Mutations in the Poliovirus 3CD Proteinase S1-Specificity Pocket Affect Substrate Recognition and RNA Binding

AbstractSequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases. In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition. Single amino acid substitutions at 3C residues Thr-142, His-161, Gly-163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3Cciscleavage activity. Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure. Anin vivoanalysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication. In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5′-terminal sequences

The Shapes of Tight Composite Knots

We present new computations of tight shapes obtained using the constrained gradient descent code RIDGERUNNER for 544 composite knots with 12 and fewer crossings, expanding our dataset to 943 knots and links. We use the new data set to analyze two outstanding conjectures about tight knots, namely that the ropelengths of composite knots are at least 4\pi-4 less than the sums of the prime factors and that the writhes of composite knots are the sums of the writhes of the prime factors.Comment: Summary text file of tight knot lengths and writhing numbers stored in anc/ropelength_data.txt. All other data freely available at http:://www.jasoncantarella.com/ and through Data Conservanc

Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library

Detecting the B-mode Polarisation of the CMB with Clover

We describe the objectives, design and predicted performance of Clover, which is a ground-based experiment to measure the faint ``B-mode'' polarisation pattern in the cosmic microwave background (CMB). To achieve this goal, clover will make polarimetric observations of approximately 1000 deg^2 of the sky in spectral bands centred on 97, 150 and 225 GHz. The observations will be made with a two-mirror compact range antenna fed by profiled corrugated horns. The telescope beam sizes for each band are 7.5, 5.5 and 5.5 arcmin, respectively. The polarisation of the sky will be measured with a rotating half-wave plate and stationary analyser, which will be an orthomode transducer. The sky coverage combined with the angular resolution will allow us to measure the angular power spectra between 20 < l < 1000. Each frequency band will employ 192 single polarisation, photon noise limited TES bolometers cooled to 100 mK. The background-limited sensitivity of these detector arrays will allow us to constrain the tensor-to-scalar ratio to 0.026 at 3sigma, assuming any polarised foreground signals can be subtracted with minimal degradation to the 150 GHz sensitivity. Systematic errors will be mitigated by modulating the polarisation of the sky signals with the rotating half-wave plate, fast azimuth scans and periodic telescope rotations about its boresight. The three spectral bands will be divided into two separate but nearly identical instruments - one for 97 GHz and another for 150 and 225 GHz. The two instruments will be sited on identical three-axis mounts in the Atacama Desert in Chile near Pampa la Bola. Observations are expected to begin in late 2009.Comment: 5 pages, 3 figures. To appear in the proceedings of the XXXXIIIrd Rencontres de Moriond "Cosmology". Figure 1 update