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. 2016 Nov 29;45(2):775–792. doi: 10.1093/nar/gkw1180

Figure 3.

Figure 3.

Validation of BbIVET-associated RNA transcripts. (A) Deep-sequencing screen shot for a Bbive-internal TSS, displaying only the sequenced 5′ nucleotide, of overlaid biological replicates treated with (TAP+) and without (TAP−) tobacco acid pyrophosphatase. Read count ranges are shown in the upper left of each frame. The chromosome nucleotide coordinates, relative orientation of the BB_0370 ORF (wide black bar), Bbive45 sequence (thin black arrow), internal TSS (blue bent arrow), processed 5′ ends (scissors), putative transcripts (broken line arrows), Northern probe locations (black and red boxes) and luciferase fusion regions (brackets) are indicated. The predicted transcripts of interest are marked with an asterisk. (B) Northern blot analyses of the Bbive45 internal TSS, as described in the Figure 1 legend. Probes located upstream (red) and downstream (black) of the putative internal TSS, are indicated. Marker nucleotide sizes are indicated to the left of the blots. (C) Bbive45 promoter activity and specificity. B. burgdorferi clones harboring specific promoter fusions were grown to mid-log phase, and incubated with 750 μM D-luciferin. Relative luciferase units (RLU) were normalized to cell density by OD600. Data represent the mean ±SD from three biological replicates shown in log scale and were analyzed relative to pJSB161 with the two-tailed Student's t-test. Unless otherwise indicated all fusion constructs demonstrated significantly greater RLUs than the promoterless control, pJSB161 (P ≤ 0.01). n.s., not significantly greater RLU relative to pJSB161. (D) Schematic representation of Bbive luciferase fusions. The bracket designates the sequence selected for promoter fusion to luciferase in pJSB161. Relative orientation of the genome region (wide black bar with white arrows), 5′ boundary of Bbive sequence (orange line) and TSS (orange bent arrow) are indicated. (E) A variety of Bbives with associated TSSs have promoter activity in culture. Spirochetes containing control promoters (flaBp, ospCp and ospAp) and specific Bbives, fused to luciferase, were grown to mid-log phase, and analyzed as described above.