Designing multiple degenerate primers via consecutive pairwise alignments

<p>Abstract</p> <p>Background</p> <p>Different algorithms have been proposed to solve various versions of degenerate primer design problem. For one of the most general cases, multiple degenerate primer design problem, very few algorithms exist, none of them satisfying the criterion of designing low number of primers that cover high number of sequences. Besides, the present algorithms require high computation capacity and running time.</p> <p>Results</p> <p>PAMPS, the method presented in this work, usually results in a 30% reduction in the number of degenerate primers required to cover all sequences, compared to the previous algorithms. In addition, PAMPS runs up to 3500 times faster.</p> <p>Conclusion</p> <p>Due to small running time, using PAMPS allows designing degenerate primers for huge numbers of sequences. In addition, it results in fewer primers which reduces the synthesis costs and improves the amplification sensitivity.</p

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

<p>Abstract</p> <p>Background</p> <p>Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in <it>Escherichia coli </it>and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices.</p> <p>Results</p> <p>Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile.</p> <p>Conclusions</p> <p>Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge.</p

Genome-wide gene expression profiling of stress response in a spinal cord clip compression injury model.

BackgroundThe aneurysm clip impact-compression model of spinal cord injury (SCI) is a standard injury model in animals that closely mimics the primary mechanism of most human injuries: acute impact and persisting compression. Its histo-pathological and behavioural outcomes are extensively similar to human SCI. To understand the distinct molecular events underlying this injury model we analyzed global mRNA abundance changes during the acute, subacute and chronic stages of a moderate to severe injury to the rat spinal cord.ResultsTime-series expression analyses resulted in clustering of the majority of deregulated transcripts into eight statistically significant expression profiles. Systematic application of Gene Ontology (GO) enrichment pathway analysis allowed inference of biological processes participating in SCI pathology. Temporal analysis identified events specific to and common between acute, subacute and chronic time-points. Processes common to all phases of injury include blood coagulation, cellular extravasation, leukocyte cell-cell adhesion, the integrin-mediated signaling pathway, cytokine production and secretion, neutrophil chemotaxis, phagocytosis, response to hypoxia and reactive oxygen species, angiogenesis, apoptosis, inflammatory processes and ossification. Importantly, various elements of adaptive and induced innate immune responses span, not only the acute and subacute phases, but also persist throughout the chronic phase of SCI. Induced innate responses, such as Toll-like receptor signaling, are more active during the acute phase but persist throughout the chronic phase. However, adaptive immune response processes such as B and T cell activation, proliferation, and migration, T cell differentiation, B and T cell receptor-mediated signaling, and B cell- and immunoglobulin-mediated immune response become more significant during the chronic phase.ConclusionsThis analysis showed that, surprisingly, the diverse series of molecular events that occur in the acute and subacute stages persist into the chronic stage of SCI. The strong agreement between our results and previous findings suggest that our analytical approach will be useful in revealing other biological processes and genes contributing to SCI pathology

