[43]. Large-scale isolation of E. coli plasmids for nucleotide sequence analysis was performed with the Plasmid Midi Kit (Qiagen Ltd., Crawley, United Kingdom) according to the manufacturer’s instructions. Constructions for E. durans were achieved using L. lactis NZ9000 as intermediate host. Plasmid and chromosomal DNA of E. durans and L. lactis were isolated and transformed as described previously [44]. All enzymes for DNA technology were used according to the manufacturer’s specifications. DNA hybridizations were performed using the non-radioactive DNA Labelling and Detection Kit (Roche Molecular

Biochemicals) following the manufacturer’s instructions. RNA manipulation and northern blot analysis of FK506 clinical trial tyrS transcripts Total

RNA was isolated from cells of E. durans IPLA655 grown in GM17-Y and GM17 + Y at pH 4.9 and pH 7.5 to exponential phase (optical density at 600 nm [OD600] of 0.6). Purified RNAs were resuspended in DEPC 0.1% (diethyl pyrocarbonate) treated water, and total concentration and yield were determined by UV spectrophotometry by measuring absorbance at 260 nm using a BioPhotometer (Eppendorf, NY). After extraction, RNA samples were treated with DNase (Fermentas, Vilnius, Lithuania), as described by the manufacturer, to eliminate any genomic contaminations. 20 μg of each sample were subjected to electrophoresis through a 1.5% agarose gel selleck chemicals containing 5% formaldehyde and 1X MOPS buffer [20 mM 3-N-morfolino-propanesulfonic acid (MOPS), 1 mM EDTA, 5 mM sodium acetate; pH 7.0]. Transfers and hybridizations were performed as described by Sambrook

et al. [43]. DNA probes were labeled with [α-32P]dATP by nick translation with the DNA polimerase/DNase I (Invitrogen A/S, Taastrup, Denmark). Primers used in the PCR-amplification Astemizole of the probes are summarized in Table 2. Table 2 Oligonucleotides used in this study Primer Function* Sequence (5′ to 3′) TDC11 RT1 tyrS probe amplification (F) tyrS probe amplification (R) TCAATTACAGATCGGTGGGG ACTTACCATCGAATGCATCAAATG TRNA2B(2) TRNA(P) TyrS prom (F) tyrS prom (R) mRNA-C quantification (F) mRNA-C quantification (R) mRNA-L quantification (F) mRNA-L quantification (R) CGTAAATTAGAAGGGCCAGAGGCAG GATCAAGCCAGATTGCGCCACCTGCAG AACAGGCAATGATCAAAACGAAGTA CATAGGCTCCTAAAATGTAATTCGC TYR2 PtyrS mapping (R) ACTTCGTTTTGATCATTGCCTG TDC123 TDC130 PtyrS Δ cloning and sequencing (F) PtyrS Δ cloning and sequencing (R) AAAACTTCCCATATGCATTGTAACG CTGCAGCATTTTATATGTTTTGTAGTAA TYS (F) TYS(R) tyrS overexpression (F) tyrS overexpression (R) ATGGGTGGTGGATTTGCTAATATTATCGATGAATTAACTT TTGGAAGTATAAATTTTCATCAACTACTTTGGCCAAAAAG *F, forward; R, reverse Quantification of tyrS expression by reverse transcription quantitative PCR (RT-qPCR) Gene expression analysis was carried out by RT-q PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using SYBR® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA).