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In JoVE (1)
Other Publications (5)
Articles by Michael Petrascheck in JoVE
Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates
Gregory M. Solis1,2, Michael Petrascheck1,2
1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute
In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
Other articles by Michael Petrascheck on PubMed
Plant Physiology. Aug, 2004 | Pubmed ID: 15299139
Plant lateral organs exhibit proximal-distal and adaxial-abaxial polarity. In Arabidopsis, abaxial cell fate is regulated in part by putative transcription factors of the YABBY family, such as FILAMENTOUS FLOWER (FIL) and INNER NO OUTER (INO), by a mechanism that currently is not fully understood. NOZZLE (NZZ) encodes a plant-specific nuclear protein. Genetic evidence has shown that NZZ is involved in the positive feedback regulation of INO, thereby acting both as a temporal and spatial repressor of INO transcription. This mechanism allows the ovule primordium to complete its proximal-distal organization, prior to the onset of adaxial-abaxial development in the chalaza. During our study, we isolated FIL in a yeast two-hybrid screen using NZZ as bait. In vitro pull-down experiments confirmed the NZZ-FIL interaction. NZZ also bound INO and YABBY3, suggesting that NZZ generally interacts with YABBY proteins in vitro. The polar-charged region of NZZ was necessary and sufficient to bind to the zinc finger of INO and to interact with its C terminus carrying the high mobility group-like domain. We suggest that NZZ coordinates proximal-distal patterning and adaxial-abaxial polarity establishment in the developing ovule by directly binding to INO.
Quenching Accumulation of Toxic Galactose-1-phosphate As a System to Select Disruption of Protein-protein Interactions in Vivo
BioTechniques. Nov, 2004 | Pubmed ID: 15560141
The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo. This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell. Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth. Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures. Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest. We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity. Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.
Nucleic Acids Research. 2005 | Pubmed ID: 16002789
Enhancers are DNA sequences that can activate gene transcription from remote positions. In yeast, regulatory sequences that are functionally equivalent to the metazoan enhancers are called upstream activating sequences (UASs). UASs show a lower degree of flexibility than their metazoan counterparts, but can nevertheless activate transcription from a distance of >1000 bp from the promoter. One of several models for the mechanism of action of transcriptional enhancers proposes that enhancer-bound activating proteins contact promoter-bound transcription factors and thereby get in close proximity to the promoter region with concomitant looping of the intervening DNA. We tested the mode of enhancer activity in yeast. A polymerase II-transcribed gene was paired with a remote, inducible enhancer. An independent reporter system was inserted next to the promoter to monitor the potential modes of enhancer activity. Our results show that the enhancer activated the reporter system only in the presence of a functional promoter. We also demonstrate that the heterologous expression of GAGA, a factor known to facilitate DNA loop formation, allows enhancer action in yeast over a distance of 3000 bp.
Nature. Nov, 2007 | Pubmed ID: 18033297
The mechanisms that determine the lifespan of an organism are still largely a mystery. One goal of ageing research is to find drugs that would increase lifespan and vitality when given to an adult animal. To this end, we tested 88,000 chemicals for the ability to extend the lifespan of adult Caenorhabditis elegans nematodes. Here we report that a drug used as an antidepressant in humans increases C. elegans lifespan. In humans, this drug blocks neural signalling by the neurotransmitter serotonin. In C. elegans, the effect of the drug on lifespan is reduced or eradicated by mutations that affect serotonin synthesis, serotonin re-uptake at synapses, or either of two G-protein-coupled receptors: one that recognizes serotonin and the other that detects another neurotransmitter, octopamine. In vitro studies show that the drug acts as an antagonist at both receptors. Testing of the drug on dietary-restricted animals or animals with mutations that affect lifespan indicates that its effect on lifespan involves mechanisms associated with lifespan extension by dietary restriction. These studies indicate that lifespan can be extended by blocking certain types of neurotransmission implicated in food sensing in the adult animal, possibly leading to a state of perceived, although not real, starvation.
Annals of the New York Academy of Sciences. Jul, 2009 | Pubmed ID: 19686215
One long-term goal of aging research is to find drugs that can delay aging and the onset of age-associated diseases. With this in mind, we screened 88,000 chemicals for the ability to increase the lifespan of Caenorhabditis elegans nematodes. We found that mianserin, a serotonin receptor antagonist used as an antidepressant in humans, can increase C. elegans lifespan when given only during adulthood. This effect is reduced or abolished by mutations that affect serotonin synthesis or serotonin reuptake at synapses. It also requires a serotonin receptor and an octopamine receptor, both of which are inhibited by the drug. Mianserin has no effect on the lifespan of animals with increased longevity due to dietary restriction or with a mutation that reduces food intake, indicating that the drug extends lifespan via mechanisms linked to dietary restriction. These studies indicate that lifespan can be increased by inhibiting certain kinds of neurotransmission previously implicated in food sensing, possibly by mimicking a physiological state associated with dietary restriction.