Journal: The Journal of Biological Chemistry
Article Title: The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply *
Figure Lengend Snippet: Noncoding RNAs connected with the flv4-2 operon. A , schematic presentation of chromosomal arrangement of the genes flv4 , sll 0218, and flv2 in the flv4-2 operon; ctpB ; the antisense transcribed sections for as1_flv4 , as2_flv4 , and as_flv2 ; and the gene for ncRNA, ncr0080. B , verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M , molecular mass marker; R , total RNA after electrophoresis. C , classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface , and the transcription start for flv4 , according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow . Predicted −10 and −35 elements are underlined . The chromosomal positions are according to Cyanobase.
Article Snippet: To characterize small RNAs, RNA samples (5 μg) were mixed with RNA loading dye (Fermentas, St. Leon-Rot, Germany), denatured for 10 min at 70 °C, separated in 10% polyacrylamide-urea gels, and transferred to Hybond-N nylon membranes (GE Healthcare) by electroblotting for 1 h. For the mRNA studies, RNA samples were mixed with denaturation solution, incubated for 10 min at 70 °C, separated in 1.3% agarose gels containing 7% formaldehyde in MOPS, and transferred to Hybond-N nylon membranes by capillary blotting with 10× SSC overnight ( ).
Techniques: Northern Blot, Isolation, Electrophoresis, Staining, Derivative Assay, In Vitro, Marker, Sequencing, Amplification