shows that the few mutants that do form colonies derive from micro-clusters of mutants on the selective plate. In addition, a mathematical model describes the fate of the wild-type and mutant populations on the selective plate.

scholarly journals Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

1994 ◽  
Vol 14 (4) ◽  
pp. 2699-2712 ◽  
Author(s):  
M Yoneda ◽  
T Miyatake ◽  
G Attardi
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Wild Type ◽  
Mitochondrial Dna Mutation ◽  
Experimental Conditions ◽  
Chloramphenicol Resistance ◽  
Dna Mutation ◽  
Mitochondrial Dnas ◽  
A Cell ◽  
Type Gene

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

scholarly journals DILUTION KINETIC STUDIES OF YEAST POPULATIONS: IN VIVO AGGREGATION OF GALACTOSE UTILIZING ENZYMES AND POSITIVE REGULATOR MOLECULES

Genetics ◽  
1974 ◽  
Vol 77 (3) ◽  
pp. 491-505
Author(s):  
Shinji Tsuyumu ◽  
Bruce G Adams
Keyword(s):  
Gene Product ◽  
Kinetic Studies ◽  
Selective Medium ◽  
Gene Products ◽  
Wild Type ◽  
Population Analyses ◽  
Leloir Pathway ◽  
A Cell

ABSTRACT By use of a selective medium containing ethidium bromide, population analyses of yeast galactose long-term adaptation mutants (gal3) in the process of deadaptation in the absence of galactose have been performed. The analysis of diploid strains homozygous for the gal3 locus but heterozygous for different combinations of the other mutant galactose loci, which thus have reduced amounts of the gene products of those loci, have demonstrated that, in addition to the two permease units determined in a previous study, a cell requires one complex of the Leloir pathway enzymes and two complexes specified by the Gal4 locus to be readily induced. From the consideration of these complexes as being aggregated molecules which are diluted out as units (i.e., if such a molecule were a dimer, it would not dissociate into monomers) during cell growth, the in vivo aggregation of these enzymes and the Gal4 gene product could be studied. The data indicate that the function of the Gal4 gene product is to activate a Leloir enzyme complex. It is postulated that the gal3 phenotype is the result of such strains' inability to actively synthesize an endogenous co-inducer which allows wild-type cells to be readily induced upon exposure to galactose

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