For the detection of excised circular intermediates of the various GIs, a series of oligonucleotide primers

was designed from the presumable ends of the respective elements which are supposed to join during circularization. In the case of the adjacent elements GI1, GI2 and GI3 we considered that also various combinations might occur by common excision events of these adjacent islands (Figure 3). The direct repeats flanking the various clc-like elements of B. petrii are shown in Figure 4. Figure 3 Schematic presentation of the genomic region comprising the genomic islands GI1, GI2 and GI3. The GIs are shown as a red lines, their flanking direct repeat regions (DR) by red boxes (dark and light red for identical or nearly identical sequences, respectively) (see also Figure 4). The sequence position of AR-13324 clinical trial the direct repeats and the approximate size of the islands are shown below the elements. The

position of tRNA genes is indicated. eFT-508 Some relevant or characteristic genes encoded by the islands are shown above the elements. The bars below the elements show the expected dimensions of the element after excision from the genome. Stars indicate predicted elements which may use alternative direct repeat sequences for excision or elements composed of more than one island. Arrows above the bars indicate the approximate position of PCR primers and their names (in blue) designed for the amplification of the respective circular intermediates of these Adenylyl cyclase elements (Tab. 3). Figure 4 The direct repeats generated by the integration of the clc -like elements in the B. petrii genome are shown. Identical sequences are indicated in red or blue letters, respectively. Sequence

identities are indicated by vertical bars. The positions of the sequences on the genome sequence are shown on the left and the right of the sequences. The core region identical in all repeats flanking the clc-like elements is indicated by the green box. In case the repeats are part of a tRNA gene, the respective gene is mentioned on the right side of the respective sequences. Table 2 shows the results of this analysis. In the case of GI1 no product could be amplified when using the primer pair GI1–1/GI1–2 which AG-881 cost should provide a product, when the excision involves the direct repeat sequences directly upstream (sequence position 1,084,006) and downstream (sequence position 1,339,485) of the island. Instead, a product was obtained when the primer pair GI1–2/GI1–3 was used which can yield a product only when ring formation involved an alternative downstream repeat sequence (sequence position 1,350,146). This alternative downstream repeat sequence has three mismatches as compared to the upstream repeat and has probably been generated by the integration of GI2, since GI2 at the downstream end is flanked by a second nearly identical copy of this direct repeat (Figure 4).