The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis

In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging. Two categories of pseudorevertants emerged. The first type had restored the MJ interaction but not necessarily the native sequence. The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI. In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling. Our results show the importance of translational control for fitness of the phage. They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Synthesis and expression of a gene encoding a 48-residue repeat in the Pseudomonas syringae ice nucleation protein

The aim of this work was to construct short analogues of the repetitive water-binding domain of the Pseudomonas syringae ice nucleation protein, InaZ. Structural analysis of these analogues might provide data pertaining to the protein-water contacts that underlie ice nucleation. An artificial gene coding for a 48-mer repeat sequence from InaZ was synthesized from four oligodeoxyribonucleotides and ligated into the expression vector, pGEX2T. The recombinant vector was cloned in Escherichia coli and a glutathione S-transferase fusion protein obtained. This fusion protein displayed a low level of ice-nucleating activity when tested by a droplet freezing assay. The fusion protein could be cleaved with thrombin, providing a means for future recovery of the 48-mer peptide in amounts suitable for structural analysis by nuclear magnetic resonance spectroscopy.

Build-up of the tendency to segregate auditory streams:resetting effects of single deviant tones

The tendency to hear a sequence of alternating low (L) and high (H) frequency tones as two streams can be increased by a preceding induction sequence, even one composed only of same-frequency tones. Four experiments used such an induction sequence (10 identical L tones) to promote segregation in a shorter test sequence comprising L and H tones. Previous studies have shown that the build-up of stream segregation is usually reduced greatly when a sudden change in acoustic properties distinguishes all of the induction tones from their test-sequence counterparts. Experiment 1 showed that a single deviant tone, created by altering the final inducer (in frequency, level, duration, or replacement with silence) reduced reported segregation, often substantially. Experiment 2 partially replicated this finding, using changes in temporal discrimination as a measure of streaming. Experiments 3 and 4 varied the size of a frequency change applied to the deviant tone; the extent of resetting varied with size only gradually. The results suggest that resetting begins to occur once the change is large enough to be noticeable. Since the prior inducers always remained unaltered in the deviant-tone conditions, it is proposed that a single change actively resets the build-up evoked by the induction sequence.

The use of ERS-1 crossovers and TOPEX/Poseidon repeat pass data for global sea surface variability studies

In previous sea-surface variability studies, researchers have failed to utilise the full ERS-1 mission due to the varying orbital characteristics in each mission phase, and most have simply ignored the Ice and Geodetic phases. This project aims to introduce a technique which will allow the straightforward use of all orbital phases, regardless of orbit type. This technique is based upon single satellite crossovers. Unfortunately the ERS-1 orbital height is still poorly resolved (due to higher air drag and stronger gravitational effects) when compared with that of TOPEX/Poseidon (T/P), so to make best use of the ERS-1 crossover data corrections to the ERS-1 orbital heights are calculated by fitting a cubic-spline to dual-crossover residuals with T/P. This correction is validated by comparison of dual satellite crossovers with tide gauge data. The crossover processing technique is validated by comparing the extracted sea-surface variability information with that from T/P repeat pass data. The two data sets are then combined into a single consistent data set for analysis of sea-surface variability patterns. These patterns are simplified by the use of an empirical orthogonal function decomposition which breaks the signals into spatial modes which are then discussed separately. Further studies carried out on these data include an analysis of the characteristics of the annual signal, discussion of evidence for Rossby wave propagation on a global basis, and finally analysis of the evidence for global mean sea level rise.

Orbit and altimetric corrections for the ERS satellites through analysis of single and dual satellite crossovers

