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1.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068004

RESUMO

Enzymes with fructan exohydrolase (FEH) activity are present not only in fructan-synthesizing species but also in non-fructan plants. This has led to speculation about their functions in non-fructan species. Here, a cell wall invertase-related Zm-6&1-FEH2 with no "classical" invertase motif was identified in maize. Following heterologous expression in Pichia pastoris and in Nicotiana benthamiana leaves, the enzyme activity of recombinant Zm-6&1-FEH2 displays substrate specificity with respect to inulin and levan. Subcellular localization showed Zm-6&1-FEH2 exclusively localized in the apoplast, and its expression profile was strongly dependent on plant development and in response to drought and abscisic acid. Furthermore, formation of 1-kestotriose, an oligofructan, was detected in vivo and in vitro and could be hydrolyzed by Zm-6&1-FEH2. In summary, these results support that Zm-6&1-FEH2 enzyme from maize can degrade both inulin-type and levan-type fructans, and the implications of the co-existence of Zm-6&1-FEH2 and 1-kestotriose are discussed.


Assuntos
Frutanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Inulina/metabolismo , Trissacarídeos/metabolismo , Zea mays/metabolismo , Glicosídeo Hidrolases/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Tabaco/genética , Tabaco/metabolismo , Zea mays/crescimento & desenvolvimento
2.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33925940

RESUMO

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.


Assuntos
Antimônio/toxicidade , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Estresse Oxidativo/genética , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Telômero/metabolismo
3.
Int J Food Microbiol ; 347: 109200, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33894461

RESUMO

The physicochemical characteristics and yeasts diversity in honey samples from 17 species of stingless bees of the genera Nannotrigona, Melipona, Plebeia, Scraptotrigona, and Tetragonisca cultivated in Southern Brazil were determined. The sugar content, moisture, water activity, pH, reducing sugars/total sugar ratio, and total yeast population varied significantly among the honey from the different bee species. The highest yeast population was found in the Plebeia's honey samples and correlated with their high water-activity. Sixteen yeast species were identified based on the nuclear large subunit (26S) ribosomal RNA partial sequences. The genera Starmerella and Zygosaccharomyces were found predominant, with a high prevalence of Starmerella sp., S. etchellsii, and S. apicola. Some yeast species were only identified in honey samples from specific bee species indicating a close relationship between the yeasts and the insects. For the first time, Wickerhamomyces sydowiorum in honey is being reported. In general, the yeast species isolated from stingless bee honey samples demonstrated high osmotolerance and low sugar assimilation.


Assuntos
Abelhas/metabolismo , Mel/microbiologia , Leveduras/classificação , Leveduras/isolamento & purificação , Animais , Biodiversidade , Brasil , Saccharomycetales/genética , Saccharomycetales/isolamento & purificação , Açúcares , Fermento Seco , Leveduras/genética
4.
Methods Mol Biol ; 2280: 231-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751439

RESUMO

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Saccharomycetales/crescimento & desenvolvimento , Oxirredutases do Álcool/metabolismo , Técnicas de Cultura Celular por Lotes , Técnicas Biossensoriais , Cromatografia por Troca Iônica , Clonagem Molecular , Formaldeído/análise , Formaldeído/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Metanol/análise , Metanol/metabolismo , Saccharomycetales/enzimologia , Saccharomycetales/genética
5.
Methods Mol Biol ; 2280: 249-260, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33751440

RESUMO

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.


Assuntos
L-Lactato Desidrogenase (Citocromo)/metabolismo , Engenharia de Proteínas/métodos , Saccharomycetales/crescimento & desenvolvimento , Técnicas Biossensoriais , Cromatografia de Afinidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , L-Lactato Desidrogenase (Citocromo)/genética , Ácido Láctico/análise , Plasmídeos/genética , Regiões Promotoras Genéticas , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transformação Genética
6.
Food Microbiol ; 97: 103750, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33653523

RESUMO

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.


