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1.
Mol Syst Biol ; 6: 393, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20664639

ABSTRACT

Complexity of cellular response to oxidative stress (OS) stems from its wide-ranging damage to nucleic acids, proteins, carbohydrates, and lipids. We have constructed a systems model of OS response (OSR) for Halobacterium salinarum NRC-1 in an attempt to understand the architecture of its regulatory network that coordinates this complex response. This has revealed a multi-tiered OS-management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids and gas vesicles, metal trafficking, and various other aspects of metabolism. Through experimental validation of interactions within the OSR regulatory network, we show that despite their inability to directly sense reactive oxygen species, general transcription factors have an important function in coordinating this response. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was earlier constructed from cellular responses to diverse environmental perturbations--this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of gamma rays.


Subject(s)
Archaeal Proteins/metabolism , Halobacterium salinarum/metabolism , Oxidative Stress , Oxygen/metabolism , Reactive Oxygen Species/metabolism , Archaeal Proteins/genetics , Carotenoids/metabolism , Catalase/metabolism , Dose-Response Relationship, Drug , Gene Expression Regulation, Archaeal , Genotype , Halobacterium salinarum/drug effects , Halobacterium salinarum/enzymology , Halobacterium salinarum/genetics , Halobacterium salinarum/growth & development , Hydrogen Peroxide/pharmacology , Models, Biological , Mutation , Oxidants/pharmacology , Oxidation-Reduction , Oxidative Stress/drug effects , Oxidative Stress/genetics , Paraquat/pharmacology , Peroxidases/metabolism , Phenotype , Protein Transport , Rhodopsins, Microbial/metabolism , Superoxide Dismutase/metabolism , Superoxides/metabolism , Time Factors , Transcription, Genetic
2.
Mol Syst Biol ; 5: 285, 2009.
Article in English | MEDLINE | ID: mdl-19536208

ABSTRACT

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.


Subject(s)
Genes, Archaeal , Operon , Promoter Regions, Genetic , Transcription Factors/genetics , Computer Simulation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Gene Regulatory Networks , Genome, Bacterial , Halobacterium salinarum/genetics , Halobacterium salinarum/physiology , Models, Genetic , Monte Carlo Method , RNA/genetics , Reproducibility of Results , Transcription Factors/metabolism , Transcription, Genetic
3.
Nat Rev Microbiol ; 7(4): 297-305, 2009 04.
Article in English | MEDLINE | ID: mdl-19252506

ABSTRACT

Technologies to synthesize and transplant a complete genome into a cell have opened limitless potential to redesign organisms for complex, specialized tasks. However, large-scale re-engineering of a biological circuit will require systems-level optimization that will come from a deep understanding of operational relationships among all the constituent parts of a cell. The integrated framework necessary for conducting such complex bioengineering requires the convergence of systems and synthetic biology. Here, we review the status of these rapidly developing interdisciplinary fields of biology and provide a perspective on plausible venues for their merger.


Subject(s)
Biotechnology/methods , Models, Biological , Systems Biology/methods
4.
Nature ; 454(7208): 1119-22, 2008 Aug 28.
Article in English | MEDLINE | ID: mdl-18668041

ABSTRACT

Natural selection dictates that cells constantly adapt to dynamically changing environments in a context-dependent manner. Gene-regulatory networks often mediate the cellular response to perturbation, and an understanding of cellular adaptation will require experimental approaches aimed at subjecting cells to a dynamic environment that mimics their natural habitat. Here we monitor the response of Saccharomyces cerevisiae metabolic gene regulation to periodic changes in the external carbon source by using a microfluidic platform that allows precise, dynamic control over environmental conditions. We show that the metabolic system acts as a low-pass filter that reliably responds to a slowly changing environment, while effectively ignoring fast fluctuations. The sensitive low-frequency response was significantly faster than in predictions arising from our computational modelling, and this discrepancy was resolved by the discovery that two key galactose transcripts possess half-lives that depend on the carbon source. Finally, to explore how induction characteristics affect frequency response, we compare two S. cerevisiae strains and show that they have the same frequency response despite having markedly different induction properties. This suggests that although certain characteristics of the complex networks may differ when probed in a static environment, the system has been optimized for a robust response to a dynamically changing environment.


Subject(s)
Environment , Gene Expression Regulation, Fungal , Metabolic Networks and Pathways/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Carbon/metabolism , Carbon/pharmacology , Culture Media/chemistry , Culture Media/pharmacology , Galactose/metabolism , Galactose/pharmacology , Glucose/metabolism , Glucose/pharmacology , Half-Life , Microfluidics , RNA Stability , RNA, Fungal/genetics , RNA, Fungal/metabolism , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/drug effects
5.
Mol Syst Biol ; 3: 127, 2007.
Article in English | MEDLINE | ID: mdl-17667949

ABSTRACT

Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.


Subject(s)
Gene Regulatory Networks , Protein Processing, Post-Translational/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/metabolism , Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Green Fluorescent Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae/cytology
6.
Mol Syst Biol ; 1: 2005.0024, 2005.
Article in English | MEDLINE | ID: mdl-16729059

ABSTRACT

Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative description of the dynamics of many important cellular processes. Such a description will require novel experimental techniques that enable the generation of time-series data for the governing regulatory proteins in a large number of individual living cells. Here, we utilize microfabrication to construct a Tesla microchemostat that permits single-cell fluorescence imaging of gene expression over many cellular generations. The device is used to capture and constrain asymmetrically dividing or motile cells within a trapping region and to deliver nutrients and regulate the cellular population within this region. We illustrate the operation of the microchemostat with Saccharomyces cerevisiae and explore the evolution of single-cell gene expression and cycle time as a function of generation. Our findings highlight the importance of novel assays for quantifying the dynamics of gene expression and cellular growth, and establish a methodology for exploring the effects of gene expression on long-term processes such as cellular aging.


Subject(s)
Gene Expression Profiling/methods , Gene Expression , Microscopy, Fluorescence/instrumentation , Saccharomyces cerevisiae Proteins/biosynthesis , Bacterial Proteins/analysis , Cell Cycle , Cell Movement , Equipment Design , Gene Expression Profiling/instrumentation , Luminescent Proteins/analysis , Microchemistry , Microscopy, Fluorescence/methods , Microspheres , Mycology/instrumentation , Mycology/methods , Recombinant Fusion Proteins/biosynthesis , Rheology , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Software , Red Fluorescent Protein
7.
J Pharm Biomed Anal ; 28(3-4): 701-9, 2002 May 15.
Article in English | MEDLINE | ID: mdl-12008150

ABSTRACT

SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr alpha and beta cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr alpha or beta) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (alpha and beta) were incubated with 1, 25, and 100 microM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both alpha and beta cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.


Subject(s)
Aniline Compounds/analysis , Aniline Compounds/metabolism , Antineoplastic Agents/metabolism , Isoxazoles/metabolism , Nitriles/analysis , Nitriles/metabolism , 3T3 Cells , Algorithms , Animals , Antineoplastic Agents/analysis , Chromatography, High Pressure Liquid , Culture Media , Isoxazoles/analysis , Leflunomide , Mass Spectrometry , Mice , Receptors, Platelet-Derived Growth Factor/biosynthesis , Reference Standards , Spectrophotometry, Ultraviolet
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