Two-Color GFP Expression System for
            <i>C. elegans</i>

Miller, David M.; Desai, Neerav; Hardin, D.C.; Piston, David W.; Patterson, George H.; Fleenor, Jamie; Xu, Sheng; Fire, Andrew

doi:10.2144/99265rr01

Cited by 88 publications

(58 citation statements)

References 2 publications

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“…1 A were cultured overnight in BHI medium. Overnight cultures were diluted 1:50 in fresh BHI medium or BHI with 40% horse serum (BHIS), and grown at 37°C for 4 h, collected, and ␤-Galactosidase assays carried out as previously described (36).…”

Section: Methodsmentioning

confidence: 99%

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis

Fox¹,

Ramesh

Stearns

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

142

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Ethanolamine, a product of the breakdown of phosphatidylethanolamine from cell membranes, is abundant in the human intestinal tract and in processed foods. Effective utilization of ethanolamine as a carbon and nitrogen source may provide a survival advantage to bacteria that inhabit the gastrointestinal tract and may influence the virulence of pathogens. In this work, we describe a unique series of posttranscriptional regulatory strategies that influence expression of ethanolamine utilization genes (eut) in Enterococcus, Clostridium, and Listeria species. One of these mechanisms requires an unusual 2-component regulatory system. Regulation involves specific sensing of ethanolamine by a sensor histidine kinase (EutW), resulting in autophosphorylation and subsequent phosphoryl transfer to a response regulator (EutV) containing a RNA-binding domain. Our data suggests that EutV is likely to affect downstream gene expression by interacting with conserved transcription termination signals located within the eut locus. Breakdown of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure different from previously established AdoCbl riboswitches. We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intrinsic transcription terminator element located within the intercistronic region. Together, these results suggest an intricate and carefully coordinated interplay of multiple regulatory strategies for control of ethanolamine utilization genes. Gene expression appears to be directed by overlapping posttranscriptional regulatory mechanisms, each responding to a particular metabolic signal, conceptually akin to regulation by multiple DNAbinding transcription factors.riboswitch ͉ 2-component system

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Section: Methodsmentioning

confidence: 99%

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis

Fox¹,

Ramesh

Stearns

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

142

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“…Plasmid constructs: To make che-12 proTGFP, the 936 bp immediately upstream of the B0024.8 start site were amplified by PCR and cloned into the SalI/BamHI restriction sites of the pPD95.75 vector (Miller et al 1999). The full cDNA of B0024.8 was amplified by PCR from a mixed-stage cDNA library (Schumacher et al 2005) and ligated into the KpnI/ AgeI sites of che-12 proTGFP, to make a translational fusion construct.…”

Section: Methodsmentioning

confidence: 99%

The Conserved Proteins CHE-12 and DYF-11 Are Required for Sensory Cilium Function in Caenorhabditis elegans

Bacaj

Shaham

2008

Genetics

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Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function.

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“…Reporter constructs and functional fusions: Plasmids containing the CFP, YFP, or GFP coding sequences were derived from the pPD95.67 plasmid (Miller et al 1999). cho-1 promoter and functional fusions with GFP were generated using a PCR-fusion approach with overlapping PCR primers (Horton et al 1989;Hobert 2002).…”

Section: Methodsmentioning

confidence: 99%

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

Mullen

Mathews

et al. 2007

Genetics

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The cho-1 gene in Caenorhabditis elegans encodes a high-affinity plasma-membrane choline transporter believed to be rate limiting for acetylcholine (ACh) synthesis in cholinergic nerve terminals. We found that CHO-1 is expressed in most, but not all cholinergic neurons in C. elegans. cho-1 null mutants are viable and exhibit mild deficits in cholinergic behavior; they are slightly resistant to the acetylcholinesterase inhibitor aldicarb, and they exhibit reduced swimming rates in liquid. cho-1 mutants also fail to sustain swimming behavior; over a 33-min time course, cho-1 mutants slow down or stop swimming, whereas wildtype animals sustain the initial rate of swimming over the duration of the experiment. A functional CHO-1TGFP fusion protein rescues these cho-1 mutant phenotypes and is enriched at cholinergic synapses. Although cho-1 mutants clearly exhibit defects in cholinergic behaviors, the loss of cho-1 function has surprisingly mild effects on cholinergic neurotransmission. However, reducing endogenous choline synthesis strongly enhances the phenotype of cho-1 mutants, giving rise to a synthetic uncoordinated phenotype. Our results indicate that both choline transport and de novo synthesis provide choline for ACh synthesis in C. elegans cholinergic neurons.

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Two-Color GFP Expression System for C. elegans

Cited by 88 publications

References 2 publications

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis

The Conserved Proteins CHE-12 and DYF-11 Are Required for Sensory Cilium Function in Caenorhabditis elegans

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

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