The ftsA probe, hybridized to the same filters, revealed three

ftsA-specific RNA bands. The fastest one LY2835219 migrated slightly less than the monogenic ftsZ RNA band, which is in keeping with the 144 bp longer coding sequence of the ftsA gene; the Selleckchem AZD8186 second ftsA-specific band colocalized with the ftsZ bicistronic transcripts; the third band in the uppermost position was broader and more intense than the other two bands, indicating that ftsA was particularly abundant in long transcripts, mostly ftsQ-ftsA-ftsZ RNA. The intensity of the uppermost band is higher when probed with ftsA than when probed with ftsZ,

indicating that a fraction of the transcripts does not contain ftsZ but carries the RNA of the murB gene, located upstream of ftsQ (Figure 1, schematics). These results show that the bulk of the ftsA and ftsZ-specific RNAs were in molecules spanning one, two and three gene units, though the low level of detection and molecular weight definition of the Northern blots required further analysis. Primer extension analysis of ftsZ, ftsA and ftsQ RNA In order to map the initiation sites of the observed GANT61 RNAs, the vegetative SIN and DX RNAs were analyzed by Primer Extension (PE) (Figure 2). FtsZ transcripts were hybridized to primer ZB (Table 1), annealing to RNA at nucleotide position +103 relative to the A of MycoClean Mycoplasma Removal Kit the first ATG codon of the ftsZ open reading frame (+1). Two cDNA bands, elongated by reverse transcriptase (RT) starting from this primer, stopped at positions −14 and −140 (Figure 2A and Additional file 1). The −140 cDNA, which mapped inside the coding sequence

of the preceding gene ftsA, was more abundant than the one at −14. The fact that the −14 position lies in the spacer region between ftsA and ftsZ, at the upper end of the ribosome binding site (RBS), suggests that this RNA may originate from a longer RNA, such as the one at −140, protected from degradation by ribosomes bound to the RBS. Figure 2 Determination of ftsZ, ftsA and ftsQ RNA 5’ ends by primer extension (PE) in B. mycoides SIN (S) and DX (D). 5’ 32P-labeled primers were hybridized to total RNA, extended by reverse transcriptase and the cDNAs separated by 6% urea-PAGE electrophoresis. The numbers on the right side of the autoradiograms indicate the position of the cDNA 3’ ends relative to the ORF first nucleotide (+1). The thick lateral bar indicates the approximate position in the gel of the next upstream gene.