7244 Nucleic Acids Research, 2008, Vol. 36, No. 22 Table 3. Analysed sRNAs sRNA ID method Starta scr1906 scr2101 scr3045 scr3261 3287 scr3558 scr3974 scr4677 scr5676 Predicted Cloned Predicted Predicted Cloned Predicted Predicted Cloned Predicted 2 040 854 2 258 135 3 334 576, 3 334 532 3 610 382 3 636 567 3 933 641, 3933612, 3 933 597 4 375 744, 46 5 108 143, 5 108 166 6 176 413 Enda 204 091, 92, 93 2 257 986 3 334 819 3 610 566 3 636 512, 3 636 506 3 933 512, 3 933 523, 3 933 531 4 375 703 5 108 202, 5 108 199 6 176 230 Length (nt)b 61–63 150 244 and 288 185 56 and 62 130, 90, 67 42 and 44 70 and 34 184 Flanking genes SCO1905/1906 SCO2100/2101 SCO3044/3045 SCO3260/3261 SCO3287 SCO3557/3558 SCO3973/3974 SCO4676/4677 SCO5675/gabT Flanking gene Orientation sRNA Orientation < < > > > > > > > < > > < < < > < < < > > < < < > < aBolded values were determined through 50- or 30-RACE mapping experiments; Bolded, italicized values were determined by cloning of the corresponding cDNA fragment. bIn absence of 50- or 30-RACE data, length estimations are based on mapped transcription start site (if determined), approximate transcript size as determined by northern blot analysis, and associated terminator sequences (if appropriate). higher levels of expression were observed for scr3045 and scr3974 on rich medium, and scr5676 was more highly expressed on minimal medium. The three transcripts observed for scr3558 were examined using the ‘primer walking’ method, and were found to have a shared common core sequence, but diﬀerent 50- and 30-ends. In addition to exhibiting distinct media-speciﬁc trans- cription patterns, each sRNA also had a unique temporal expression proﬁle that could be correlated with particular stages of the S. coelicolor life cycle (Figure 2). Nearly all sRNAs exhibited decreased expression during later spor- ulation (64 or 72 h), apart from scr3558, whose expression was strongest at this time on both media types. Relatively constitutive expression was observed during vegetative growth and aerial hyphae formation for scr3045, scr3261 and scr5676. scr3974 was also expressed constitutively on minimal medium, albeit at low levels; however, on rich medium, it was expressed most highly during vegetative growth (24 h), before transcript levels decreased to nearly undetectable levels by late sporulation. scr1906 had an equally distinctive expression proﬁle. The scr1906 trans- cript was observed exclusively during aerial hyphae formation and sporulation on minimal medium. Cloning of sRNAs To complement our bioinformatic approach to identifying candidate sRNAs, we also conducted a small-scale cDNA cloning experiment to identify novel sRNAs. This is a technique we had used previously in our discovery of a unique population of tRNA ‘halves’ (30). In addition to identifying tRNA cleavage products (30), we detected a variety of sequences that corresponded to functional RNAs (Supplementary Table S4). These included the RNA component of the signal recognition particle (4.5S RNA); 5S, 16S and 23S rRNA degradation pro- ducts; and a number of sequences that corresponded to protein coding genes. We also successfully cloned a number of sequences that appeared to represent novel sRNA candidates, based upon their position within inter- genic regions or in antisense orientations relative to pro- tein coding genes. We focussed our attention on three of these sequences (Table 3), and conducted northern blot hybridization experiments to verify gene expression, using oligonucleotide probes complementary to the cloned cDNA sequences. We detected transcripts from IG regions between SCO2100-2101 (scr2101) and SCO4676-4677 (scr4677), and antisense to the protein- coding gene SCO3287 ( 3287). Hybridization to the scr2101 sequence revealed a transcript of 150 nt, which was constitutively present on rich and minimal media, apart from the ﬁnal timepoint on rich medium where transcript abundance decreased (Figure 3), as observed for many of the previously examined sRNAs (Figure 2). This transcript was larger than the cloned cDNA frag- ment, suggesting that the cloned cDNA represented a degradation product of the larger transcript. For scr4677, two transcripts were detected: the larger tran- script ( 70 nt) was constitutively expressed during growth on both media types (again, apart from the ﬁnal timepoint on rich medium), while the smaller transcript was observed exclusively on rich medium. This smaller transcript ( 35 nt) likely corresponded to the cloned cDNA sequence (34 nt), and may be a processed version of the larger transcript, as its expression proﬁle closely mirrored that of the larger transcript. The single SCO3287 antisense transcript was detected only during growth on rich medium, where its expression increased slightly during aerial hyphae formation and sporulation. Like scr2101, the 3287 transcript was larger than the cloned cDNA product, as determined by the mapping of both 50- and 30-transcript ends. Intriguingly, mapping of the 30-end of the 3287 transcript reproducibly revealed multiple ends (Supplementary Table S3); this could explain the relatively diﬀuse banding pattern observed on the northern blot (Figure 3), and may suggest that termination of 3287 transcription is not a precise event. Expression of sRNAs in developmental mutants Given that many sRNAs exhibited expression proﬁles that varied throughout the S. coelicolor developmental cycle, we wanted to determine whether the expression of any of these was aﬀected by mutations in developmental genes. In S. coelicolor, developmental mutants are divided into two classical groups: the bld mutants and the whi mutants.