RESUMEN
Dosage-sensitive modifier screening is a powerful tool for linking genes to biological processes. Use of chromosomal deletions permits sampling the effects of removing groups of genes related by position on the chromosome. Here, we explore the use of inducible microRNA transgenes as a complement to deficiency-based modifier screens. miRNAs are predicted to have hundreds of targets. miRNA overexpression provides an efficient means to reduces expression of large gene sets. A collection of transgenes was prepared to allow overexpression of 89 miRNAs or miRNA clusters. These transgenes and a set of genomic deficiencies were screened for their ability to modify the bristle phenotype of the cell-cycle regulator minus. Sixteen miRNAs were identified as dominant suppressors, while the deficiency screen uncovered four genomic regions that contain a dominant suppressor. Comparing the genes uncovered by the deletions with predicted miRNA targets uncovered a small set of candidate suppressors. Two candidates were identified as suppressors of the minus phenotype, Cullin-4 and CG5199/Cut8. Additionally, we show that Cullin-4 acts through its substrate receptor Cdt2 to suppress the minus phenotype. We suggest that inducible microRNA transgenes are a useful complement to deficiency-based modifier screens.
Asunto(s)
Drosophila/genética , MicroARNs/genética , Transgenes , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Dosificación de Gen , Expresión Génica , Orden Génico , Vectores Genéticos/genética , Proteínas de Choque Térmico/genética , Ligasas/genética , Ligasas/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , FenotipoRESUMEN
Energy homeostasis depends on insulin signaling in metazoans. Insulin levels reflect the nutritional status of the animal to control levels of circulating sugar and regulate storage of resources in the form of glycogen and fat. Over the past several years, evidence has begun to accumulate that insulin production and secretion, as well as cellular responsiveness to insulin, are subject to regulation by microRNAs. Here we present evidence that miR-14 acts in the insulin-producing neurosecretory cells in the adult Drosophila brain to control metabolism. miR-14 acts in these cells through its direct target, sugarbabe. sugarbabe encodes a predicted zinc finger protein that regulates insulin gene expression in the neurosecretory cells. Regulation of sugarbabe levels by nutrients and by miR-14 combines to allow the fly to manage resource mobilization in a nutritionally variable environment.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/metabolismo , Insulina/metabolismo , MicroARNs/fisiología , Factores de Transcripción/fisiología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Drosophila/genética , Metabolismo Energético , Homeostasis , Insulina/biosíntesis , Neurosecreción , Dedos de ZincRESUMEN
A large number of CHO glycosylation mutants were isolated by Ricinus communis agglutinin-I (RCA-I). Complementation tests revealed that all these mutant lines possessed a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. Sequencing analyses on the GnT I cDNAs isolated from 16 mutant lines led to the identification of nine different single base pair mutations. Some mutations result in a premature stop codon whereas others cause a single amino acid substitution in the GnT I protein. Interestingly, expression of the normal GnT I cDNA in mutant cells resulted in enhanced sialylation of N-glycans. The sialylation of recombinant erythropoietin (EPO) produced in mutant cells that were co-transfected with GnT I was enhanced compared to that of EPO produced in wild type CHO cells. The enhanced sialylation of EPO produced by JW152 cells in the presence of GnT I over CHO-K1 cells is a result of increased sialylated glycan structures with higher antennary branching. These findings represent a new strategy that may be utilized by the biotechnology industry to produce highly sialylated therapeutic glycoproteins.
Asunto(s)
Eritropoyetina/metabolismo , N-Acetilglucosaminiltransferasas/genética , Ácido N-Acetilneuramínico/metabolismo , Lectinas de Plantas/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Glicosilación , Mutación Puntual , Proteínas RecombinantesRESUMEN
A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.
Asunto(s)
Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Aparato de Golgi/metabolismo , Transportadores de Anión Orgánico/metabolismo , Simportadores/metabolismo , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , ADN Complementario/genética , ADN Complementario/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Galectinas/metabolismo , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Transportadores de Anión Orgánico/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Simportadores/genética , TransfecciónRESUMEN
In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures.
Asunto(s)
Inhibidores de Caspasas , Clorometilcetonas de Aminoácidos/farmacología , Animales , Reactores Biológicos , Células CHO , Caspasa 8/análisis , Caspasa 8/genética , Caspasa 8/metabolismo , Caspasa 9/análisis , Caspasa 9/genética , Caspasa 9/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Clonación Molecular , Cricetinae , Cricetulus , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Biblioteca de Genes , Humanos , Mutación , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Bcl-2 family proteins play a crucial role in the regulation of the mitochondrial pathway that leads to apoptosis. Members of the Bcl-2 family can be divided into the anti-apoptotic proteins such as Bcl-2 and Bcl-X(L), and the pro-apoptotic proteins such as Bax and Bak and the BH3-only proteins. In this study, siRNA constructs to silence the Bax and Bak genes in Chinese hamster ovary (CHO) cells were generated. Stable CHO cell lines in which the expression of Bax and Bak were significantly knocked down were screened by Western blot analysis and confirmed by RT-PCR. CHO cells with both Bax and Bak knocked down showed a clear resistance against cytotoxic lectins and UV irradiation-induced apoptosis. Compared to original CHO-K1 cells, these cells also survived longer when cultured under extreme conditions such as complete nutrient depletion or in high-osmolality medium. CHO cells with both Bax and Bak genes knocked down displayed an extended lifespan as well as higher viable cell densities in fed-batch cultures, both in adherent form on microcarrier beads and in suspension. The IFN-gamma productivity by a rCHO IFN-gamma cell line in which both Bak and Bax were knocked down increased by 35% compared to the control cells. These results indicate that the genetic inactivation of Bax and Bak in recombinant CHO cells can be an effective strategy in delaying the onset of apoptosis in batch and fed-batch cultures.
Asunto(s)
Supervivencia Celular/genética , Interferencia de ARN , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de la radiación , Western Blotting , Células CHO , Técnicas de Cultivo de Célula , Clonación Molecular , Cricetinae , Cricetulus , Cartilla de ADN , Datos de Secuencia Molecular , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rayos Ultravioleta , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
The activation of caspases represents a crucial turning point during a batch or a fed-batch culture of mammalian cells. It not only affects the quantity but also the quality of the recombinant glycoprotein produced. In this study, the activation of various caspases, the release of intracellular sialidase and the changes in sialylation pattern of a recombinant product, erythropoietin (EPO), in the culture medium were analyzed in both batch and fed-batch cultures. In both setups, all caspase activities peaked at the culture time point at which decline of cell viability was most pronounced. In addition, the release of intracellular lactate dehydrogenase (LDH) was also tracked during these cultures. The increase in LDH activity in the medium coincided with the increase of intracellular caspase activities, the release of sialidase and the observed decline in cell viability, suggesting that the LDH activity in the medium can be used as an indirect indicator of apoptotic cell death in bioreactors. Isoelectric focusing (IEF) coupled with double blotting was employed to analyze the changes in sialylation pattern of the recombinant EPO. This assay resulted in a prompt resolution of secreted EPO isoforms in a time course format. IEF profile of batch culture showed relatively consistent product sialylation compared to fed-batch culture, which showed gradual band shifts towards the isoforms with fewer sialic acid as the culture progressed. These data provided a guideline for the optimal time point to terminate the culture and collect products in batch and fed-batch cultures.