Computational protocol: Organelles in Blastocystis that Blur the Distinction between Mitochondria and Hydrogenosomes

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Protocol publication

[…] Total RNA from strain NandII was used for library construction by Amplicon Express (Washington). Complementary DNAs (cDNAs) were sequenced from the 5′ end to generate ESTs and where necessary 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed to obtain full-length genes. ESTs were assembled into clusters and incorporated into the TBestDB database . Three strains of Blastocystis were used for DNA isolation: NandII, BT-1, and DMP/02-328. Amplification of portions of the organellar genome was carried out for all three strains. SSU ribosomal RNA gene sequences obtained by PCR were used to construct specific primers, which were then employed in PCR-based primer walking along the genome. Phylogenetic analyses were carried out after alignment of the sequences by using ClustalX followed by manual editing in which ambiguously aligned sites were excluded. All organellar genome-encoded protein sequences were used in a concatenated analysis for phylogenetic reconstruction. Trees were calculated by using maximum-likelihood (ML) and Bayesian methods. ML bootstrap analyses were derived to estimate statistical support for each node. A homologous antibody raised against the Blastocystis [FeFe] hydrogenase was used in confocal immunofluorescence microscopy for localization of the protein within cells. DAPI and MitoTracker staining were carried out to visualize DNA and organelles, and as a negative control cells were stained with CellTracker. An in silico analysis of all ESTs sequenced from the cDNA library was carried out to classify the transcripts as orthologs of human/yeast mitochondrial or nuclear/cytosolic proteomes. In addition, the KEGG Automatic Annotation Server was used to generate KEGG pathways. A more detailed description of all strain material and methods used is provided in the . […]

Pipeline specifications

Software tools Clustal W, KAAS
Databases TBestDB KEGG
Application Phylogenetics
Organisms Homo sapiens
Diseases Blastocystis Infections
Chemicals Iron, Tricarboxylic Acids