Cyclone phosphor system

Total RNA was isolated from 30 mL samples of Synechocystis cultures in the mid-exponential growth phase (3 to 4 µg chlorophyll mL⁻¹). Extractions were performed by vortexing cells in presence of phenol-chloroform and acid-washed baked glass beads (0.25–0.3 mm diameter) as previously described [47] (link). 5 µg of total RNA was loaded per lane and electrophoresed in 1.2% agarose denaturing formaldehyde gels [90] and transferred to nylon membranes (Hybond N-Plus; GE Healthcare). Prehybridization, hybridization, and washes were in accordance with GE Healthcare instruction manuals. Probes for Northern blot hybridization were synthesized by PCR using oligonucleotide pairs: petEF-petER, petJF-petJR, copM1F-copM1R, copBF-copBR, slr2015F-slr2015R, slr1667F-slr1667R, nrsBF-nrsBR, NRP3-NRP1, sufRF-sufRR, sufBF-sufBR (see Table S7) for petE, petJ, copM, copB, slr2015, slr1667, nrsB, 5′nrsD, sufR and sufB, respectively. As a control, in all cases the filters were stripped and re-probed with a 580-bp HindIII-BamHI probe from plasmid pAV1100 containing the constitutively expressed RNase P RNA gene (rnpB) from Synechocystis (Vioque, 1992). DNA probes were ³²P labeled with a random-primer kit (Amersham Biosciences) using [α-³²P] dCTP (3,000 Ci/mmol). Hybridization signals were quantified with a Cyclone Phosphor System (Packard). Each experiment was performed at least two independent times.

Total RNA was isolated from 25 ml samples of Synechocystis cultures at the early-exponential phase (3–4 μg chlorophyll/ml), except for experiments involving different growth phases. RNA extraction was performed by vortexing cells in the presence of phenol:chloroform and acid washed baked glass beads (0.25–0.3 mm diameter; Braun, Melsungen, Germany) as previously described [36 (link)]. RNA blotting to nylon membranes (Hybond N-plus; Amersham), prehybridization, hybridization and washes were in accordance with Amersham instruction manuals. A 655 bp EcoRI-BamHI fragment from plasmid pGEM-lrtA was used as lrtA probe for Northern blotting experiments. In order to quantify signals, in all the cases the filters were reprobed with a HindIII-BamHI 580 bp probe from plasmid pAV1100 that contains the constitutively expressed rnpB gene from Synechocystis [37 (link)]. To determine cpm of radioactive areas in Northern blot hybridizations either an InstantImager Electronic Autoradiography apparatus (Packard Instrument Company, Meriden, CT) or a Cyclone Phosphor System (Packard) were used.

Total RNA was isolated from 30 mL samples of Anabaena cultures in the mid-exponential growth phase (3–5 μg chlorophyll mL^-1). Extractions were performed by vortexing cells in the presence of phenol-chloroform and acid-washed baked glass beads (0,25–0,3 mm diameter) as previously described (García-Domínguez and Florencio, 1997 (link)). Five micrograms of total RNA was loaded per lane and electrophoresed in 1,2% agarose denaturing formaldehyde gels and transferred to nylon membranes (Hybond N-Plus; Amersham, GE Healthcare, Buckinghamshire, England). Prehybridization, hybridization, and washes were in accordance with Amersham instruction manuals. All probes were synthesized by PCR and oligonucleotide pairs used are described in (Supplementary Table S1). All filters were stripped and re-hybridized with the constitutively expressed rnpB gene from Anabaena. Hybridization signals were quantified with a Cyclone Phosphor System (Packard, Meriden, CT, US). The histograms depicted in the Figures 3D, 4D and 5D correspond to relative mRNA levels, calculated by quantifying the radioactive signals and normalizing them to rnpB signal. The data showed represent the average of three independent experiments.