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1.
Methods ; 191: 87-94, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32717290

RESUMO

Cre-loxP recombination system is a powerful tool for genome engineering. One of its applications is found in genetic mouse models that often require to induce Cre recombination in preimplantation embryos. Here, we describe a technically simple, affordable and highly efficient protocol for Cre protein delivery into mouse zygotes by electroporation. We show that electroporation based delivery of Cre has no negative impact on embryo survival and the method can be easily combined with in vitro fertilization resulting in a significantly faster generation of desired models. Lastly, we demonstrate that Cre protein electroporation is suitable for allelic conversion in primary cells derived from conditional mouse models.


Assuntos
Zigoto , Alelos , Animais , Eletroporação , Integrases/genética , Camundongos
2.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916973

RESUMO

NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) is a member of a gene family with a profound effect on health/disease status. NME7 is an established member of the ciliome and contributes to the regulation of the microtubule-organizing center. We aimed to create a rat model to further investigate the phenotypic consequences of Nme7 gene deletion. The CRISPR/Cas9 nuclease system was used for the generation of Sprague Dawley Nme7 knock-out rats targeting the exon 4 of the Nme7 gene. We found the homozygous Nme7 gene deletion to be semi-lethal, as the majority of SDNme7-/- pups died prior to weaning. The most prominent phenotypes in surviving SDNme7-/- animals were hydrocephalus, situs inversus totalis, postnatal growth retardation, and sterility of both sexes. Thinning of the neocortex was histologically evident at 13.5 day of gestation, dilation of all ventricles was detected at birth, and an external sign of hydrocephalus, i.e., doming of the skull, was usually apparent at 2 weeks of age. Heterozygous SDNme7+/- rats developed normally; we did not detect any symptoms of primary ciliary dyskinesia. The transcriptomic profile of liver and lungs corroborated the histological findings, revealing defects in cell function and viability. In summary, the knock-out of the rat Nme7 gene resulted in a range of conditions consistent with the presentation of primary ciliary dyskinesia, supporting the previously implicated role of the centrosomally located Nme7 gene in ciliogenesis and control of ciliary transport.


Assuntos
Transtornos da Motilidade Ciliar/genética , Genes Letais , Predisposição Genética para Doença , Núcleosídeo-Difosfato Quinase/deficiência , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/diagnóstico , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Genótipo , Imuno-Histoquímica , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transcriptoma , Microtomografia por Raio-X
3.
BMC Genomics ; 17: 662, 2016 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-27544903

RESUMO

BACKGROUND: Exposure of the budding Saccharomyces cerevisiae to an alkaline environment produces a robust transcriptional response involving hundreds of genes. Part of this response is triggered by an almost immediate burst of calcium that activates the Ser/Thr protein phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor (TF) Crz1, which moves to the nucleus and binds to calcineurin/Crz1 responsive gene promoters. In this work we present a genome-wide study of the binding of Crz1 to gene promoters in response to high pH stress. RESULTS: Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 and 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 140 genes likely responsive to Crz1 regulation. Gene Ontology analysis confirmed the relevant impact of calcineurin/Crz1 on a set of genes involved in glucose utilization, and uncovered novel targets, such as genes responsible for trehalose metabolism. We also identified over a dozen of genes encoding TFs that are likely under the control of Crz1, suggesting a possible mechanism for amplification of the signal at the transcription level. Further analysis of the binding sites allowed refining the consensus sequence for Crz1 binding to gene promoters and the effect of chromatin accessibility in the timing of Crz1 recruitment to promoters. CONCLUSIONS: The present work defines at the genomic-wide level the kinetics of binding of Crz1 to gene promoters in response to alkaline stress, confirms diverse previously known Crz1 targets and identifies many putative novel ones. Because of the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involve calcium signaling, such as exposition to sexual pheromones or saline stress.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Transdução de Sinais , Estresse Fisiológico
4.
Cell Mol Biol Lett ; 20(5): 773-87, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26540224

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.