MANNICH BASES OF CYCLOALKANONES AND RELATED COMPOUNDS AS CYTOTOXIC AGENTS

Cancer chemotherapy is primarily limited by the non-selective action of drugs and hence toxicity to normal cells. Therefore one of the most important features of new drugs should be selectivity towards tumor cells. Mannich bases have been found to possess some degree of selective antineoplastic activity. These compounds demonstrate a greater affinity for the thiol groups than amino and hydroxy functions in biomolecules. Hence carcinogenic and mutagenic side effects may be avoided. The aim of the present investigation was to design and prepare a number of Mannich bases which could be screened against P388-D1 leukemia cells and also in the NCI screen. Thus the following series of compounds were designed and synthesized: • Mono Mannich bases of cycloalkanones which are potentially cytotoxic through conversion to the corresponding a,ß-unsaturated ketones. • Bis Mannich bases of cycloalkanones. These were designed to examine the theory of sequential cytotoxicity. • Quaternary ammonium salts of mono and bis Mannich bases with a view to attain a faster rate of deamination and hence greater cytotoxicity than the corresponding tertiary amines. • 2-(Arylmethylene)cycloalkanones and the corresponding Mannich bases. The Mannich bases of cyclopentanone and cyclohexanone generally demonstrated significant cytotoxicity against P388-D1 leukemia cells. However the activity is decreased with further increases in the size of the cycloalkanering. Thus the cyclododecanone mono and bis Mannich bases were in general far less active. The bis Mannich bases of cyclohexanone were found to be more cytotoxic in the P388-D1 screen than the corresponding mono derivatives. In order to investigate whether there is a correlation between the cytotoxicity and the basicity of the amino function and hence the rate of deamination, various series of mono and bis Mannich bases of cyclopentanone, cyclohexanone and cyclododecanone with different amino moieties having varying log Kb values were synthesized and evaluated. However, no correlation was observed between the predicted rates of deamination and cytotoxicity. A number of mono and bis Mannich bases of cycloalkanones synthesized in this project showed greater cytotoxicity and selectivity than melphalan in the NCI screen. 2-(Arylmethylene)cycloalkanone Mannich bases demonstrated greater cytotoxicity against P388-D1 leukemia cells than the corresponding 2-(aryl-methylene)cycloalkanones. The activities of the Mannich bases in which the aryl ring was unsubstituted or contained a 4-methyl group were similar to that of melphalan

MANNICH BASES OF CYCLOALKANONES AND RELATED COMPOUNDS AS CYTOTOXIC AGENTS

Cancer chemotherapy is primarily limited by the non-selective action of drugs and hence toxicity to normal cells. Therefore one of the most important features of new drugs should be selectivity towards tumor cells. Mannich bases have been found to possess some degree of selective antineoplastic activity. These compounds demonstrate a greater affinity for the thiol groups than amino and hydroxy functions in biomolecules. Hence carcinogenic and mutagenic side effects may be avoided. The aim of the present investigation was to design and prepare a number of Mannich bases which could be screened against P388-D1 leukemia cells and also in the NCI screen. Thus the following series of compounds were designed and synthesized: • Mono Mannich bases of cycloalkanones which are potentially cytotoxic through conversion to the corresponding a,ß-unsaturated ketones. • Bis Mannich bases of cycloalkanones. These were designed to examine the theory of sequential cytotoxicity. • Quaternary ammonium salts of mono and bis Mannich bases with a view to attain a faster rate of deamination and hence greater cytotoxicity than the corresponding tertiary amines. • 2-(Arylmethylene)cycloalkanones and the corresponding Mannich bases. The Mannich bases of cyclopentanone and cyclohexanone generally demonstrated significant cytotoxicity against P388-D1 leukemia cells. However the activity is decreased with further increases in the size of the cycloalkanering. Thus the cyclododecanone mono and bis Mannich bases were in general far less active. The bis Mannich bases of cyclohexanone were found to be more cytotoxic in the P388-D1 screen than the corresponding mono derivatives. In order to investigate whether there is a correlation between the cytotoxicity and the basicity of the amino function and hence the rate of deamination, various series of mono and bis Mannich bases of cyclopentanone, cyclohexanone and cyclododecanone with different amino moieties having varying log Kb values were synthesized and evaluated. However, no correlation was observed between the predicted rates of deamination and cytotoxicity. A number of mono and bis Mannich bases of cycloalkanones synthesized in this project showed greater cytotoxicity and selectivity than melphalan in the NCI screen. 2-(Arylmethylene)cycloalkanone Mannich bases demonstrated greater cytotoxicity against P388-D1 leukemia cells than the corresponding 2-(aryl-methylene)cycloalkanones. The activities of the Mannich bases in which the aryl ring was unsubstituted or contained a 4-methyl group were similar to that of melphalan

