RESUMEN
Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.
Asunto(s)
Núcleo Celular/genética , Sondas de ADN/metabolismo , ADN de Forma Z/aislamiento & purificación , Animales , Células CHO , Línea Celular , Inmunoprecipitación de Cromatina , Cricetinae , Cricetulus , ADN de Forma Z/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Células MCF-7RESUMEN
BACKGROUND: The optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability. RESULTS: In this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction. CONCLUSIONS: Our data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production.
Asunto(s)
Proteínas de Unión al ADN/genética , Inmunoglobulina G/biosíntesis , Factores de Transcripción/genética , Animales , Células CHO , Cricetulus , Medios de Cultivo , Doxiciclina , Retículo Endoplásmico , Humanos , Plásmidos , Isoformas de Proteínas/genética , Factores de Transcripción del Factor Regulador X , Temperatura , Transfección , Proteína 1 de Unión a la X-BoxRESUMEN
In the natural photosynthesis process, light harvesting complexes (LHCs) absorb light and pass excitation energy to photosystem I (PSI) and photosystem II (PSII). In this study, we have used nanocrystalline quantum dots (NQDs) as an artificial LHC by integrating them with PSI to extend their spectral range. We have performed photoluminescence (PL) and ultrafast time-resolved absorption measurements to investigate this process. Our PL experiments showed that emission from the NQDs is quenched, and the fluorescence from PSI is enhanced. Transient absorption and bleaching results can be explained by fluorescence resonance energy transfer (FRET) from the NQDs to the PSI. This nonradiative energy transfer occurs in â¼6 ps. Current-voltage (I-V) measurements on the composite NQD-PSI samples demonstrate a clear photoresponse.
Asunto(s)
Transferencia de Energía , Fenómenos Ópticos , Complejo de Proteína del Fotosistema I/química , Puntos Cuánticos , Absorción , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Chlamydomonas reinhardtii/enzimología , Transporte de Electrón , Transferencia Resonante de Energía de Fluorescencia , Luz , Complejo de Proteína del Fotosistema I/metabolismoRESUMEN
Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Farmacorresistencia Fúngica Múltiple , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Rotenona/farmacología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Mitocondrias/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Delta mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Delta cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Delta cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Delta cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.
Asunto(s)
Carboxiliasas/metabolismo , Citocinesis , Fosfatidiletanolaminas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Carboxiliasas/química , Carboxiliasas/genética , Medios de Cultivo/metabolismo , Datos de Secuencia Molecular , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de SecuenciaRESUMEN
We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His(6)-tagged PS1 could be purified with a yield of 80-90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His(6)-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain.