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Both M. pneumoniae (M129, broth passage 28, ATCC 29342) and M. genitalium (G-37, ATCC 33530) cultures were grown at 37 C in 150 ml cell culture flasks containing 100 ml of modified Hayflick medium (3) supplemented with 20% horse serum. RNA was extracted with the Roti-Quick-Kit (Roth, Karlsruhe, Germany). The kit consists of three solutions: solution 1 contains guanidiniumthiocyanate, solution 2 includes a phenol/chloroform mixture and solution 3 contains isopropanol. After a growth period of 96 hours ( M. pneumoniae) or 168 hours ( M. genitalium), surface-attached cells were washed twice with phosphate-buffered saline (PBS: 0.15 M NaCl, 10 mM sodium phosphate, pH 7.4) and immediately lysed in the culture flasks by adding 2 ml of the solution 1. The mixture was then put on ice and homogenized by repeated passage through a 0.9 x 40 mm gauge needle. After adding 2.6 ml of solution 2 was and 10 min of incubation on ice the probes were centrifuged at 10,000 x g and 4 C for 15 min. The RNA was precipitated from the water phase with 2 ml of solution 3 and 40 min of incubation at -20 C. After 15 min of centrifugation at 10,000 x the supernatant was discarded and the pellet resuspended in 600 l of solution 1 and 600 l of solution 3. The RNA was again precipitated at -20 C for 20 min and collected by centrifugation at 20,000 x for 15 min. The pellet was washed thoroughly several times in 75% ethanol and suspended in 200 l of water treated with 0.1% DEPC.
10 g of total RNA were separated by denaturing polyacrylamide gel electrophoresis. The 8% acrylamide gel contained 7 M Urea and was prepared with 1 x TBE (0.09 M Tris-borate, 0.002 M EDTA, pH 8.3). The RNA was diluted 1:1 with an 8 M urea loading solution, denaturated for 5 min at 95 C, immediately cooled down on ice and then loaded onto the gel. Both bands were cut out separately and eluted for 12 hours at 37 C in a buffer consisting of 0.1% SDS, 2 mM EDTA and 0.5 M Tris-HCl pH 7.0, precipitated with 2 vol of 100% ethanol and washed thoroughly three times with 75% ethanol.
Twenty six cosmids covering the complete genome of M. pneumoniae (8) were digested with endonuclease EcoRI, separated on a 0.8% agarose gel, blotted onto a positively charged nylon membrane (Pall Biodyne B) and UV-crosslinked.
The isolated #921 RNA was treated with shrimp alkaline phophatase (Roche Diagnostics, Mannheim) and radioactively labelled by T4 polynucleotide kinase (Roche Diagnostics, Mannheim) in the presence of [-P]ATP. The membrane was hybridized overnight under non-stringent conditions in 5 x (1 x SSC: 150 mM NaCl and 15 mM sodium citrate, pH 7.2) and 1 % SDS with the labelled #921 RNA at 37 C. After hybridization the membrane was washed twice with 2 x SSC and 0.05% SDS for 15 min at 55 C, and exposed to a PhosphorImager screen (Molecular Dynamics) for 3 h. Based on the results of this experiment, overlapping 1 kb long PCR products covering the total area of the EcoRI Fragment F11_28.0 were synthesized with Taq-polymerase (Promega, Madison, USA) and checked on an 0.8% agarose gel. To 1.5 l of each PCR product 1.5l of 0.4 N NaOH was added and the total mixture was spotted onto a positively charged nylon membrane. The PCR products were covalently bound to the membrane by autocrosslinking with UV-light (Stratalinker, Stratagene) and hybridized against the same solution of radioactively labelled RNAs used in the previous experiment. Hybridization was carried out overnight at 50 C, the membrane was washed twice with 2 x SSC and 0.1% SDS and the membrane was subsequently exposed to a PhosphorImager screen for 75 min.
Approximately 10 cpm (Cerenkov) of [P] 5'-end labelled oligonucleotide (ca. 5 x 10 cpm/pmol), and 5g of total RNA were precipitated with ethanol and sodium acetate as described (7). The labelled RNA was added to 30l hybridization buffer [1 M NaCl, 166.6 mM Hepes (pH 7.5), 0.33 mM EDTA (pH 7.6)] and hybridized overnight at 30 C. The mixture was precipitated and the material was dissolved in 25l primer extension reaction mix [1.4 mM dNTP, 1 x Avian Myeblastosis Virus Reverse Transcriptase (AMV-RT) buffer, 500 U RNase inhibitor (Roche Diagnostics, Mannheim)]. The reverse transcription was started by adding 20 U of AMV-RT (Roche Diagnostics, Mannheim), and the reaction mix was incubated at 42 C for 1 h. The nucleic acids were precipitated and washed thoroughly with 75% ethanol. The pellet was dissolved in standard formamide gel loading buffer (7). The length of the cDNA was determined by a 6% denaturing sequencing gel containing 7 M urea, with a sequencing band pattern as a molecular weight marker.
Sequencing reactions were done with the ThermoSequenase radiolabelled terminator cycle sequencing kit (Amersham). The reaction mixture contained ddNTPs and [P] 5'-end labelled oligonucleotides as primers. For preparing the sequencing gel and as running buffer 1 x TBE was used.
Polyacrylamide gels. A 2 mm thick and 10 cm long 6% polyacrylamide gel containing 1 x TBE and 7 M urea was electroblotted in semi-dry conditions for 1 hour at 15 V with the BioRad TransBlot semi-dry transfer cell onto a positively charged nylon membrane and covalently linked by UV-crosslinking. The RNA was stained with methylene blue as described (7) and photographed. The membrane was prehybridized for 4 hours with a solution containing 5 x SSC, 10 x Denhards, 0.05% SDS, 18% formamide. The hybridization was done overnight at 30 C in 10 ml buffer containing 5 x SSC, 10 x Denhardts, 0.05% SDS, 18% formamide and 1.5 x 10 cpm (Cerenkov) of [P] 5'-end labelled primer.
Agarose gels. Total RNA was separated on a standard denaturing 1.2 % agarose gel as described (7). The RNA was blotted onto a positively charged nylon membrane and covalently linked by UV-crosslinking. Hybridization signals were detected by exposing the membrane to a PhosphorImager screen (Molecular Dynamics).
For direct determination of the 3' end, the isolated RNA species were labelled with pCp as described (2). The 3' labelled RNAs were further purified by denaturating polyacrylamide gel electrophoresis and used for two-dimensional mobility shift analysis. The 3' end group of each RNA was determined after complete hydrolysis with RNase T2 (Roche Diagnostics, Mannheim) and two-dimensional thin layer chromatography with comigrating non-radioactive standards (5).
Sequence analysis was performed with the the BLAST program package (1), found on the NCBI BLAST server (http://www.ncbi.nlm.nih.gov:80/BLAST/). The program package ``mfold'' (version 2.0, default parameters) (4) was used for secondary structure prediction of the RNA.
Parameters used: blastn - expect=10; blastp - expect=1000, matrix=pam30; tblastn - expect=1000, matrix=pam30 ; blastx - expect=10.
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