Assuntos
Sistemas CRISPR-Cas/genética , Vetores Genéticos/metabolismo , Alelos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Técnicas de Introdução de Genes/normas , Vetores Genéticos/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Células-Tronco Embrionárias Murinas , Mutagênese Insercional , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Controle de Qualidade , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo
5.
Biochim Biophys Acta ; 1828(2): 623-31, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23022132

RESUMO

Cation-Cl(-) cotransporters (CCCs) are integral membrane proteins which catalyze the coordinated symport of Cl(-) with Na(+) and/or K(+) ions in plant and mammalian cells. Here we describe the first Saccharomyces cerevisiae CCC protein, encoded by the YBR235w open reading frame. Subcellular localization studies showed that this yeast CCC is targeted to the vacuolar membrane. Deletion of the YBR235w gene in a salt-sensitive strain (lacking the plasma-membrane cation exporters) resulted in an increased sensitivity to high KCl, altered vacuolar morphology control and decreased survival upon hyperosmotic shock. In addition, deletion of the YBR235w gene in a mutant strain deficient in K(+) uptake produced a significant growth advantage over the parental strain under K(+)-limiting conditions, and a hypersensitivity to the exogenous K(+)/H(+) exchanger nigericin. These results strongly suggest that we have identified a novel yeast vacuolar ion transporter mediating a K(+)-Cl(-) cotransport and playing a role in vacuolar osmoregulation. Considering its identified function, we propose to refer to the yeast YBR235w gene as VHC1 (vacuolar protein homologous to CCC family 1).


Assuntos
Cátions/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Simportadores/química , Vacúolos/química , Cloretos/química , DNA/química , Eletroquímica/métodos , Genótipo , Concentração de Íons de Hidrogênio , Transporte de Íons , Potenciais da Membrana , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Nigericina/farmacologia , Oligonucleotídeos/química , Fases de Leitura Aberta , Pressão Osmótica , Filogenia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Sorbitol/química , Simportadores/fisiologia
6.
Fungal Genet Biol ; 53: 1-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23454581

RESUMO

Yeast flocculation and invasive growth are processes of great interest in fundamental biology and also relevant in biotechnology and medicine. Hal3 and Vhs3 are moonlighting proteins acting in Saccharomyces cerevisiae both as inhibitors of the Ppz protein phosphatases and as components of a catalytic step in CoA biosynthesis. The double hal3 vhs3 mutant is not viable but, under semi-permissive conditions, the tetO:HAL3 vhs3 strain shows a flocculent phenotype, invasive growth and increased expression of the flocculin-encoding FLO11 gene. We show here that all these effects are caused by hyperactivation of Ppz1 as a result of depletion of its natural inhibitors. The evidence indicates that hyperactivation of Ppz1 would impair potassium transport through the Trk1/Trk2 transporters, thus resulting in a decrease in the intracellular pH and a subsequent increase in the levels of cAMP. Mutation of the TPK2 isoform of protein kinase A blocks the increase in FLO11 expression, and eliminates the flocculent and invasive phenotypes produced by depletion of Hal3 and Vhs3. Interestingly, mutation of RIM101 also significantly decreases FLO11 expression under these conditions. Cells lacking Trk1,2 display an invasive phenotype that is abolished by deletion of FLO8 or by increasing the potassium concentration in the medium. Therefore, our results support a model in which hyperactivation of Ppz phosphatases would result in alteration of potassium transport, activation of Tpk2 and signaling to the FLO11 promoter by means of the Flo8 transcription factor, thus modulating flocculation and invasive growth. This model highlights an unsuspected link between potassium homeostasis and these important morphogenetic events.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Homeostase , Mutação , Potássio/metabolismo , Leveduras/genética , Leveduras/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/genética , Regulação Fúngica da Expressão Gênica , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Leveduras/patogenicidade
7.
Yeast ; 30(10): 395-402, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24251329

