Role of Global and Local Topology in the Regulation of Gene Expression in Streptococcus pneumoniae
Figure 2
Construction of R6-derivative strains with Pt
ccat insertions. (A) Organization of the S. pneumoniae R6 chromosome in topology-related domains. Circles (outside to inside) represent: % GC (values above the average in purple); DNA topoisomerase genes (dark blue curved arrows); topology-responsive domains. The chromosome is organized in domains up-regulated (U, red boxes) or down-regulated (D, blue boxes) in response to DNA relaxation; and flanking regions (F, green boxes). Representation has been performed with Artemis and DNA plotter software at www.sanger.ac.uk/resources/software/artemis [45]. (B) Insertion of the Ptccat cassette in diverse supercoiling domains. The cat cassette (green drawing), promoters (curved arrows) and transcription terminators (stem and loop structures) and oligonucleotides with their restriction targets are indicated. (C) Gene organization of the strains indicated. R6 genes (spr numbering) are indicated by white arrows. The cat cassette, promoters and transcription terminators are shown as in B, N, NcoI targets and predicted sizes of fragments, N* indicate that the target is outside the region shown. Distances between NcoI targets are shown above the dashed lines. Stripped box at the bottom of the figure represents the cat probe used for Southern analysis and its position inside the cat gene. (D) Southern blot hybridization of R6-CAT strains. Chromosomal DNA from the strains was cut with NcoI, separated by agarose gel electrophoresis, transferred to a nylon membrane and hybridized with the biothynilated cat probe shown in C.