The secondary structures in the MITEs were predicted employing the RNAfold webserver. RM methods A protein database of RM system genes was locally pre pared by collecting data from your REBASE. All anno tated proteins initially searched against this database, and protein hits of blastP have been picked as candi dates for manual checking. RM techniques have been confirmed with the linked presence of restriction enzymes, DNA methylase as well as other RM genes or domains. Sep arate RM genes have been stored even though the E value was 1e 10 or less. The disrupted RM genes had been reconfirmed by manually inspecting the alignment in the remaining elements. Metabolic pathway analysis A pathway/genome database of Anabaena sp. 90 was constructed working with Pathway Equipment from your annota tion. Pathway Hole Filler was even further used for retriev ing the missing enzymes. Hassallidin DNA extraction and PCR Both Anabaena sp.
90 and also a knockout mutant con structed in 1999 have been grown as previously described. Genomic DNA through the two Anabaena strains was extracted using either the DNeasyW Plant Mini Kit or even the E. Z. N. A SP Plant DNA selleckchem Mini Kit. PCR amplifications of DNA from Anabaena have been performed in iCycler working with the primers hasV fw which were built to amplify throughout the web site of deletion inside the hasV gene. The primer pairs a1 and hasV fw had been created to enable particular detection of deletion and full length versions of hasV, respectively. Every PCR was carried out in one ? DyNAzyme buffer with 100 uM of each dNTP, 0. 25 uM of each primer, 0. 4 U DyNAzyme II DNA polymerase and ten 170 ng of template DNA inside a ultimate volume of 20 ul. The next PCR ther mocycle was applied to amplify the hasV gene, preliminary de naturation at 94 C for 3 min, 32 cycles of 94 C for thirty s, 57 C for 30 s and 72 C for 1. five min, in addition to a final extension at 72 C for ten min.
For detection in the deletion, a similar PCR programme was utilised, together with the exception of annealing at 64 C for thirty s and elongation at 72 C for one min. Chemical analysis Cells on the cyanobacterial strains were collected in the twenty 40 ml cultures Sunitinib by centrifugation at 7000 ? g for seven min and freeze dried. The dried cells had been extracted with one ml of methanol in two ml plastic tubes containing glass beads using a FastPrep cell disrupter for ten s at a speed of six. five m s 1. The extracts had been centrifuged at ten 000 ? g 5 min just before LC/MS examination. The LC/MS analyses of extracts had been carried out in an Agilent 1100 Series LC/MSD Trap XCT Plus Program implementing a Phenomenex Luna C18 LC column. The column was protected which has a C18 precolumn. The LC/MS parameters had been optimized with hassallidin ion m/z 1862 with detrimental mode of polarity. The mobile phase consisted of 0. 1% aqueous formic acid and acetonitrile. A gradient from 10% to 100% was run over 30 min at a flow charge of 0.