Functional and interacting domains of the yeast and human Mre11

In an effort to understand the molecular mechanisms of DNA alkylation repair, I initiated molecular cloning of a novel gene, ' NGS1'. The Saccharomyces cerevisiae ngs1-1 mutant was previously identified by its enhanced sensitivity to simple DNA alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of 'NGS1' as a putative DNA alkylation repair gene revealed that it is identical to 'MRE11'/'RAD58', a gene that is involved in meiotic recombination and DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, I determined the nucleotide sequence alterations of a number of 'mre11' mutant alleles, including 'ngs1-1', 'mre11-1' ('ts'), 'mre11-2', 'mre11-3' and 'mre11-58'. The location of various 'ngs1'/' mre11'/'rad58' mutations combined with the deletion analysis indicates that the functional domain(s) resides in the highly conserved N-terminus of Mre11. I also investigated various roles of Mre11 in spontaneous and DNA damage-inducedmitotic recombination. The assays used in this study show that the mre11D mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, 'MRE11' appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination. Physical interactions between members of the 'RAD52' epistasis group have been detected genetically and biochemically. These protein interactions also appear to be important at the early stage of meiotic recombination. Mre11 has been shown to interact with itself, Rad50 and Xrs2 in a yeast two-hybrid system. Preliminary studies employing deletion analysis predicted that the self-interaction domain of Mre11 resided at the N-terminus of the protein. To determine domain(s) required for Mre11 complex formation, and to elucidate the relationship between this complex formation and 'MRE11' DNA repair function, I employed a combined yeast two-hybrid and functional analyses. My results indicate that both Mre11 dimerization and interaction with Rad50 are essential for recombinational repair. I found that the N-terminus of the Mre11 protein constitutes the core homodimerization and heterodimerization domain and is sufficient for Mre11 DNA repair activity. Collectively, these studies support the hypothesis that Mre11 self-association as well as its assembly into a multi-protein complex consisting of Mre11 and Rad50 are essential for effective DNA recombinational repair. Using the sequence of yeast 'MRE11', isolated the full-length ' hMRE11B' cDNA from a human HeLa cell cDNA library. Compared to the previously identified 'hMRE11', 'hMRE11B' contains an additional 84 base pair sequence that results in a 28 amino acid insertion close to the C-terminus. Overexpression of 'hMRE11B' does not complement the alkylation sensitivity of the 'mre11' null and temperature sensitive mutant strains. My results suggest that species-specific protein interaction determines the functional specificity of 'MRE11 ' and that the participation of the C-terminus of Mre11 protein plays an important role in this regard. (Abstract shortened by UMI.

MAD-DPD: designing highly degenerate primers with maximum amplification specificity

This work introduces minimum accumulative degeneracy, a variant of the degenerate primer design problem, which is particularly useful when a large number of sequences are to be covered by a set of restricted number of primers. A primer set, which is designed on a minimum accumulative degeneracy basis, especially helps to reduce nonspecific PCR amplification of undesired DNA fragments, as fewer primer species are present in PCR. A Boltzmann machine is designed to solve the minimum accumulative degeneracy degenerate primer design problem, called the MAD-DPD Boltzmann machine. This algorithm shows great flexibility, as it can be determined either to solve the problem with strict fidelity to covering all input sequences or to exclude some input sequences if it results in less degenerate primers. This Boltzmann machine is successfully implemented in designing a new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers more diverse antibody sequences than currently available primers. The MAD-DPD Boltzmann machine is available online at bioinf.cs.ipm.ir/download/MAD_DPD08172007.zip

An example of merging two non-gapped aligned sequences into a single sequence

<p><b>Copyright information:</b></p><p>Taken from "Designing multiple degenerate primers via consecutive pairwise alignments"</p><p>http://www.biomedcentral.com/1471-2105/9/55</p><p>BMC Bioinformatics 2008;9():55-55.</p><p>Published online 27 Jan 2008</p><p>PMCID:PMC2253518.</p><p></p> Bases that differ in the two sequences are underlined