Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac</it> transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase) itself. A thorough understanding of just how <it>piggyBac</it> works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative ZnF (ZnF).</p> <p>Results</p> <p>We fused the <it>piggyBac</it> TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the <it>Drosophila melanogaster</it> inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of <it>piggyBac</it> localization with a scanning confocal microscope 48 hours after induction with copper sulphate.</p> <p>Conclusion</p> <p>Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the <it>piggyBac</it> TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.</p

Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac </it>mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of <it>piggyBac</it>, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for <it>piggyBac </it>at amino acid positions D268, D346, and D447.</p> <p>Results</p> <p>This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the <it>piggyBac </it>transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the <it>piggyBac </it>transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.</p> <p>Conclusion</p> <p>We found all the designated DDD aspartates reside in clusters of amino acids that conserved among <it>piggyBac </it>family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.</p

Constraints on Nucleon Decay via "Invisible" Modes from the Sudbury Neutrino Observatory

Data from the Sudbury Neutrino Observatory have been used to constrain the lifetime for nucleon decay to ``invisible'' modes, such as n -> 3 nu. The analysis was based on a search for gamma-rays from the de-excitation of the residual nucleus that would result from the disappearance of either a proton or neutron from O16. A limit of tau_inv > 2 x 10^{29} years is obtained at 90% confidence for either neutron or proton decay modes. This is about an order of magnitude more stringent than previous constraints on invisible proton decay modes and 400 times more stringent than similar neutron modes.Comment: Update includes missing efficiency factor (limits change by factor of 2) Submitted to Physical Review Letter

Electron Antineutrino Search at the Sudbury Neutrino Observatory

Upper limits on the \nuebar flux at the Sudbury Neutrino Observatory have been set based on the \nuebar charged-current reaction on deuterium. The reaction produces a positron and two neutrons in coincidence. This distinctive signature allows a search with very low background for \nuebar's from the Sun and other potential sources. Both differential and integral limits on the \nuebar flux have been placed in the energy range from 4 -- 14.8 MeV. For an energy-independent \nu_e --> \nuebar conversion mechanism, the integral limit on the flux of solar \nuebar's in the energy range from 4 -- 14.8 MeV is found to be \Phi_\nuebar <= 3.4 x 10^4 cm^{-2} s^{-1} (90% C.L.), which corresponds to 0.81% of the standard solar model 8B \nu_e flux of 5.05 x 10^6 cm^{-2} s^{-1}, and is consistent with the more sensitive limit from KamLAND in the 8.3 -- 14.8 MeV range of 3.7 x 10^2 cm^{-2} s^{-1} (90% C.L.). In the energy range from 4 -- 8 MeV, a search for \nuebar's is conducted using coincidences in which only the two neutrons are detected. Assuming a \nuebar spectrum for the neutron induced fission of naturally occurring elements, a flux limit of Phi_\nuebar <= 2.0 x 10^6 cm^{-2} s^{-1}(90% C.L.) is obtained.Comment: submitted to Phys. Rev.

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion