A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved.

Recombination Drives Genetic Diversification of Streptococcus dysgalactiae Subspecies equisimilis in a Region of Streptococcal Endemicity

Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential.

Site Characterization of Bangalore using Geophysical Method

A number of geophysical methods have been proposed for near-surface site characterization and measurement of shear wave velocity by using a great variety of testing configurations, processing techniques,and inversion algorithms. In particular, two widely-used techniques are SASW (Spectral Analysis of SurfaceWaves) and MASW (Multichannel Analysis of SurfaceWaves). MASW is increasingly being applied to earthquake geotechnical engineering for the local site characterization, microzonation and site response studies.A MASW is a geophysical method, which generates a shear-wave velocity (Vs) profile (i.e., Vs versus depth)by analyzing Raleigh-type surface waves on a multichannel record. MASW system consisting of 24 channels Geode seismograph with 24 geophones of 4.5 Hz frequency have been used in this investigation. For the site characterization program, the MASW field experiments consisting of 58 one-dimensional shear wave velocity tests and 20 two-dimensional shear wave tests have been carried out. The survey points have been selected in such a way that the results supposedly represent the whole metropolitan Bangalore having an area of 220 km2.The average shear wave velocity of Bangalore soils have been evaluated for depths of 5m, 10m, 15m, 20m, 25m and 30 m. The subsoil site classification has been made for seismic local site effect evaluation based on average shear wave velocity of 30m depth (Vs30) of sites using National Earthquake Hazards Reduction Program (NEHRP) and International Building Code (IBC) classification. Soil average shearwave velocity estimated based on overburden thickness from the borehole information is also presented. Mapping clearly indicates that the depth of soil obtained from MASW is closely matching with the soil layers in bore logs. Among total 55 locations of MASW survey carried out, 34 locations were very close to the SPT borehole locations and these are used to generate correlation between Vs and corrected “N” values. The SPT field “N” values are corrected by applying the NEHRP recommended corrections.

Tuning of dielectric properties and magnetism of SrTiO(3) by site-specific doping of Mn

Combining experiments with first-principles calculations, we show that site-specific doping of Mn into SrTiO(3) has a decisive influence on the dielectric properties of these doped systems. We find that phonon contributions to the dielectric constant invariably decrease sharply on doping at any site. However, a sizable, random dipolar contribution only for Mn at the Sr site arises from a strong off-centric displacement of Mn in spite of Mn being in a non-d(0) state; this leads to a large dielectric constant at higher temperatures and gives rise to a relaxor ferroelectric behavior at lower temperatures. We also investigate magnetic properties in detail and critically reevaluate the possibility of a true multiglass state in such systems.

Endonuclease Active Site Plasticity Allows DNA Cleavage with Diverse Alkaline Earth and Transition Metal Ions

A majority of enzymes show a high degree of specificity toward a particular metal ion in their catalytic reaction. However, Type II restriction endonuclease (REase) R.KpnI, which is the first member of the HNH superfamily of REases, exhibits extraordinary diversity in metal ion dependent DNA cleavage. Several alkaline earth and transition group metal ions induce high fidelity and promiscuous cleavage or inhibition depending upon their concentration. The metal ions having different ionic radii and co-ordination geometries readily replace each other from the enzyme's active site, revealing its plasticity. Ability of R KpnI to cleave DNA with both alkaline earth and transition group metal ions having varied ionic radii could imply utilization of different catalytic site(s). However, mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all of the cofactors, indicating that the enzyme follows a single metal ion catalytic mechanism for DNA cleavage. Indispensability of His in nucleophile activation together with broad cofactor tolerance of the enzyme indicates electrostatic stabilization function of metal ions during catalysis. Nevertheless, a second metal ion is recruited at higher concentrations to either induce promiscuity or inhibit the DNA cleavage. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The active site plasticity of R.KpnI opens up avenues for redesigning cofactor specificities and generation of mutants specific to a particular metal ion.

Remote detection and distinction of ants using nest-site specific LISS-derived Normalised Difference Vegetation Index

This study in Western Ghats, India, investigates the relation between nesting sites of ants and a single remotely sensed variable: the Normalised Difference Vegetation Index (NDVI). We carried out sampling in 60 plots each measuring 30 x 30 m and recorded nest sites of 13 ant species. We found that NDVI values at the nesting sites varied considerably between individual species and also between the six functional groups the ants belong to. The functional groups Cryptic Species, Tropical Climate Specialists and Specialist Predators were present in regions with high NDVI whereas Hot Climate Specialists and Opportunists were found in sites with low NDVI. As expected we found that low NDVI values were associated with scrub jungles and high NDVI values with evergreen forests. Interestingly, we found that Pachycondyla rufipes, an ant species found only in deciduous and evergreen forests, established nests only in sites with low NDVI (range = 0.015 - 0.1779). Our results show that these low NDVI values in deciduous and evergreen forests correspond to canopy gaps in otherwise closed deciduous and evergreen forests. Subsequent fieldwork confirmed the observed high prevalence of P. rufipes in these NDVI-constrained areas. We discuss the value of using NDVI for the remote detection and distinction of ant nest sites.

