Computational protocol: Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

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Protocol publication

[…] A putative desmosterol reductase gene sequence for Bombyx mori was obtained by the following bioinformatic procedure. The human 3β-hydroxysterol Δ24 reductase (DHCR24) sequence was used to search B. mori protein sequences at Silkdb (http://silkworm.genomics.org.cn/) with BLAST . This identified two homologues with identifiers BGIBMGA005735 and BGIBMGA012624. An alignment of the human and the two potential B. mori sequences was produced using Clustal X. Each homologue was used to further query the nr database and sequences of close relatives retrieved. These were supplemented with sequences obtained at genome resource webpages for Meloidogyne incognita (http://www.inra.fr/meloidogyne_incognita/) and M. hapla (http://www.pngg.org/cbnp/) . Since relatives of the Arabidopsis DIMINUTO gene product were not the prime focus of this work only a selection of these was included. These, along with a more distantly related plant cytokinin dehydrogenase to serve as an outgroup in phylogenetic analysis, were aligned with MUSCLE . Phylogenetic analysis was done with MEGA 4 as follows. The evolutionary history was inferred using the Minimum Evolution method employing 500 replicates generated by bootstrapping . The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm at a search level of 1 and the Neighbor-joining algorithm was used to generate the initial tree. All positions containing gaps and missing data were eliminated. Subcellular localization was predicted using TargetP . The 5′ upstream regions of candidate B. mori genes were searched against the TRANSFAC database at http://www.biobase.de. […]

Pipeline specifications

Software tools Clustal W, MUSCLE, MEGA, TargetP, Biobase
Databases TRANSFAC SilkDB Meloidogyne
Applications Miscellaneous, Phylogenetics, Protein sequence analysis, Nucleotide sequence alignment
Organisms Bombyx mori, Saccharomyces cerevisiae
Chemicals Cholesterol, Desmosterol, Flavin-Adenine Dinucleotide, NAD, Phytosterols, Sterols