Specific primers to the sequence BGIBMGA005735 were used to amplify the DNA from midgut cDNA and the DNA was cloned into pYES2, a galactose-inducible yeast expression vector. invertebrate species dealkylate phytosterols obtained from the plant, yielding cholesterol or a closely related ring-modified C27 sterol [3]. The invertebrate phyla/species which have been demonstrated to be capable of phytosterol dealkylation, include phytophagous insect species [2], [3], yellow fever mosquito, and are also the same as in insects [8]. All the foregoing steps of dealkylation have been demonstrated to occur in insect midgut and, with the exception of the first step, have also been achieved using microsomal (endoplasmic reticulum) preparations [2]. In both insects and cholesterol biosynthetic pathways [21]. We now report the cloning, recombinant expression and characterization of the cDNA encoding desmosterol reductase from the silkworm, sterol biosynthesis, as in vertebrates, but instead in the transformation (dealkylation) of C29 and C28 plant sterols into C27 sterols. Materials Mouse monoclonal to CD48.COB48 reacts with blast-1, a 45 kDa GPI linked cell surface molecule. CD48 is expressed on peripheral blood lymphocytes, monocytes, or macrophages, but not on granulocytes and platelets nor on non-hematopoietic cells. CD48 binds to CD2 and plays a role as an accessory molecule in g/d T cell recognition and a/b T cell antigen recognition and Methods AV-412 All chemicals were obtained from Sigma. Bioinformatic analysis A putative desmosterol reductase gene sequence for was obtained by the following bioinformatic procedure. The human 3-hydroxysterol 24 reductase (DHCR24) sequence [22] was used to search protein sequences at Silkdb (http://silkworm.genomics.org.cn/) with BLAST [23]. This identified two homologues with identifiers BGIBMGA005735 and BGIBMGA012624. An alignment of the human and the two potential sequences was produced using Clustal X. Each homologue was used to further query the nr database [24] and sequences of close relatives retrieved. These were supplemented with sequences obtained at genome resource webpages for (http://www.inra.fr/meloidogyne_incognita/) [25] and (http://www.pngg.org/cbnp/) [26]. 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 [27]. Phylogenetic analysis was done with MEGA 4 [28] as follows. The evolutionary history was inferred using the Minimum Evolution method [29] employing 500 replicates generated by bootstrapping [30]. The evolutionary distances were computed using the p-distance method [31] 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 [31] at a search level of 1 and the Neighbor-joining algorithm [32] was used to generate the initial tree. All positions containing gaps and missing data were eliminated. Subcellular AV-412 localization was predicted using TargetP [33]. The 5 upstream regions of candidate genes were searched against the TRANSFAC database [34] at http://www.biobase.de. Cloning of cDNA Total RNA was extracted from the midgut of day 5, 5th instar larvae. First strand DNA was synthesised using a Bioline cDNA kit. Double-stranded DNA was generated using Pfu polymerase (Stratagene) from the first strand DNA in a PCR reaction using the primers and eggs [four-way polyhybrid strain (12657)(7079)] and artificial diet were obtained from Professor Silvia Cappellozza, CRA- Unit di Ricerca di Api-Bachcioltura, sede di Padova, 35143 Padova, Italy. Eggs were incubated at 27C for approximately 5 days prior to hatching and then kept at 27C and fed on artificial diet [36].The larvae progress through all developmental instars in 30 days under these conditions. Insects were AV-412 taken from various stages of growth and immobilized by chilling on ice before dissecting midgut and various other tissues. The tissues were kept on ice in buffer I (37 mM HEPES/NaOH, pH 7, 300 mM sucrose) during the dissection procedure. The midguts were sliced longitudinally and rinsed repeatedly in buffer I to remove gut contents. Preparation of microsomes Larval tissues were resuspended in a minimal volume of buffer I and homogenised in a Potter- Elvehjem mechanical.
