Expression of both Chlamydia pneumoniae RNase HIIs in Escherichia coli

Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39

“…The CP0654 and CP0782 fragments were amplified from pET28a-CP0654 and pET28a-CP0782 (Pei et al, 2005; Table 1) with primers CP0654-F/CP0654-R (F: 59-GGGGGGCTCGAGatgaatacttctatttctgaaattc-39; R: 59-GGGGGGGCATGCtcatacaatagcacacatttgc-39) and CP0782-F/CP0782-R (F: 59-GGGGGGCTCGAGatgtcctgcatgccgccacc-39; R: 59-GGGGGGGCATGCttatttccccgaacaaatttcgtca-39). These fragments were treated with XhoI/SphI and ligated with predigested pUC-G, producing pUC-rnhA, pUC-rnhB, pUC-CP0654 and pUC-CP0782, respectively (Table 1).…”

“…Recombinant Cpn-RNase Hs were expressed and purified, and the enzymic activities were measured as before (Pei et al, 2005). Unless specified otherwise, assays were performed in reaction buffer containing 10 mM Tris/HCl, pH 9.0, 50 mM NaCl, 1 mM b-mercaptoethanol, 10 mM MgCl 2 and 10 mg bovine serum albumin ml 21 .…”

“…The complete genome sequence of C. pneumoniae AR39 offered much genetic information about this microbe (Read et al, 2000). Two prospective RNase HII and HIII genes, CP0654 and CP0782, have been isolated from C. pneumoniae AR39 and expressed in E. coli previously (Pei et al, 2005). In this study, the enzymic properties of these two RNase Hs (CpnRNase HII and HIII) were analysed further and their functional complementation of RNase H-deficient E. coli mutants was demonstrated in vivo.…”

Detection of Single Nucleotide Polymorphism by RNase H-Cleavage Mediated Allele-Specific Extension Method

“…it has been demonstrated by many results that type 2 Rnase h can hydrolyze DnA-rn 1 -DnA/DnA substrates (rn 1 = one ribonucleotide) at the DNA-RNA junction (5'-side of the single ribonucleotide) (4,19,20). Two of type 2 RNases H from Chlamydia pneumoniae, Rnase hii (cpRnase hii) and RNase HIII (CpRNase HIII) were successfully expressed and purified in our lab previously (17). Furthermore, our research demonstrated that cpRnase hii could cleave DnA-rn 1 -DnA/ DNA duplex effectively and the cleavage rates of CpRNase HII were negatively affected by mismatches carried by the DnA-rn 1 -DNA/DNA duplex (5).…”

“…It wass confirmed that the cleavage efficiencies of perfectly matched DNA duplexes are 4.2-to 11.1-fold greater than those of mismatched duplexes. Based on the CpRNase HII-cleavage and allele specific extension, we developed a novel and effective method for SnP detection -Rnase h-cleavage mediated allele- CpRNase HII catalyzed reaction CpRNase HII were prepared as described by Pei et al (17) and their catalyzed reactions were performed at 37 °C for 10-30 min in 10 μl solution containing 1× reaction buffer (10 mM tris-hcl, ph 8.0, 50 mM nacl, 10 mM Mgcl 2 , 1 mM b-mercaptoethanol, 10 μg/ml bovine serum albumin), specified amounts of labeled or non-labeled DnA-rn 1 -DnA/DnA substrates and 10 ng CpRNase HII (≈1.8 × 10 -3 units). the specific activity of CpRNase HII was determined as described by Pei et al (17).…”