Dataset Information

GFP Loss-of-Function Mutations in Arabidopsis thaliana.

ABSTRACT: Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded ?-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence and create derivatives with novel properties. However, loss-of-function mutations that would aid in understanding GFP protein folding and chromophore formation have not been fully cataloged. Here we report a collection of ethyl methansulfonate-induced GFP loss-of-function mutations in the model plant Arabidopsis thaliana. Mutations that alter residues important for chromophore maturation, such as Arg96 and Ser205, greatly reduce or extinguish fluorescence without dramatically altering GFP protein accumulation. By contrast, other loss-of-fluorescence mutations substantially diminish the amount of GFP protein, suggesting that they compromise protein stability. Many mutations in this category generate substitutions of highly conserved glycine residues, including the following: Gly67 in the chromogenic tripeptide; Gly31, Gly33, and Gly35 in the second ?-strand; and Gly20, Gly91, and Gly127 in the lids of the ?-barrel scaffold. Our genetic analysis supports conclusions from structural and biochemical studies and demonstrates a critical role for multiple, highly conserved glycine residues in GFP protein stability.

SUBMITTER: Fu JL

PROVIDER: S-EPMC4555221 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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GFP Loss-of-Function Mutations in Arabidopsis thaliana.

Fu Jason L JL Kanno Tatsuo T Liang Shih-Chieh SC Matzke Antonius J M AJ Matzke Marjori M

G3 (Bethesda, Md.) 20150706 9

Green fluorescent protein (GFP) and related fluorescent proteins are widely used in biological research to monitor gene expression and protein localization in living cells. The GFP chromophore is generated spontaneously in the presence of oxygen by a multi-step reaction involving cyclization of the internal tripeptide Ser65 (or Thr65)-Tyr66-Gly67, which is embedded in the center of an 11-stranded β-barrel structure. Random and site-specific mutagenesis has been used to optimize GFP fluorescence ...[more]

PMID: 26153075

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Project description:BACKGROUND: Plant microRNAs (miRNAs) have been revealed to play important roles in developmental control, hormone secretion, cell differentiation and proliferation, and response to environmental stresses. However, our knowledge about the regulatory mechanisms and functions of miRNAs remains very limited. The main difficulties lie in two aspects. On one hand, the number of experimentally validated miRNA targets is very limited and the predicted targets often include many false positives, which constrains us to reveal the functions of miRNAs. On the other hand, the regulation of miRNAs is known to be spatio-temporally specific, which increases the difficulty for us to understand the regulatory mechanisms of miRNAs. DESCRIPTION: In this paper we present miRFANs, an online database for Arabidopsis thalianamiRNA function annotations. We integrated various type of datasets, including miRNA-target interactions, transcription factor (TF) and their targets, expression profiles, genomic annotations and pathways, into a comprehensive database, and developed various statistical and mining tools, together with a user-friendly web interface. For each miRNA target predicted by psRNATarget, TargetAlign and UEA target-finder, or recorded in TarBase and miRTarBase, the effect of its up-regulated or down-regulated miRNA on the expression level of the target gene is evaluated by carrying out differential expression analysis of both miRNA and targets expression profiles acquired under the same (or similar) experimental condition and in the same tissue. Moreover, each miRNA target is associated with gene ontology and pathway terms, together with the target site information and regulating miRNAs predicted by different computational methods. These associated terms may provide valuable insight for the functions of each miRNA. CONCLUSION: First, a comprehensive collection of miRNA targets for Arabidopsis thaliana provides valuable information about the functions of plant miRNAs. Second, a highly informative miRNA-mediated genetic regulatory network is extracted from our integrative database. Third, a set of statistical and mining tools is equipped for analyzing and mining the database. And fourth, a user-friendly web interface is developed to facilitate the browsing and analysis of the collected data.

Dataset Information

GFP Loss-of-Function Mutations in Arabidopsis thaliana.

Publications

GFP Loss-of-Function Mutations in Arabidopsis thaliana.

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