Assuntos
Enterococcaceae/crescimento & desenvolvimento , Etanol/metabolismo , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Enterococcaceae/química , Enterococcaceae/genética , Enterococcaceae/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Saccharomycetales/química , Saccharomycetales/genética , Propriedades de Superfície
7.
Biochim Biophys Acta Gen Subj ; 1865(6): 129893, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33731300

RESUMO

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has now spread worldwide to infect over 110 million people, with approximately 2.5 million reported deaths. A safe and effective vaccine remains urgently needed. METHOD: We constructed three variants of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (residues 331-549) in yeast as follows: (1) a "wild type" RBD (RBD219-WT), (2) a deglycosylated form (RBD219-N1) by deleting the first N-glycosylation site, and (3) a combined deglycosylated and cysteine-mutagenized form (C538A-mutated variant (RBD219-N1C1)). We compared the expression yields, biophysical characteristics, and functionality of the proteins produced from these constructs. RESULTS AND CONCLUSIONS: These three recombinant RBDs showed similar secondary and tertiary structure thermal stability and had the same affinity to their receptor, angiotensin-converting enzyme 2 (ACE-2), suggesting that the selected deletion or mutations did not cause any significant structural changes or alteration of function. However, RBD219-N1C1 had a higher fermentation yield, was easier to purify, was not hyperglycosylated, and had a lower tendency to form oligomers, and thus was selected for further vaccine development and evaluation. GENERAL SIGNIFICANCE: By genetic modification, we were able to design a better-controlled and more stable vaccine candidate, which is an essential and important criterion for any process and manufacturing of biologics or drugs for human use.


Assuntos
/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , /imunologia , Saccharomycetales/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Domínios Proteicos , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
8.
Nat Microbiol ; 6(2): 196-208, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33398099

RESUMO

Microbial communities often undergo intricate compositional changes yet also maintain stable coexistence of diverse species. The mechanisms underlying long-term coexistence remain unclear as system-wide studies have been largely limited to engineered communities, ex situ adapted cultures or synthetic assemblies. Here, we show how kefir, a natural milk-fermenting community of prokaryotes (predominantly lactic and acetic acid bacteria) and yeasts (family Saccharomycetaceae), realizes stable coexistence through spatiotemporal orchestration of species and metabolite dynamics. During milk fermentation, kefir grains (a polysaccharide matrix synthesized by kefir microorganisms) grow in mass but remain unchanged in composition. In contrast, the milk is colonized in a sequential manner in which early members open the niche for the followers by making available metabolites such as amino acids and lactate. Through metabolomics, transcriptomics and large-scale mapping of inter-species interactions, we show how microorganisms poorly suited for milk survive in-and even dominate-the community, through metabolic cooperation and uneven partitioning between grain and milk. Overall, our findings reveal how inter-species interactions partitioned in space and time lead to stable coexistence.


Assuntos
Bactérias/metabolismo , Kefir/microbiologia , Interações Microbianas , Microbiota/fisiologia , Saccharomycetales/metabolismo , Ácido Acético/metabolismo , Bactérias/classificação , Bactérias/genética , Fermentação , Ácido Láctico/metabolismo , Metabolômica , Microbiota/genética , Filogenia , Saccharomycetales/classificação , Saccharomycetales/genética
9.
Protein Expr Purif ; 177: 105750, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32920041

RESUMO

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.


Assuntos
Proteínas de Insetos/imunologia , Leishmania/imunologia , Vacinas contra Leishmaniose/imunologia , Phlebotomus/química , Proteínas e Peptídeos Salivares/imunologia , Animais , Clonagem Molecular , Feminino , Fermentação , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Leishmania/química , Vacinas contra Leishmaniose/genética , Vacinas contra Leishmaniose/metabolismo , Leishmaniose Cutânea/prevenção & controle , Peso Molecular , Phlebotomus/fisiologia , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo
10.
Methods Mol Biol ; 2191: 29-48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865737

RESUMO

For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.


Assuntos
Channelrhodopsins/genética , Biologia Molecular/métodos , Nanocompostos/química , Saccharomycetales/genética , Fenômenos Biofísicos , Channelrhodopsins/química , Chlamydomonas/química , Regulação da Expressão Gênica/genética , Luz , Proteínas de Plantas/química , Espectroscopia de Infravermelho com Transformada de Fourier
11.
Methods Mol Biol ; 2196: 15-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32889709

RESUMO

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Mutação , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/genética , Ordem dos Genes , Vetores Genéticos/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transformação Genética
12.
Methods Mol Biol ; 2196: 167-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32889720

RESUMO

Budding yeast, as a eukaryotic model organism, has well-defined genetic information and a highly efficient recombination system, making it a good host to produce exogenous chemicals. Since most metabolic pathways require multiple genes to function in coordination, it is usually laborious and time-consuming to construct a working pathway. To facilitate the construction and optimization of multicomponent exogenous pathways in yeast, we recently developed a method called YeastFab Assembly, which includes three steps: (1) make standard and reusable genetic parts, (2) construct transcription units from characterized parts, and (3) assemble a complete pathway. Here we describe a detailed protocol of this method.


