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Abstract Background There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R). Conclusions These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation.

Abstract Background The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

Background: There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. Results: We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R). Conclusions: These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation. [ABSTRACT FROM AUTHOR]

Background: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. Results: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. Conclusion: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs. [ABSTRACT FROM AUTHOR]

The neurotoxin 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH(2)-MPTP) damages forebrain serotonin (5-HT) and norepinephrine (NE) nerve terminals while sparing striatal dopaminergic innervation. Previous studies suggest that 2'-NH(2)-MPTP acts by a mechanism that involves uptake by the plasma membrane 5-HT and NE transporters. The present investigation further explores the molecular mechanism of 2'-NH(2)-MPTP with regard to cellular transport and effects on body temperature. Mice with genetically controlled decreases in serotonin transporter (SERT) expression were studied to corroborate pharmacologic evidence implicating SERT in 2'-NH(2)-MPTP-induced serotonin neurotoxicity. To investigate whether sequestration by the intracellular vesicular monoamine transporter type 2 (VMAT2) occurs, mice with reduced VMAT2 expression or mice receiving the VMAT2 inhibitor Ro 4-1284 (2-hydroxy-2-ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydrobenzo[alpha]chinolizin hydrochloride) were treated with 2'-NH(2)-MPTP. Body temperature was measured as a function of reduced SERT or VMAT2 expression. 2'-NH(2)-MPTP caused a 2 degrees C drop in temperature that was attenuated by decreased SERT but not VMAT2. In addition, complete loss of SERT attenuated cortical and hippocampal depletions in 5-HT but not NE. In contrast, mice with a 50% reduction in VMAT2 exhibited similar 5-HT and NE toxicity when compared with wild-type mice at higher doses of 2'-NH(2)-MPTP, whereas a slight potentiation of toxicity was observed at very low doses of 2'-NH(2)-MPTP. Pharmacologic inhibition of VMAT2 caused minimal potentiation of neurotransmitter depletions in response to moderate doses of 2'-NH(2)-MPTP. Thus, 2'-NH(2)-MPTP seems to be similar to MPTP in its requirement for selective plasma membrane transport and the expression of acute hypothermia; however, unlike MPTP, VMAT2 does not appear to play a major role in the toxic mechanism of 2'-NH(2)-MPTP.