coli BL21. Growth temperature were 37°C, except where indicated and growth rates were estimated by measuring the increase in OD600. Origin of the immunoreactive MS2/28 DNA fragment Isolation and characterization of the M. synoviae DNA fragment MS2/28 [GenBank: MSU66315] was previously described [18]. MS2/28 contains two partial ORFs, referred to as MS2/28.1 (5′ end) and MS2/28.2 (3′ end). Reverse transcription and polymerase chain reaction (RT-PCR) The total RNA of M. synoviae strain WVU 1853 was isolated from a

24-h culture, using a protocol recommended for Gram-positive bacteria [23]. Genomic M. synoviae DNA was eliminated from the RNA preparation using DNAse I (2,5 mg/ml) digestion for a 1-h period at 37°C. DNAse I-treated see more total RNA of M. synoviae was prepared as described above. Reverse transcription was performed at 55°C in a 20 μl reaction mixture containing 2 μg of total RNA, 4 μl of dNTP at 20 mM each, 12.5 μM of the reverse primer 2/28.1Rev (5′-GGGCGGCCGCCTACACTTGCAGTACTTGGCG-3′), 20 units of AMV reverse transcriptase and 2 μl of 10 × buffer reaction (50 mM Tris-Cl, 8 mM MgCl2, 30 mM KCl, 1 mM dithiotreitol, pH = 8). The first strand cDNA synthesis was allowed to proceed

for 1 h followed by inactivation at 65°C during 10 min. PCR amplification was next performed using 2/28.1Rev coupled to the PromF primer (5′-GTCGACGAAATTAAGTAAATTATTAAAG-3′) which anneals to the 5′ end region (-120 to -98) of the expected vlhA1-derived transcript. The amplification Hippo pathway inhibitor reaction consisted of 30 cycles of 94°C for 120 s, 55°C for 120 s and 72°C for 120 s, followed by an extension of 72°C for 7 min. Cloning and sequencing of the RT-PCR

product The 1.934 kb RT-PCR product was purified and ligated into NotI/SalI-digested pBluescript II KS+ plasmid. The ligation product was used to transform E. coli HB101 cells and recombinant clones were screened using restriction analysis. Determination nearly of the nucleotide sequence was performed with the Prism Ready Reaction Dye Deoxy Terminator Cycle sequencing Kit on an ABI PRISM 377 DNA sequencer (Applied Biosystems). The cloned amplicon was selleck compound sequenced in both orientations from two different plasmid clones using sequence-specific internal and plasmid-anchored primers. The sequence data were edited and aligned using the software programs BioEdit [24] and ClustalW [25]. Confirmation of the position of the completed MS2/28.1 gene sequence relative to the unique vlhA1 promoter Using genomic DNA extracted from single colonies as template, PCR amplifications were performed, combining EXpro (5′-CAAATTTAGTTAATTCACTTA-3′), a sense primer placed in the vlhA1 promoter region (-213 to -193), with either vlhA1 R (5′-TATTGTTTTCGGCATTATTTGCTACGTC-3′), a vlhA1-specific reverse primer, or ORF5.1R (5′-GCCTCCACTTCCATCTCCGCTTTCACT-3′), the MS2/28.1-specific reverse primer. To ensure that the full-length MS2/28.