Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Identification of conformational B-cell Epitopes in an antigen from its primary sequence

Background

One of the major challenges in the field of vaccine design is to predict conformational B-cell epitopes in an antigen. In the past, several methods have been developed for predicting conformational B-cell epitopes in an antigen from its tertiary structure. This is the first attempt in this area to predict conformational B-cell epitope in an antigen from its amino acid sequence.

Results

All Support vector machine (SVM) models were trained and tested on 187 non-redundant protein chains consisting of 2261 antibody interacting residues of B-cell epitopes. Models have been developed using binary profile of pattern (BPP) and physiochemical profile of patterns (PPP) and achieved a maximum MCC of 0.22 and 0.17 respectively. In this study, for the first time SVM model has been developed using composition profile of patterns (CPP) and achieved a maximum MCC of 0.73 with accuracy 86.59%. We compare our CPP based model with existing structure based methods and observed that our sequence based model is as good as structure based methods.

Conclusion

This study demonstrates that prediction of conformational B-cell epitope in an antigen is possible from is primary sequence. This study will be very useful in predicting conformational B-cell epitopes in antigens whose tertiary structures are not available. A web server CBTOPE has been developed for predicting B-cell epitope http://www.imtech.res.in/raghava/cbtope/.     

Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase systems and the protein-polyelectrolyte complex formation

Complexes formation between two model proteins (catalase and chymotrypsin) and polyelectrolytes (polyvinyl sulphonate and polyacrilic acid) and a non-charged flexible chain polymer (PCF) as polyethylene propylene oxide (molecular mass 8400) was studied by a spectroscopy technique combination: UV absorption, fluorescence emission and circular dichroism. All the polymers increase the protein surface hydrophobicity (S(0)) parameter value as a proof of the modification of the protein surface exposed to the solvent. Chymotrypsin showed an increase in its biological activity in polymer presence, which suggests a change in the superficial microenvironment. The decrease in the biological activity of catalase might be due to a competition between the polymer and the substrate. This result agrees with the polymer effect on the catalase superficial hydrophobic area. It was found that, when flexible chain polymers increase protein stability and the enzymatic activity they could be used to isolate this enzyme without inducing loss of protein enzymatic activity. Our findings suggest that the interactions are dependent on the protein physico-chemical parameters such as: isoelectric pH, hydrophobic surface area, etc.     

Support Vector Machine-based method for predicting subcellular localization of mycobacterial proteins using evolutionary information and motifs

Background

Annotations that describe the function of sequences are enormously important to researchers during laboratory investigations and when making computational inferences. However, there has been little investigation into the data quality of sequence function annotations. Here we have developed a new method of estimating the error rate of curated sequence annotations, and applied this to the Gene Ontology (GO) sequence database (GOSeqLite). This method involved artificially adding errors to sequence annotations at known rates, and used regression to model the impact on the precision of annotations based on BLAST matched sequences.

Results

We estimated the error rate of curated GO sequence annotations in the GOSeqLite database (March 2006) at between 28% and 30%. Annotations made without use of sequence similarity based methods (non-ISS) had an estimated error rate of between 13% and 18%. Annotations made with the use of sequence similarity methodology (ISS) had an estimated error rate of 49%.

Conclusion

While the overall error rate is reasonably low, it would be prudent to treat all ISS annotations with caution. Electronic annotators that use ISS annotations as the basis of predictions are likely to have higher false prediction rates, and for this reason designers of these systems should consider avoiding ISS annotations where possible. Electronic annotators that use ISS annotations to make predictions should be viewed sceptically. We recommend that curators thoroughly review ISS annotations before accepting them as valid. Overall, users of curated sequence annotations from the GO database should feel assured that they are using a comparatively high quality source of information.     

A simple method of chymotrypsin concentration and purification from pancreas homogenate using Eudragit® L100 and Eudragit® S100

The condition of chymotrypsin (ChTRP)–Eudragit^® (Eu) insoluble complex formation was studied with the aim of applying this information to the separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate. The optimal pH of the complex precipitation was 4.60 for ChTRP–Eudragit^® L100 and 5.40 for ChTRP–Eudragit^® S100. The polyelectrolyte concentration necessary for the commercial enzyme precipitation was lower than 0.1% (w/v). The complex formation was inhibited by NaCl for both polyelectrolytes. The method was applied to recover the enzyme from bovine homogenate; ChTRP was precipitated by Eudragit^® addition. The non-soluble complexes were separated by simple centrifugation and re-dissolved by a pH change to 8.20. The best conditions to recover ChTRP brought about a purification factor of around 4 and 90% yield.     

Correlation and prediction of gene expression level from amino acid and dipeptide composition of its protein

Background  

A large number of papers have been published on analysis of microarray data with particular emphasis on normalization of data, detection of differentially expressed genes, clustering of genes and regulatory network. On other hand there are only few studies on relation between expression level and composition of nucleotide/protein sequence, using expression data. There is a need to understand why particular genes/proteins express more in particular conditions. In this study, we analyze 3468 genes of Saccharomyces cerevisiae obtained from Holstege et al., (1998) to understand the relationship between expression level and amino acid composition.