Three-Dimensional Site Characterization Model Using Artificial Neural Networks

This research is designed to develop a new technique for site characterization in a three-dimensional domain. Site characterization is a fundamental task in geotechnical engineering practice, as well as a very challenging process, with the ultimate goal of estimating soil properties based on limited tests at any half-space subsurface point in a site.In this research, the sandy site at the Texas A&M University's National Geotechnical Experimentation Site is selected as an example to develop the new technique for site characterization, which is based on Artificial Neural Networks (ANN) technology. In this study, a sequential approach is used to demonstrate the applicability of ANN to site characterization. To verify its robustness, the proposed new technique is compared with other commonly used approaches for site characterization. In addition, an artificial site is created, wherein soil property values at any half-space point are assumed, and thus the predicted values can compare directly with their corresponding actual values, as a means of validation. Since the three-dimensional model has the capability of estimating the soil property at any location in a site, it could have many potential applications, especially in such case, wherein the soil properties within a zone are of interest rather than at a single point. Examples of soil properties of zonal interest include soil type classification and liquefaction potential evaluation. In this regard, the present study also addresses this type of applications based on a site located in Taiwan, which experienced liquefaction during the 1999 Chi-Chi, Taiwan, Earthquake.

Roles of Salmonella enterica serovar Typhimurium encoded Peptidase N during systemic infection of Ifnγ(-/-) mice.

Pathogen encoded peptidases are known to be important during infection; however, their roles in modulating host responses in immunocompromised individuals are not well studied. The roles of S. typhimurium (WT) encoded Peptidase N (PepN), a major aminopeptidase and sole M1 family member, was studied in mice lacking Interferon-γ (IFNγ), a cytokine important for immunity. S. typhimurium lacking pepN (ΔpepN) displays enhanced colony forming units (CFU) compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ(-/-) mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ΔpepN. Concomitantly, reintroduction of pepN in ΔpepN (ΔpepN/pepN) reduces CFU, demonstrating pepN-dependence. Interestingly, expression of a catalytically inactive PepN (ΔpepN/E298A) also lowers CFU, demonstrating that the decrease in CFU is independent of the catalytic activity of PepN. In addition, three distinct differences are observed between infection of C57BL/6 and Ifnγ(-/-) mice: First, serum amounts of TNFα and IL1β post infection are significantly lower in Ifnγ(-/-) mice. Second, histological analysis of C57BL/6 mice reveals that damage in spleen and liver upon infection with WT or ΔpepN is greater compared to ΔpepN/pepN or ΔpepN/E298A. On the other hand, Ifnγ(-/-) mice are highly susceptible to organ damage by all strains of S. typhimurium used in this study. Finally, greater survival of C57BL/6, but not Ifnγ(-/-) mice, is observed upon infection with ΔpepN/pepN or ΔpepN/E298A. Overall, the roles of the host encoded IFNγ during infection with S. typhimurium strains with varying degrees of virulence are highlighted.

Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation

Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (as: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro. (C) 2011 Elsevier Inc. All rights reserved.

Dimethyl sulfoxide induced structural transformations and non-monotonic concentration dependence of conformational fluctuation around active site of lysozyme

Experimental studies have observed significant changes in both structure and function of lysozyme (and other proteins) on addition of a small amount of dimethyl sulfoxide (DMSO) in aqueous solution. Our atomistic molecular dynamic simulations of lysozyme in water-DMSO reveal the following sequence of changes on increasing DMSO concentration. (i) At the initial stage (around 5% DMSO concentration) protein's conformational flexibility gets markedly suppressed. From study of radial distribution functions, we attribute this to the preferential solvation of exposed protein hydrophobic residues by the methyl groups of DMSO. (ii) In the next stage (10-15% DMSO concentration range), lysozome partially unfolds accompanied by an increase both in fluctuation and in exposed protein surface area. (iii) Between 15-20% concentration ranges, both conformational fluctuation and solvent accessible protein surface area suddenly decrease again indicating the formation of an intermediate collapse state. These results are in good agreement with near-UV circular dichroism (CD) and fluorescence studies. We explain this apparently surprising behavior in terms of a structural transformation which involves clustering among the methyl groups of DMSO. (iv) Beyond 20% concentration of DMSO, the protein starts its final sojourn towards the unfolding state with further increase in conformational fluctuation and loss in native contacts. Most importantly, analysis of contact map and fluctuation near the active site reveal that both partial unfolding and conformational fluctuations are centered mostly on the hydrophobic core of active site of lysozyme. Our results could offer a general explanation and universal picture of the anomalous behavior of protein structure-function observed in the presence of cosolvents (DMSO, ethanol, tertiary butyl alcohol, dioxane) at their low concentrations. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3694268]