RESUMO

Cation­chloride co-transporters serve to transport Cl­ and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation­chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation­chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali­metal cation exporters Nha1 and Ena1-5 and the vacuolar cation­chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali­metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation­chloride co-transporters.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cátions/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Deleção de Genes , Humanos , Transporte de Íons , Cloreto de Lítio/metabolismo , Fenótipo , Cloreto de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/genética , Especificidade por Substrato , Simportadores/genética
8.
Eukaryot Cell ; 10(9): 1241-50, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21724935

RESUMO

Potassium homeostasis is crucial for living cells. In the yeast Saccharomyces cerevisiae, the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H(+)-ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism. Here, we describe a genomewide screen for mutants that present altered tolerance to hygromycin B, spermine, and tetramethylammonium. Two hundred twenty-six mutant strains displayed altered tolerance to all three drugs (202 hypersensitive and 24 hypertolerant), and more than 50% presented a strong or moderate growth defect at a limiting potassium concentration (1 mM). Functional groups such as protein kinases and phosphatases, intracellular trafficking, transcription, or cell cycle and DNA processing were enriched. Essentially, our screen has identified a substantial number of genes that were not previously described to play a direct or indirect role in potassium homeostasis. A subset of 27 representative mutants were selected and subjected to diverse biochemical tests that, in some cases, allowed us to postulate the basis for the observed phenotypes.


Assuntos
Proteínas de Transporte de Cátions/genética , Mutação/genética , Potássio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Homeostase , Higromicina B/farmacologia , Potenciais da Membrana , Fenótipo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Compostos de Amônio Quaternário/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermina/farmacologia
9.
Nat Commun ; 13(1): 1866, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35387987

RESUMO

Type 2 diabetes mellitus represents a major health problem with increasing prevalence worldwide. Limited efficacy of current therapies has prompted a search for novel therapeutic options. Here we show that treatment of pre-diabetic mice with mitochondrially targeted tamoxifen, a potential anti-cancer agent with senolytic activity, improves glucose tolerance and reduces body weight with most pronounced reduction of visceral adipose tissue due to reduced food intake, suppressed adipogenesis and elimination of senescent cells. Glucose-lowering effect of mitochondrially targeted tamoxifen is linked to improvement of type 2 diabetes mellitus-related hormones profile and is accompanied by reduced lipid accumulation in liver. Lower senescent cell burden in various tissues, as well as its inhibitory effect on pre-adipocyte differentiation, results in lower level of circulating inflammatory mediators that typically enhance metabolic dysfunction. Targeting senescence with mitochodrially targeted tamoxifen thus represents an approach to the treatment of type 2 diabetes mellitus and its related comorbidities, promising a complex impact on senescence-related pathologies in aging population of patients with type 2 diabetes mellitus with potential translation into the clinic.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Idoso , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/metabolismo , Humanos , Camundongos , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico
10.
FEMS Yeast Res ; 10(5): 508-17, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20491939

RESUMO

A new YNB medium containing very low concentrations of alkali metal cations has been developed to carry out experiments to study potassium homoeostasis. Physiological characterization of Saccharomyces cerevisiae BY4741 strain and the corresponding mutant lacking the main potassium uptake systems (trk1 trk2) under potassium nonlimiting and limiting concentrations was performed, and novel important differences between both strains were found. At nonlimiting concentrations of KCl, the two strains had a comparable cell size and potassium content. Nevertheless, mutants were hyperpolarized, had lower pH and extruded fewer protons compared with the BY4741 strain. Upon transfer to K(+)-limiting conditions, cells of both strains became hyperpolarized and their cell volume and K(+) content diminished; however, the decrease was more relevant in BY4741. In low potassium, trk1 trk2 cells were not able to accomplish the cell cycle to the same extent as in BY4741. Moreover, K(+) limitation triggered a high-affinity K(+)/Rb(+) uptake process only in BY4741, with the highest affinity being reached as soon as 30 min after transfer to potassium-limiting conditions. By establishing basic cellular parameters under standard growth conditions, this work aims to establish a basis for the investigation of potassium homoeostasis at the system level.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Deleção de Genes , Potássio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte de Cátions/genética , Ciclo Celular , Membrana Celular/fisiologia , Meios de Cultura/química , Citoplasma/química , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Rubídio/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
11.
Cell Rep ; 33(12): 108548, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33357424