Due to the failure of PRARE the orbital accuracy of ERS-1 is typically 10-15 cm radially as compared to 3-4cm for TOPEX/Poseidon. To gain the most from these simultaneous datasets it is necessary to improve the orbital accuracy of ERS-1 so that it is commensurate with that of TOPEX/Poseidon. For the integration of these two datasets it is also necessary to determine the altimeter and sea state biases for each of the satellites. Several models for the sea state bias of ERS-1 are considered by analysis of the ERS-1 single satellite crossovers. The model adopted consists of the sea state bias as a percentage of the significant wave height, namely 5.95%. The removal of ERS-1 orbit error and recovery of an ERS-1 - TOPEX/Poseidon relative bias are both achieved by analysis of dual crossover residuals. The gravitational field based radial orbit error is modelled by a finite Fourier expansion series with the dominant frequencies determined by analysis of the JGM-2 co-variance matrix. Periodic and secular terms to model the errors due to atmospheric density, solar radiation pressure and initial state vector mis-modelling are also solved for. Validation of the dataset unification consists of comparing the mean sea surface topographies and annual variabilities derived from both the corrected and uncorrected ERS-1 orbits with those derived from TOPEX/Poseidon. The global and regional geographically fixed/variable orbit errors are also analysed pre and post correction, and a significant reduction is noted. Finally the use of dual/single satellite crossovers and repeat pass data, for the calibration of ERS-2 with respect to ERS-1 and TOPEX/Poseidon is shown by calculating the ERS-1/2 sea state and relative biases.

A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterisation of type 1 cell surface-associated antigens

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64?% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Chromatic dispersion compensation using full-field maximum-likelihood sequence estimation

We investigate full-field detection-based maximum-likelihood sequence estimation (MLSE) for chromatic dispersion compensation in 10 Gbit/s OOK optical communication systems. Important design criteria are identified to optimize the system performance. It is confirmed that approximately 50% improvement in transmission reach can be achieved compared to conventional direct-detection MLSE at both 4 and 16 states. It is also shown that full-field MLSE is more robust to the noise and the associated noise amplifications in full-field reconstruction, and consequently exhibits better tolerance to nonoptimized system parameters than full-field feedforward equalizer. Experiments over 124 km spans of field-installed single-mode fiber without optical dispersion compensation using full-field MLSE verify the theoretically predicted performance benefits.

Build-up of auditory stream segregation induced by tone sequences of constant or alternating frequency and the resetting effects of single deviants

A sequence of constant-frequency tones can promote streaming in a subsequent sequence of alternating-frequency tones, but why this effect occurs is not fully understood and its time course has not been investigated. Experiment 1 used a 2.0-s-long constant-frequency inducer (10 repetitions of a low-frequency pure tone) to promote segregation in a subsequent, 1.2-s test sequence of alternating low- and high-frequency tones. Replacing the final inducer tone with silence substantially reduced reported test-sequence segregation. This reduction did not occur when either the 4th or 7th inducer was replaced with silence. This suggests that a change at the induction/test-sequence boundary actively resets build-up, rather than less segregation occurring simply because fewer inducer tones were presented. Furthermore, Experiment 2 found that a constant-frequency inducer produced its maximum segregation-promoting effect after only three tones—this contrasts with the more gradual build-up typically observed for alternating-frequency sequences. Experiment 3 required listeners to judge continuously the grouping of 20-s test sequences. Constant-frequency inducers were considerably more effective at promoting segregation than alternating ones; this difference persisted for ~10 s. In addition, resetting arising from a single deviant (longer tone) was associated only with constant-frequency inducers. Overall, the results suggest that constant-frequency inducers promote segregation by capturing one subset of test-sequence tones into an ongoing, preestablished stream, and that a deviant tone may reduce segregation by disrupting this capture. These findings offer new insight into the dynamics of stream segregation, and have implications for the neural basis of streaming and the role of attention in stream formation. (PsycINFO Database Record (c) 2013 APA, all rights reserved)

Toward bacterial protein sub-cellular location prediction:single-class discrimminant models for all gram- and gram+ compartments

Based on Bayesian Networks, methods were created that address protein sequence-based bacterial subcellular location prediction. Distinct predictive algorithms for the eight bacterial subcellular locations were created. Several variant methods were explored. These variations included differences in the number of residues considered within the query sequence - which ranged from the N-terminal 10 residues to the whole sequence - and residue representation - which took the form of amino acid composition, percentage amino acid composition, or normalised amino acid composition. The accuracies of the best performing networks were then compared to PSORTB. All individual location methods outperform PSORTB except for the Gram+ cytoplasmic protein predictor, for which accuracies were essentially equal, and for outer membrane protein prediction, where PSORTB outperforms the binary predictor. The method described here is an important new approach to method development for subcellular location prediction. It is also a new, potentially valuable tool for candidate subunit vaccine selection.

