Sequences were compared to A. fumigatus Af293 genomic sequence (Nierman et al., mTOR inhibitor 2005) using the blast function on the cadre database (Mabey Gilsenan et al., 2009).
Both flanking regions were located in the genomic sequence and used to pinpoint the insertion site. Colony radial growth experiments were carried out as described previously (Robson et al., 1995) using 2% glucose in agar plates containing Vogel’s salts. Four thousand transformants were isolated and screened for altered susceptibility to ITR. After overlay with ITR containing agar, 19 transformants that displayed either continued or completely arrested growth were selected, of which eight had at least a fourfold difference in ITR susceptibility relative to the parental strain (Table 2). All eight transformants displayed normal growth rate colony morphology and sporulation compared to the parental strain. These eight transformants were selected for further analysis. The eight transformants (termed REMI-11, REMI-14D, REMI-56, REMI-85, REMI-101, REMI-102, REMI-103 and REMI-116) were characterised further to determine the nature of the REMI insertion. PCR using primers directed against the AmpR gene in pUC19 confirmed that all of them had at least one integrated copy of pPyrG. Restriction digestion followed by Southern hybridisation with the pUC19 vector fragment of pPyrG
was carried out to determine the nature of the plasmid integrations. XhoI digests established whether or not ‘perfect’ Interleukin-2 receptor REMI that retained the XhoI sequence at the site of insertion had selleck screening library occurred: a single 4.8 kb hybridising band, which represents pPyrG, indicated such an event (Fig. 1). REMI-11, REMI-56 and REMI-101 all give 4.8 kb bands expected from a single insertion. REMI-85,
REMI-14D, REMI-103 and REMI-102 give single bands larger than 4.8 kb and REMI-116 gives two bands. This data were combined with sequence from the insertion site and flanking regions to determine whether the REMI event had occurred at a genomic XhoI site. In REMI-85, REMI-14D, REMI-102, REMI-104 and REMI-116, the rescued plasmids had partial XhoI sites flanking the insertion suggesting that integration occurred in an imperfect manner. REMI-11, REMI-56 and REMI-101 all contained intact XhoI sites at the insertional locus. Combining the Southern blot data and the flanking sequence, we were able to categorise the REMI insertion into perfect or imperfect (Table 2) and determine the insertional copy number. 7/8 RMI isolates had one single plasmid insertion in the genome, three which were perfect REMI. One of them, REMI-116, had multiple insertions and was not investigated further. The site of plasmid insertion was successfully determined by plasmid rescue in all REMI transformants.