RESUMO

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Íntrons/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Camundongos
12.
Front Cell Dev Biol ; 7: 155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31440507

RESUMO

Congenital heart defects, dysmorphic facial features and intellectual developmental disorders (CHDFIDD) syndrome in humans was recently associated with mutation in CDK13 gene. In order to assess the loss of function of Cdk13 during mouse development, we employed gene trap knock-out (KO) allele in Cdk13 gene. Embryonic lethality of Cdk13-deficient animals was observed by the embryonic day (E) 16.5, while live embryos were observed on E15.5. At this stage, improper development of multiple organs has been documented, partly resembling defects observed in patients with mutated CDK13. In particular, overall developmental delay, incomplete secondary palate formation with variability in severity among Cdk13-deficient animals or complete midline deficiency, kidney failure accompanied by congenital heart defects were detected. Based on further analyses, the lethality at this stage is a result of heart failure most likely due to multiple heart defects followed by insufficient blood circulation resulting in multiple organs dysfunctions. Thus, Cdk13 KO mice might be a very useful model for further studies focused on delineating signaling circuits and molecular mechanisms underlying CHDFIDD caused by mutation in CDK13 gene.

13.
Cell Biosci ; 9: 21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834109

RESUMO

Zinc finger 644 (Zfp644 in mouse, ZNF644 in human) gene is a transcription factor whose mutation S672G is considered a potential genetic factor of inherited high myopia. ZNF644 interacts with G9a/GLP complex, which functions as a H3K9 methyltransferase to silence transcription. In this study, we generated mouse models to unravel the mechanisms leading to symptoms associated with high myopia. Employing TALEN technology, two mice mutants were generated, either with the disease-carrying mutation (Zfp644 S673G ) or with a truncated form of Zfp644 (Zfp644 Δ8 ). Eye morphology and visual functions were analysed in both mutants, revealing a significant difference in a vitreous chamber depth and lens diameter, however the physiological function of retina was preserved as found under the high-myopia conditions. Our findings prove that ZNF644/Zfp644 is involved in the development of high-myopia, indicating that mutations such as, Zfp644 S673G and Zfp644 Δ8 are causative for changes connected with the disease. The developed models represent a valuable tool to investigate the molecular basis of myopia pathogenesis and its potential treatment.

14.
Mitochondrion ; 8(2): 117-29, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18024212

RESUMO

Valinomycin and nigericin are potassium ionophores acting selectively on the mitochondrial inner membrane of Saccharomyces cerevisiae [Kovac, L., Bohmerova, E., Butko, P., 1982a. Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae. Biochim. Biophys. Acta 721, 341-348]. However, the molecular mechanism of their action is not understood. Here we show that their selective effect on mitochondrial membranes is not caused by the pleiotropic drug resistance system. To identify the molecular components mediating the action of ionophores we isolated several mutants specifically resistant to valinomycin and/or nigericin. In contrast to the parental strain, these mutants do not form respiratory-deficient cells in the presence of ionophores. Moreover, all mutants harbor extensively fragmented mitochondria and these morphological defects can be alleviated by the ionophores. Interestingly, we observed that these mitochondrial defects may be accompanied by changes in vacuolar dynamics. Our results demonstrate that the classical genetic approach can provide a starting point for the analysis of components involved in the action of ionophores on mitochondria-related processes in eukaryotic cell.


Assuntos
Farmacorresistência Fúngica/genética , Ionóforos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Nigericina/farmacologia , Saccharomyces cerevisiae/genética , Valinomicina/farmacologia , Membranas Mitocondriais/ultraestrutura , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Raios Ultravioleta
15.
Genetics ; 171(2): 517-26, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16020778

RESUMO

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.