Assuntos
Clonagem Molecular , Vetores Genéticos/genética , Engenharia Metabólica , Leveduras/genética , Leveduras/metabolismo , Clonagem Molecular/métodos , Expressão Gênica , Ordem dos Genes , Engenharia Metabólica/métodos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Saccharomycetales/genética , Saccharomycetales/metabolismo , Biologia Sintética/métodos , Transformação Genética
13.
Methods Mol Biol ; 2178: 479-503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33128767

RESUMO

In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.


Assuntos
Celulases , Escherichia coli , Lignina/química , Proteínas Recombinantes de Fusão , Saccharomycetales , Celulases/biossíntese , Celulases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomycetales/enzimologia , Saccharomycetales/genética
14.
Cell Mol Life Sci ; 78(6): 2665-2681, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33341920

RESUMO

Messenger RNA (mRNA) localisation enables a high degree of spatiotemporal control on protein synthesis, which contributes to establishing the asymmetric protein distribution required to set up and maintain cellular polarity. As such, a tight control of mRNA localisation is essential for many biological processes during development and in adulthood, such as body axes determination in Drosophila melanogaster and synaptic plasticity in neurons. The mechanisms controlling how mRNAs are localised, including diffusion and entrapment, local degradation and directed active transport, are largely conserved across evolution and have been under investigation for decades in different biological models. In this review, we will discuss the standing of the field regarding directional mRNA transport in light of the recent discovery that RNA can hitchhike on cytoplasmic organelles, such as endolysosomes, and the impact of these transport modalities on our understanding of neuronal function during development, adulthood and in neurodegeneration.


Assuntos
Neurônios/metabolismo , RNA Mensageiro/metabolismo , Animais , Axônios/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Humanos , Proteínas Motores Moleculares/metabolismo , Neurônios/citologia , Oócitos/metabolismo , Transporte de RNA , Saccharomycetales/genética , Saccharomycetales/metabolismo
15.
Sci Rep ; 10(1): 21779, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311634

RESUMO

The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by high-performance liquid chromatography, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2.


Assuntos
/química , Glicoproteína da Espícula de Coronavírus/química , Humanos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomycetales/química , Saccharomycetales/genética , Saccharomycetales/metabolismo , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/genética
16.
Mol Cell Biol ; 40(22)2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32900821

RESUMO

The rRNA gene, which consists of tandem repetitive arrays (ribosomal DNA [rDNA] repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast Saccharomyces cerevisiae is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in aging mammalian cells. Using quantitative single-cell analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to levels in 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice, and two of them were found in the conserved region in budding yeast. We established yeast strains with the old-mouse-specific mutations and found that they shortened the life span of the cells. Our findings suggest that rDNA is also fragile in mammalian cells and that alterations within this region have a profound effect on cellular function.


Assuntos
Envelhecimento/genética , Variações do Número de Cópias de DNA , Metilação de DNA , DNA Ribossômico/metabolismo , Envelhecimento/metabolismo , Animais , Células da Medula Óssea/metabolismo , Senescência Celular/genética , Sequência Conservada , Análise Mutacional de DNA , Sequência de DNA Instável , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Saccharomycetales/genética , Análise de Célula Única , Especificidade da Espécie , Transcrição Genética
17.
BMC Evol Biol ; 20(1): 109, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32842959

