Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication-9
Nthesis 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 (, , , , , , , , , ) or down-regulated genes () in the intracellular environment. Molecular weight DNA ladders were run in parallel. (B) Limited transcriptional analysis. A partial AI-restricted genomic library from the strain B1940 was constructed. Individual plasmid clones were digested with AI and a Southern blot analysis was performed using P-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 , up-regulated by intracellular meningococci. Asterisks mark the 983 and 1123 bp-long 3AI 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 () are indicated with respect to the available genetic map of MC58. Open rectangles with asterisks locate the 983 and 1123 bp-long 3AI fragments contained in the plasmid clone shown in panel B.<p><b>Copyright information:</b></p><p>Taken from "Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of and its role in intracellular replication"</p><p>http://www.biomedcentral.com/1471-2180/8/131</p><p>BMC Microbiology 2008;8():131-131.</p><p>Published online 29 Jul 2008</p><p>PMCID:PMC2527323.</p><p></p