Build-up of auditory stream segregation induced by tone sequences of constant or alternating frequency and the resetting effects of single deviants

A sequence of constant-frequency tones can promote streaming in a subsequent sequence of alternating-frequency tones, but why this effect occurs is not fully understood and its time course has not been investigated. Experiment 1 used a 2.0-s-long constant-frequency inducer (10 repetitions of a low-frequency pure tone) to promote segregation in a subsequent, 1.2-s test sequence of alternating low- and high-frequency tones. Replacing the final inducer tone with silence substantially reduced reported test-sequence segregation. This reduction did not occur when either the 4th or 7th inducer was replaced with silence. This suggests that a change at the induction/test-sequence boundary actively resets build-up, rather than less segregation occurring simply because fewer inducer tones were presented. Furthermore, Experiment 2 found that a constant-frequency inducer produced its maximum segregation-promoting effect after only three tones—this contrasts with the more gradual build-up typically observed for alternating-frequency sequences. Experiment 3 required listeners to judge continuously the grouping of 20-s test sequences. Constant-frequency inducers were considerably more effective at promoting segregation than alternating ones; this difference persisted for ~10 s. In addition, resetting arising from a single deviant (longer tone) was associated only with constant-frequency inducers. Overall, the results suggest that constant-frequency inducers promote segregation by capturing one subset of test-sequence tones into an ongoing, preestablished stream, and that a deviant tone may reduce segregation by disrupting this capture. These findings offer new insight into the dynamics of stream segregation, and have implications for the neural basis of streaming and the role of attention in stream formation. (PsycINFO Database Record (c) 2013 APA, all rights reserved)

Microstructural development in aluminide diffusion coatings on nickel-base superalloy single crystals

An investigation has been made of the microstructural stability of aluminide diffusion coatings during post-coating thermal exposure. This study has employed edge-on transmission electron microscopy to examine high-activity pack aluminised single crystals of a gamma prime strengthened nickel-base superalloy. The influence of exposure temperature, duration and atmosphere as well as the initial coating thickness has been assessed. Two major processes have been found to contribute to microstructural changes in the coating. These are, firstly, the transformation of the coating matrix (β-phase, nominally NiAl) to other Ni-Al based phases, especially γ' (nominally Ni3(Al, Ti)) and, secondly, the precipitation of chromium containing phases. The work has enabled the roles of three processes contributing to γ formation, namely: oxidation of the coating surface, interdiffusion with the substrate and ageing of the coating, to be understood. In addition, the factors leading to the formation of a sequence of chromium-containing phases have been identified.

Build-up of auditory stream segregation induced by tone sequences of constant or alternating frequency and the resetting effects of single deviants

Three experiments investigated the dynamics of auditory stream segregation. Experiment 1 used a 2.0-s constant-frequency inducer (10 repetitions of a low-frequency pure tone) to promote segregation in a subsequent, 1.2-s test sequence of alternating low- and high-frequency tones. Replacing the final inducer tone with silence reduced reported test-sequence segregation substantially. This reduction did not occur when either the 4th or 7th inducer was replaced with silence. This suggests that a change at the induction/test-sequence boundary actively resets buildup, rather than less segregation occurring simply because fewer inducer tones were presented. Furthermore, Experiment 2 found that a constant-frequency inducer produced its maximum segregation-promoting effect after only 3 tone cycles - this contrasts with the more gradual build-up typically observed for alternating sequences. Experiment 3 required listeners to judge continuously the grouping of 20-s test sequences. Constant-frequency inducers were considerably more effective at promoting segregation than alternating ones; this difference persisted for ∼10 s. In addition, resetting arising from a single deviant (longer tone) was associated only with constant-frequency inducers. Overall, the results suggest that constant-frequency inducers promote segregation by capturing one subset of test-sequence tones into an on-going, pre-established stream and that a deviant tone may reduce segregation by disrupting this capture. © 2013 Acoustical Society of America.