Assuntos
Cátions/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Primers do DNA , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Dilatação Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/genética , Mutação/genética , Nigericina/toxicidade , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
16.
PLoS One ; 11(6): e0158424, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27362362

RESUMO

Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.


Assuntos
Calcineurina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae , ATPase Trocadora de Sódio-Potássio/genética , Estresse Fisiológico/genética , Fatores de Transcrição/fisiologia , Transporte Ativo do Núcleo Celular/genética , Sítios de Ligação/genética , Calcineurina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Organismos Geneticamente Modificados , Regiões Promotoras Genéticas , Transporte Proteico , Elementos Reguladores de Transcrição , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Transcrição/metabolismo
17.
Mol Cell Biol ; 34(24): 4420-35, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25266663

RESUMO

The yeast Saccharomyces cerevisiae has two main high-affinity inorganic phosphate (Pi) transporters, Pho84 and Pho89, that are functionally relevant at acidic/neutral pH and alkaline pH, respectively. Upon Pi starvation, PHO84 and PHO89 are induced by the activation of the PHO regulon by the binding of the Pho4 transcription factor to specific promoter sequences. We show that PHO89 and PHO84 are induced by alkalinization of the medium with different kinetics and that the network controlling Pho89 expression in response to alkaline pH differs from that of other members of the PHO regulon. In addition to Pho4, the PHO89 promoter is regulated by the transcriptional activator Crz1 through the calcium-activated phosphatase calcineurin, and it is under the control of several repressors (Mig2, Nrg1, and Nrg2) coordinately regulated by the Snf1 protein kinase and the Rim101 transcription factor. This network mimics the one regulating expression of the Na(+)-ATPase gene ENA1, encoding a major determinant for Na(+) detoxification. Our data highlight a scenario in which the activities of Pho89 and Ena1 are functionally coordinated to sustain growth in an alkaline environment.


Assuntos
Regulação Fúngica da Expressão Gênica , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Calcineurina/metabolismo , Meios de Cultura/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Fúngico , Concentração de Íons de Hidrogênio , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Simportadores de Próton-Fosfato/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Adv Microb Physiol ; 64: 1-63, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24797924

RESUMO

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.


Assuntos
Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Sódio/metabolismo , Biologia de Sistemas , Transporte Biológico , Cátions Monovalentes/metabolismo , Homeostase , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Fungal Biol ; 114(2-3): 144-50, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20960970

RESUMO

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.


Assuntos
Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/fisiologia , Tolerância ao Sal/fisiologia , Transporte Biológico , Cátions , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Regulação Fúngica da Expressão Gênica , Homeostase , Higromicina B/farmacologia , Lítio/metabolismo , Lítio/farmacologia , Potenciais da Membrana , Mutação , Potássio/metabolismo , Potássio/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Especificidade da Espécie
20.
Can J Microbiol ; 53(4): 533-5, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17612610

RESUMO

The cellular functions are strongly influenced by the composition of the environment. In particular, phenotypes of microbial strains are modulated by concentrations of ions in the culture medium, and differences in element levels may be responsible for a phenotypic variability observed when microbial strains are grown on synthetic versus complex media. In this report, we analyzed the levels of nine elements (magnesium, potassium, sodium, calcium, iron, copper, manganese, zinc, and phosphorus) and sulphate ions in commercially available peptone and yeast extract and compared them with those in yeast nitrogen base routinely used for preparation of synthetic minimal media. We observed that whereas some elements are present at similar levels, the levels of others differ by a factor as high as 20. The observed differences should be taken into account when interpreting different phenotypes observed for microbial strains grown on synthetic versus complex media.


Assuntos
Meios de Cultura/química , Leveduras/genética , Elementos Químicos , Fenótipo , Espectrofotometria , Leveduras/crescimento & desenvolvimento
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