These results indicated that a basic locus for pWTY27 replication was pWTY27.1c (designated repA), pWTY27.2c (repB) and a 300-bp (from 321 to 620 bp) ncs. Figure 1 Identification of a pWTY27 locus required for replication in Streptomyces lividans. (a). Identification of a replication locus. Plasmids were constructed in E. coli (see Methods and A-1210477 nmr Table 1), and introduced by transformation into S. lividans ZX7. Positions of these cloned fragments on pWTY27 and transformation frequencies are shown. The ncs is indicated by striped boxes, relevant genes by open arrowheads and the two replication genes by filled arrowheads. (b). RT-PCR of a transcript
overlapping the consecutive replication genes. RNA of strain Y27 was isolated and reverse-transcribed into cDNA. The cDNA, RNA and Y27 genomic DNA were used as templates for PCR amplification and their products were electrophoresed in 1.5% agarose gel at 20 V/cm for 1 h. pWT26 was introduced XAV939 by conjugation from E. coli Repotrectinib ET12567 (pUZ8002) into 10 randomly-selected endophytic Streptomyces strains (different 16S rRNA sequences, e.g. Y22, Y45, Y19,
Y24, Y8, Y51, Y10, Y31, Y72 and Y3), and apramycin resistant transconjugants were obtained from eight of them, indicating a wide host range for this plasmid. RepA protein binds specifically to intact IR2 of the iteron sequence in vitro The pWTY27 RepB was predicted to be a DNA primase/polymerase and RepA a hypothetical protein. The 300-bp ncs was predicted as an iteron containing five direct repeats of 8 bp (DR1, GTGGGAAC), five direct repeats of 7 bp (DR2, TTCCCAC) and three pairs of inverted repeats (IR1–IR3, Figure 2a). To see if there was an interaction between the RepA protein and this iteron sequence, electrophoretic mobility shift assays for DNA-protein complex formation were employed. The 6His-tagged RepA protein was incubated with a [γ-32P]ATP-labeled iteron DNA, and then electrophoresed and autoradiographed. tuclazepam As shown in Figure 2b, the “shifted” DNA bands were visualized by adding RepA protein, indicating
that the RepA protein could bind to the DNA probe to form a DNA-protein complex. Formation of this complex was inhibited by adding a 15-fold excess of unlabeled probe but was not affected by adding even a 1000-fold excess of polydIdC DNA as a non-specific competitor, indicating that the binding reaction of the RepA protein with iteron DNA was highly specific. Figure 2 Characterization of the binding reaction of Rep1A protein with iteron DNA by EMSA and footprinting. (a). Iteron of pWTY27. Possible iteron sequences from 338 to 606 bp on pWTY27 and AT-rich regions are shown. DR: direct repeat; IR: inverted repeat. The RepA binding sequences determined by DNA footprinting are boxed. The binding sequences of RepA protein are indicated by shading. (b). Detection of the binding activity of RepA protein with the iteron by EMSA.