8.16% 3% 6.12% 20.29% 62.42% S. coelicolor Streptomyces Actinomycetes Bacteria Bacteria and Eukaryotes Figure 1. Sequence similarity of 833 core intergenic regions >200 nt in S. coelicolor. IG regions were grouped on the basis of their sequence similarity proﬁles into one of ﬁve diﬀerent categories: S. coelicolor- speciﬁc (white), Streptomyces-speciﬁc (black), actinomycetes-speciﬁc (grey), bacteria-speciﬁc (white with dots) or widely conserved in bacteria and eukaryotes (grey with dots). Oryza sativa; none of these have a characterized role or function in any organism (Figure 1). To focus our attention on those sequences most likely to encode sRNAs, we excluded regions where conserva- tion was directly adjacent to the start of ﬂanking coding genes (within 40 nt), as these could represent conserved promoter or operator regions. We also excluded segments containing rRNAs, tRNAs and transposase remnants. Of the remaining sequences, we concentrated on those conserved in at least three Streptomyces species. Finally, we compared the sites of sequence conservation with sites of predicted sRNA genes, as determined using the sRNAFinder program (13). Based upon these analyses and the sRNAFinder predictions, 114 IG regions were identiﬁed as containing possible sRNA genes (Supplemen- tary Table S1). Experimental identification of sRNAs in S. coelicolor To experimentally validate our sRNA predictions, we selected 20 sequences for examination using northern blot analysis (Table 2). We have designated these regions ‘scr’, for Streptomyces coelicolor RNA, followed by the gene number of the adjacent annotated protein-coding gene. These sequences were conserved in syntenous regions in other Streptomyces species, were unlikely to represent operator or promoter regions given their posi- tioning relative to adjacent open reading frames, and did not appear to encode small peptides as they lacked appro- priately positioned start and stop codons. Many of these sequences were also ﬂanked by inverted repeats that could represent rho-independent transcription terminators in Streptomyces, where poly U tails are not required for eﬀective transcription termination (34–36). For each puta- tive sRNA region, we designed two complementary oligo- nucleotide primers corresponding to 25–30 nt of the most highly conserved portion of the sequence. Using the sRNA scr1906b scr2094 scr2736 scr3000 scr3045b scr3124 scr3261b scr3558b scr3974b scr4069 scr4389 scr4701 scr4784 scr4797 scr4868 scr4908 scr5028 scr5334 scr5541 scr5676b 0.759 0.901 0.887 0.994 0.988 0.693 0.975 0.997 0.278 0.278 0.852 0.937 0.421 0.919 0.719 0.648 0.855 0.919 0.827 0.862 IG length 499 317 643 503 390 213 698 663 246 307 333 501 347 443 243 350 1062 519 335 400 < < > > > < < < > > > > > > < > > < > > > > > > > < < > > < < > < > < < < < > < 326–353 79–102 179–201 333–357 206–233 124–152 299–323 459–482 79–96 161–185 161–184 178–200 181–203 233–256 89–112 194–218 802–826 155–180 225–248 166–192 Nucleic Acids Research, 2008, Vol. 36, No. 22 7243 Table 2. Putative sRNA-containing intergenic regions examined by northern blot analysis sRNAFinder prediction score Flanking gene orientation Probe location in IG regiona aProbe location is given relative to the start (+1) of each intergenic region (sequences are given in Supplementary Table S1). bConﬁrmed sRNAs as determined by northern blot analysis and 50-RACE mapping. complementary 32P-end labelled primers as probes, we examined sRNA expression in RNA samples isolated over an 3-day time course from S. coelicolor grown under two very diﬀerent nutritional conditions: rich, glucose-containing medium (RM) and minimal medium supplemented with mannitol as carbon source (MM). This time course encompassed the full S. coelicolor life cycle (vegetative hyphae formation ! aerial hyphae formation/antibiotic production ! sporulation) on both media types. Upon transcript detection, northern blots were probed with individually labelled oligonucleotide primers to determine the DNA strand from which the putative sRNA was expressed. sRNA transcript ends were then delineated using 50- and 30-RACE experiments (see Supplementary Table S3) or, where this was not pos- sible due to RNA secondary structure, by sequential northern blot analysis using ‘primer walking’ with over- lapping oligonucleotide probes. We successfully identiﬁed transcripts corresponding to sRNAs in six diﬀerent intergenic regions (Table 3 and Figure 2). The candidate regions that did not yield detect- able transcripts may contain sRNA genes that are either expressed at low levels, or are simply not transcribed under the experimental conditions examined here. Alter- natively, they may represent conserved DNA sequences having an as yet unknown function. Of the six identiﬁed sRNA genes, two were transcribed in a medium-speciﬁc manner: scr1906 (61–63 nt) was expressed primarily on minimal medium, while scr3261 transcripts ( 185 nt) were observed only on rich medium. In contrast, scr3045 (244 and 288 nt), scr3558 ( 67, 90 and 130 nt), scr3974 (42 and 44 nt) and scr5676 (184 nt) transcripts were observed on both minimal and rich media, although