Figure 1.
Schematic illustration of the arrangement of the P-SSP7 genome.
(A) Sequencing of the ends of the P-SSP7 genome extracted directly from phage particles. Arrows, and numbers under the arrows, indicate the sequences acquired: Blue from the entire genome and green from end fragments produced by digestion of the genome with the BamHI and PmeI restriction enzymes. The positions of the primers used for sequencing are shown in black type at the beginning of the arrows. Genome numbering for the primers and sequences is that for the originally published sequence [5]. The purple line denotes the 728 bp region found to be upstream of ORF1 in this study, but positioned downstream of ORF54 in the originally published sequence. The repeat regions are shown in red at both ends of the genome. (B) Diagram showing the arrangement of the P-SSP7 genome as originally published (GenBank accession numbers: AY939843.1, [5] and GU071093 [16]. (C) Diagram of the revised genome arrangement based on the results from this study (updated GeneBank submission, accession number: AY939843.2).
Figure 2.
Digestion and Southern analyses of the P-SSP7 genome.
(A) Schematic genome map showing the positions of the restriction enzyme cleavage sites (red) and the expected fragment sizes after digestion with BamHI alone (top) and both BamHI and PmeI (bottom) based on the revised genome arrangement shown in Fig. 1C. (B) Restriction digestion of the P-SSP7 genome extracted from phage particles (lanes 3 and 4) and the genome cloned into a fosmid (lanes 5 and 6), with BamHI alone (lanes 3 and 5) or with BamHI and PmeI (lanes 4 and 6), separated by pulse field gel electrophoresis. Note that the only difference for digestion of the cloned genome is the presence of an additional fragment corresponding to the size of the fosmid vector. Fragments corresponding to the expected sizes shown in (A) are marked with the appropriate letter designations (a to f). Fragment size markers (M): 1 kb DNA ladder (lane 1) and Lambda DNA cut with HindIII (lane 2), are shown. (C) Southern analyses of the restriction digested DNA in (B) using 4 probes (denoted above the lanes) show that the repeat region appears twice on the genome on the same fragments as the first and last ORFs. The positions of the gene probes on the genome are shown as light blue boxes and the repeat region probe as green boxes in the top panel of (A). Lane numbering and fragment designations are the same as in (B).
Table 1.
PCR and sequencing primers used in this study.