Application of a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme for transcription start site mapping [METHOD]
Changes in the 5' leader of an mRNA can have profound effects on its translational efficiency with little effect on abundance. Sequencing-based methods to accurately map the 5' leader by identifying the first transcribed nucleotide rely on enzymatic removal of the 5' eukaryotic cap structure by tobacco acid pyrophosphatase (TAP). However, commercial TAP production has been problematic and has now been discontinued. RppH, a bacterial enzyme that can also cleave the 5' cap, and Cap-Clip, a plant-derived enzyme, have been marketed as TAP replacements. We have engineered a Schizosaccharomyces pombe Edc1-fused Dcp1–Dcp2 decapping enzyme that functions as a superior TAP replacement. It can be purified from E. coli overexpression in high yields using standard biochemical methods. This constitutively active enzyme is four orders of magnitude more catalytically efficient than RppH at 5' cap removal, compares favorably to Cap-Clip, and the 5' monophosphorylated RNA product is suitable for standard RNA cloning methods. This engineered enzyme is a better replacement for TAP treatment than the current marketed use of RppH and can be produced cost-effectively in a general laboratory setting, unlike Cap-Clip.
David R. Paquette; Jeffrey S. Mugridge; David E. Weinberg; John D. Gross
We tested the proposal that medial temporal lobe (MTL) structures support not just memory but also high-level object perception. In one task, participants decided whether a line drawing could represent an object in three-dimensional space and, in another task, they saw the components of an ... mehr
Fear memory is a highly stable and durable form of memory, even over vast (remote) time frames. Nevertheless, some elements of fear memory can be forgotten, resulting in generalization. The purpose of this study is to determine how cued fear memory generalizes over time and measure underlyi ... mehr
A person sees an object once, and then seconds, minutes, hours, days, or weeks later, she sees it again. How is the person's visual memory for that object changed, improved, or degraded by the second encounter, compared to a situation in which she will have only seen the object once? The an ... mehr