Interactions between important regulatory proteins and human alphaB crystallin

Protein pin arrays assessed interactions between alphaB crystallin and 12 regulatory proteins, including EGF, FGF-2, IGF-1, NGF-beta, TGF-beta, VEGF, insulin, beta-catenin, caspase-3, caspase-8, Bcl-2, and Bcl-xL, which are important in cellular differentiation, proliferation, signaling, cytoskeletal assembly, and apoptosis. FGF-2, NGF-beta, VEGF, insulin, and beta-catenin had strong interactions with human alphaB crystallin peptides, and the alphaB crystallin interactive sequences for these proteins were identified. The seven remaining proteins (EGF, IGF-1, TGF-beta, caspase-3, caspase-8, BCl-2, and Bcl-xL) did not interact with alphaB crystallin. The alphaB crystallin sequences that interacted with FGF-2, NGF-beta, VEGF, insulin, and beta-catenin overlapped with sequences that selectively interact with partially unfolded proteins, suggesting a common function for alphaB crystallin in chaperone activity and the regulation of cell growth and differentiation. Chaperone assays conducted with full-length alphaB crystallin and synthetic alphaB crystallin peptides confirmed the ability of alphaB crystallin to protect against the aggregation of FGF-2 and VEGF, suggesting that alphaB crystallin protects these proteins against unfolding and aggregation under conditions of stress. This is the first report in which sequences involved in interactions with regulatory proteins, including FGF-2, NGF-beta, VEGF, insulin, and beta-catenin, were identified in a small heat shock protein.     

RAPD Analysis of Indian Isolates of Rice Sheath Blight Fungus Rhizoctonia solani

Rice sheath blight fungus Rhizoctonia solani has a wide host range and is highly variable in pathogenecity, sclerotial production and cultural characteristics. In India, breeding for sheath blight resistant cultivars has been a priority area of research. However, lack of adequate information about the genetic variability of the fungal populations occurring in India, non-availability of appropriate markers and the non-availability of resistant donors are some of the limiting factors to achieve this objective. To assess the genetic variability in sheath blight fungus, 18 isolates collected from different rice growing regions of India were analyzed by using random amplified polymorphic DNA (RAPD) markers.The similarity values of RAPD profiles ranged from 0.41 to 0.85 with an average of 0.66 among all the isolates. The percentage polymorphism detected per primer varied from 79.2 to 100%. All the primers could be used to fingerprint the individual isolates. The cluster analysis using unweighted paired group method with arithmetic averages could distinguish between R. solani isolates as well as the virulent and avirulent isolates on rice.     

Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.     

Calibrating excitation light fluxes for quantitative light microscopy in cell biology

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp.     

Linkage Disequilibrium in Salt Tolerant Genotypes of Rice (Oryza sativa L)

Genome wide linkage disequilibrium (LD) was investigated in a set of 32 genotypes representing salt tolerant improved varieties and landraces and six salt sensitive genotypes of rice with 64 microsatellite markers to identify the genomic regions that are associated with salt tolerance in rice. Out of 64 markers analyzed, 36% SSR pairs exhibited significant LD at 0.05. A few regions were identified as targets of selection in 10 chromosomes with high r ² values. The model-based groups from Bayesian clustering analysis are largely consistent with known pedigrees of the lines. The increased percentage of association of SSR loci in the improved varieties indicated the role of selection in linkage disequilibrium especially for salt tolerance. LD was extended as far as 100 cM in the present study. Most of the markers (43.8%) with significant LD values were observed in the genomic regions of reported QTL for salt tolerance in rice.     

Visualization of mRNA translation in living cells

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.