Dataset Information

Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

ABSTRACT:

Background

Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.

Results

We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in Escherichia coli. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.

Conclusions

The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.

SUBMITTER: de Jong H

PROVIDER: S-EPMC2877006 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Publications

Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

de Jong Hidde H Ranquet Caroline C Ropers Delphine D Pinel Corinne C Geiselmann Johannes J

BMC systems biology 20100429

<h4>Background</h4>Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.<h4>Results</h4>We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validat ...[more]

PMID: 20429918

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Project description:BACKGROUND: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. RESULTS: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. CONCLUSIONS: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

Project description:BackgroundThe development of quantitative models of signal transduction, as well as parameter estimation to improve existing models, depends on the ability to obtain quantitative information about various proteins that are part of the signaling pathway. However, commonly-used measurement techniques such as Western blots and mobility shift assays provide only qualitative or semi-quantitative data which cannot be used for estimating parameters. Thus there is a clear need for techniques that enable quantitative determination of signal transduction intermediates.ResultsThis paper presents an integrated modeling and experimental approach for quantitatively determining transcription factor profiles from green fluorescent protein (GFP) reporter data. The technique consists of three steps: (1) creating data sets for green fluorescent reporter systems upon stimulation, (2) analyzing the fluorescence images to determine fluorescence intensity profiles using principal component analysis (PCA) and K-means clustering, and (3) computing the transcription factor concentration from the fluorescence intensity profiles by inverting a model describing transcription, translation, and activation of green fluorescent proteins. We have used this technique to quantitatively characterize activation of the transcription factor NF-kappaB by the cytokine TNF-alpha. In addition, we have applied the quantitative NF-kappaB profiles obtained from our technique to develop a model for TNF-alpha signal transduction where the parameters were estimated from the obtained data.ConclusionThe technique presented here for computing transcription factor profiles from fluorescence microscopy images of reporter cells generated quantitative data on the magnitude and dynamics of NF-kappaB activation by TNF-alpha. The obtained results are in good agreement with qualitative descriptions of NF-kappaB activation as well as semi-quantitative experimental data from the literature. The profiles computed from the experimental data have been used to re-estimate parameters for a NF-kappaB model and the results of additional experiments are predicted very well by the model with the new parameter values. While the presented approach has been applied to NF-kappaB and TNF-alpha signaling, it can be used to determine the profile of any transcription factor as long as GFP reporter fluorescent profiles are available.

Dataset Information

Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

Background

Results

Conclusions

Publications

Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria.

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