RESUMO

BACKGROUND: For decades, codon usage has been used as a measure of adaptation for translational efficiency and translation accuracy of a gene's coding sequence. These patterns of codon usage reflect both the selective and mutational environment in which the coding sequences evolved. Over this same period, gene transfer between lineages has become widely recognized as an important biological phenomenon. Nevertheless, most studies of codon usage implicitly assume that all genes within a genome evolved under the same selective and mutational environment, an assumption violated when introgression occurs. In order to better understand the effects of introgression on codon usage patterns and vice versa, we examine the patterns of codon usage in Lachancea kluyveri, a yeast which has experienced a large introgression. We quantify the effects of mutation bias and selection for translation efficiency on the codon usage pattern of the endogenous and introgressed exogenous genes using a Bayesian mixture model, ROC SEMPPR, which is built on mechanistic assumptions about protein synthesis and grounded in population genetics. RESULTS: We find substantial differences in codon usage between the endogenous and exogenous genes, and show that these differences can be largely attributed to differences in mutation bias favoring A/T ending codons in the endogenous genes while favoring C/G ending codons in the exogenous genes. Recognizing the two different signatures of mutation bias and selection improves our ability to predict protein synthesis rate by 42% and allowed us to accurately assess the decaying signal of endogenous codon mutation and preferences. In addition, using our estimates of mutation bias and selection, we identify Eremothecium gossypii as the closest relative to the exogenous genes, providing an alternative hypothesis about the origin of the exogenous genes, estimate that the introgression occurred ∼6×108 generation ago, and estimate its historic and current selection against mismatched codon usage. CONCLUSIONS: Our work illustrates how mechanistic, population genetic models like ROC SEMPPR can separate the effects of mutation and selection on codon usage and provide quantitative estimates from sequence data.


Assuntos
Uso do Códon , Genética Populacional , Modelos Genéticos , Saccharomycetales/genética , Seleção Genética , Teorema de Bayes , Mutação
18.
Nucleic Acids Res ; 48(15): 8243-8254, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32720681

RESUMO

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes - pho1, pho84, and tgp1 - that are repressed under phosphate-replete conditions by 5' flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor 'precocious' lncRNA 3'-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.


Assuntos
Regulação Fúngica da Expressão Gênica , RNA Longo não Codificante/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Transcrição Genética , Homeostase , Nutrientes/metabolismo , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , RNA Mensageiro , Regulon , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
19.
RNA ; 26(10): 1464-1480, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32631843

RESUMO

Many eukaryotes use RNA processing, including alternative splicing, to express multiple gene products from the same gene. The budding yeast Saccharomyces cerevisiae has been successfully used to study the mechanism of splicing and the splicing machinery, but alternative splicing in yeast is relatively rare and has not been extensively studied. Alternative splicing of SKI7/HBS1 is widely conserved, but yeast and a few other eukaryotes have replaced this one alternatively spliced gene with a pair of duplicated, unspliced genes as part of a whole genome doubling (WGD). We show that other examples of alternative splicing known to have functional consequences are widely conserved within Saccharomycotina. A common mechanism by which alternative splicing has disappeared is by replacement of an alternatively spliced gene with duplicate unspliced genes. This loss of alternative splicing does not always take place soon after duplication, but can take place after sufficient time has elapsed for speciation. Saccharomycetaceae that diverged before WGD use alternative splicing more frequently than S. cerevisiae, suggesting that WGD is a major reason for infrequent alternative splicing in yeast. We anticipate that WGDs in other lineages may have had the same effect. Having observed that two functionally distinct splice-isoforms are often replaced by duplicated genes allowed us to reverse the reasoning. We thereby identify several splice isoforms that are likely to produce two functionally distinct proteins because we find them replaced by duplicated genes in related species. We also identify some alternative splicing events that are not conserved in closely related species and unlikely to produce functionally distinct proteins.


Assuntos
Processamento Alternativo/genética , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Evolução Molecular , Duplicação Gênica/genética , Genoma/genética , Isoformas de Proteínas/genética
20.
Nat Commun ; 11(1): 3494, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661402

RESUMO

Cellular processes are inherently noisy, and the selection for accurate responses in presence of noise has likely shaped signalling networks. Here, we investigate the trade-off between accuracy of information transmission and its energetic cost for a mitogen-activated protein kinase (MAPK) signalling cascade. Our analysis of the pheromone response pathway of budding yeast suggests that dose-dependent induction of the negative transcriptional feedbacks in this network maximizes the information per unit energetic cost, rather than the information transmission capacity itself. We further demonstrate that futile cycling of MAPK phosphorylation and dephosphorylation has a measurable effect on growth fitness, with energy dissipation within the signalling cascade thus likely being subject to evolutionary selection. Considering optimization of accuracy versus the energetic cost of information processing, a concept well established in physics and engineering, may thus offer a general framework to understand the regulatory design of cellular signalling systems.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
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