FIG 8.

Identification of the transcriptional start of ndvB by 5′ RACE. (A) RNA was extracted from PA14 or PA14/pSC01, and first-strand cDNA synthesis was performed with a gfp-specific primer. First-strand cDNA was incubated with dATP without or with TdT (−TdT and +TdT, respectively). The dA-tailed cDNA was subsequently amplified by two rounds of PCR as described in Materials and Methods. The 5′ RACE products were analyzed by electrophoresis on a 1.2% agarose gel. Two 5′ RACE products (major and minor) were obtained. Note that 5′ RACE products were observed only for the reaction that used RNA from PA14/pSC01 and that had been incubated with TdT, demonstrating the specificity of the gfp gene-specific primers and the dependence of the obtained the products on the poly(dA) tail added by TdT. NTC, no-template control. (B) Three pUC18 plasmids carrying the major 5′ RACE product (RACE1, -2, and -3) were sequenced using the M13R primer (StemCore Laboratories, Ottawa Hospital Research Institute), and the obtained sequences were aligned to that of pSC01 using Clustal Omega (80). Sequence identity is shown by asterisks. The GTCGACTAGTAC(T)₁₇ sequence is derived from the 3′ RACE adapter primer. The −10 RpoS element is shown in blue, the TSS is shown in yellow with a +1, and the gfp coding sequence is in green. (C) Two pUC18 plasmids carrying the minor 5′ RACE product (RACE4 and -5) were analyzed as described for the major product.