Figure 1
RT-PCR-DD and limited transcriptional analysis of meningococcal transcripts up- and down-regulated during the intracellular phase. (A) RT-PCR-DD analysis. Oligonucleotides DUS-IN, DUS-OUT, RS3-IN, RS3-OUT or oligonucleotide mixtures 26L-IN/27L-IN, 26L-OUT/27L-OUT were used as primers in first-strand cDNA synthesis using total RNAs from intracellular meningococci (strain B1940) recovered from saponin-lysed HeLa cells after 7 h of infection (intracellular) or control bacteria grown for 7 h in cell culture medium without HeLa cells (control) as templates. Then second-strand cDNAs synthesis was carried out using the corresponding oligonucleotides and a mixture of random hexamers as primers, and the PCR products were analyzed by polyacrylamide gel electrophoresis. In lanes 1 and 6 molecular weight ladders, whose sizes are indicated on the left of each panel, were run in parallel. The arrows indicate bands corresponding to either up-regulated (a, c, d, e, f, g, l, m, n, o) or down-regulated genes (b, h, i) in the intracellular environment. Molecular weight DNA ladders were run in parallel. (B) Limited transcriptional analysis. A partial Sau3AI-restricted genomic library from the strain B1940 was constructed. Individual plasmid clones were digested with Sau3AI and a Southern blot analysis was performed using 32P-labeled cDNA probes derived from intracellular bacteria after 8 h of infection of HeLa cells or control bacteria grown for 8 h in cell culture medium. In the figure only a limited number of clones (22 clones out of 3000) on duplicate filters are shown. The arrow indicates the cloned NMB0551 ORF corresponding to priA, up-regulated by intracellular meningococci. Asterisks mark the 983 and 1123 bp-long Sau3AI fragments contained in the plasmid clone. (C) Genetic map of meningococcal genes up-regulated in the intracellular environment. The positions of the cDNAs (closed rectangles) corresponding to up-regulated genes that were identified by RT-PCR-DD (a, c, g, f, l, n) are indicated with respect to the available genetic map of MC58. Open rectangles with asterisks locate the 983 and 1123 bp-long Sau3AI fragments contained in the priA plasmid clone shown in panel B.