7246 Nucleic Acids Research, 2008, Vol. 36, No. 22 Figure 3. Northern blot analysis of sRNAs identiﬁed through cDNA cloning. As for Figure 2, panels on the right indicate the positioning of each sRNA (shown in black) relative to surrounding protein-coding genes (shown in grey), whilst panels on the left show northern blots probed with labelled DNA oligonucleotides. The scr4677 transcript initiates 15 nt upstream of the SCO4676 start codon, while the 3287 transcript starts 239 nt downstream of the SCO3287 translation start site, and terminates 56 or 62 nt later (184 or 178 nt downstream of the SCO3287 translation start, respectively). RNA was harvested at the timepoints indicated (in hours) above each blot, and 5S rRNA was used as a positive control for loading levels and integrity of RNA samples. Shown are representative results of northern blots that were repeated at least three times with independent RNA samples. development on minimal medium; however, both this media dependence and temporally regulated expres- sion were lost in the bldA and bldB mutant strains (Figure 4A). Both bld mutant strains exhibited wild-type expression of scr1906 on minimal medium (data not shown), while much higher expression levels were detected during growth on rich medium (Figure 4A)—a condition where little expression was typically seen in the wild-type. In contrast, there was no detectable scr1906 expres- sion under any growth condition in a whiG mutant (Figure 4A) (wild-type expression proﬁles were observed in a whiB mutant, data not shown). This suggests that scr1906 expression is completely dependent upon the product of whiG (a sporulation-speciﬁc sigma factor), and that the medium-speciﬁc expression of scr1906 is contingent upon functional bld gene activity. Like scr1906, scr3558 was expressed most highly during aerial hyphae formation and sporulation in wild-type S. coelicolor, but unlike scr1906, transcription was observed on both rich and minimal media (Figure 2). scr3558 expression was unaﬀected by whiB or whiG muta- tions (data not shown), but was aﬀected by both bld mutations. Overall expression levels for scr3558 were maintained in a bldA mutant; however, the relative propor- tion of each transcript was diﬀerent than had been observed in the wild-type strain (Figures 2 and 4B). In contrast, in a bldB mutant, transcription levels were signif- icantly decreased under all growth conditions (Figure 4B). The bldB mutation had the converse eﬀect on scr3974, where increased expression was seen during growth on rich medium, relative to wild-type (Figure 4C); a similar increase in transcript abundance was observed in both whiB and whiG mutant strains (data not shown). The bldA mutation conferred increased expression for two of the sRNAs identiﬁed through cDNA cloning (scr4677 and scr2101). This increased expression was speciﬁc to growth on rich medium (Figure 4D and E), although for scr4677, only expression of the larger trans- cript was aﬀected. The expression proﬁle of scr2101 was also altered in bldB and whiG mutants, where it was no longer constitutively expressed, as had been observed in the wild-type strain, but instead, accumulated later in development on minimal medium in the bldB mutant, and earlier in development on rich medium in the whiG mutant (Figure 4E). These diﬀering expression proﬁles may reﬂect altered stability of scr2101 sRNA transcripts in these mutant backgrounds. In all instances, sRNA expression in the developmental mutants was compared with wild-type expression proﬁles using identical experi- mental conditions. DISCUSSION sRNA detection in S. coelicolor Our bioinformatic prediction of sRNAs in S. coelicolor involved the combined analysis of IG sequences using BLAST, to determine the extent of sequence conservation in other organisms, and the sRNAFinder program, to evaluate both primary sequence and structural conserva- tion of putative sRNAs (13). This two-tiered approach led us to successfully identify six sRNAs that were expressed under deﬁned conditions. We complemented our in silico sRNA predictions with a direct cloning approach to detecting sRNAs. This enabled us to identify a further three sRNAs, two of which would have been excluded from our bioinformatic search due to their close proximity to adjacent coding sequences (scr4677), and antisense orientation relative to a protein-coding gene ( 3287). A previous bioinformatic screen for sRNAs in the S. coelicolor genome had predicted 32 candidate sRNA sequences, and veriﬁed expression using RT-PCR and