Figure 7 shows that the genes of the urease gene cluster are transcribed as a single transcript. Control assays confirmed that the purified RNA was free of contaminating DNA (Figure 7, lanes b). Figure 7 Reverse transcriptase PCR of urease operon. Ethidium bromide stained agarose gel showing results of reverse transcriptase PCR with H. influenzae strain 11P6H. Lanes: a) purified RNA with reverse transcriptase and Taq DNA polymerase; b) purified RNA with Taq DNA polymerase (negative control); c) purified DNA with Taq DNA polymerase (positive

control). Lane H2O is a water control. Oligonucleotide Selleckchem NVP-LDE225 primers were designed to span adjacent genes in the gene cluster as noted at the top of the gel (See Table 1). Molecular size markers are noted in base pairs on the right. Presence of urease operon in clinical

isolates To determine whether the urease operon is present in clinical isolates of H influenzae, 20 clinical isolates, including 10 otitis media strains and 10 COPD strains were studied by PCR. Primers corresponding to genes located in the 5′ region (ureA), central region (ureC) and 3′ region (ureH) of the Roxadustat ic50 operon were designed. Amplicons of identical size were obtained from 20 of 20 clinical isolates with all 3 sets of primers (Figure 8). These results indicate that the urease operon is present in all strains tested and that no variation was observed in the lengths of these genes in diverse strains tested. Figure 8 Urease operon in clinical isolates. Ethidium bromide stained agarose gels showing amplicons of genes in the urease gene cluster as noted on the left. Lanes a through j, amplicons from COPD sputum

isolates 6P8H1, 14P14H1, 24P17H1, 27P5H1, 33P18H1, 43P2H1, 55P3H1, 66P33H1, 74P16H1, 91P18H1, respectively. Lanes k through t, middle ear fluid aspirate isolates 1749, 1826, 6699, 6700, 4R, 17R, 26R, 47R, P86, P113, respectively. Molecular size standards are noted on the right in kilobases. A BLASTn search with the sequence corresponding to the urease operon was performed to determine which strains of H. influenzae whose genomes are available in ZD1839 ic50 GenBank contained the urease operon. Five of 6 strains whose complete genome has been sequenced contain the urease operon. A high degree of sequence similarity in the urease operon is present among the 5 strains. In strain R2866, which is urease negative, the urease operon is replaced by a single gene with homology to the gonococcal mtrF gene [40]. Sequence analysis of the same region of 9 additional urease negative strains revealed sequence that is very similar to that of strain R2866 [40]. Transcription of the ureC during growth in pooled human sputum To assess expression of urease in conditions that simulate conditions in the human respiratory tract in COPD, transcription of ureC was measured in H.