Comparative modeling of DNA and RNA polymerases from Moniliophthora perniciosa mitochondrial plasmid

<p>Abstract</p> <p>Background</p> <p>The filamentous fungus <it>Moniliophthora perniciosa </it>(Stahel) Aime & Phillips-Mora is a hemibiotrophic Basidiomycota that causes witches' broom disease of cocoa (<it>Theobroma cacao </it>L.). This disease has resulted in a severe decrease in Brazilian cocoa production, which changed the position of Brazil in the market from the second largest cocoa exporter to a cocoa importer. Fungal mitochondrial plasmids are usually invertrons encoding DNA and RNA polymerases. Plasmid insertions into host mitochondrial genomes are probably associated with modifications in host generation time, which can be involved in fungal aging. This association suggests activity of polymerases, and these can be used as new targets for drugs against mitochondrial activity of fungi, more specifically against witches' broom disease. Sequencing and modeling: DNA and RNA polymerases of <it>M. perniciosa </it>mitochondrial plasmid were completely sequenced and their models were carried out by Comparative Homology approach. The sequences of DNA and RNA polymerase showed 25% of identity to 1XHX and 1ARO (pdb code) using BLASTp, which were used as templates. The models were constructed using Swiss PDB-Viewer and refined with a set of Molecular Mechanics (MM) and Molecular Dynamics (MD) in water carried out with AMBER 8.0, both working under the ff99 force fields, respectively. Ramachandran plots were generated by Procheck 3.0 and exhibited models with 97% and 98% for DNA and RNA polymerases, respectively. MD simulations in water showed models with thermodynamic stability after 2000 ps and 300 K of simulation.</p> <p>Conclusion</p> <p>This work contributes to the development of new alternatives for controlling the fungal agent of witches' broom disease.</p

Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma

<p>Abstract</p> <p>Background</p> <p>Human Disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC).</p> <p>Methods</p> <p>We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1.</p> <p>Results</p> <p>Decrease or absent of <it>DAB2 </it>transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant <it>DAB2 </it>promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways.</p> <p>Conclusions</p> <p>We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells.</p