coli core enzyme saturated with E. chaffeensis Selleckchem GF120918 recombinant σ70 subunit (Figure 7). Figure 6 Transcriptional analysis of recombinant E. chaffeensis -σ 70 using pRG198 transcriptional template. C, transcription products by E. coli core enzyme alone; σ70, transcription products by the recombinant E. chaffeensis σ70 protein; N, transcription products by purified E. chaffeensis RNAP; C + σ70, transcription products by by E. coli core enzyme saturated with recombinant
E. chaffeensis σ70; N + σ70, transcription products by native purified enzyme saturated with recombinant E. chaffeensis-σ70. Figure 7 Transcription of pRG198 with varying potassium acetate concentrations showing transcription by E. chaffeensis RNAP GSK2118436 datasheet saturated with the recombinant σ 70 and Selleckchem ACP-196 by E. coli core RNAP reconstituted with recombinant σ 70 . Modulation of E. chaffeensis RNAP activity by whole-cell protein We evaluated the effect of E. chaffeensis whole-cell protein lysate, prepared from the bacteria grown in macrophage cell line, on transcription of p28-Omp14 and p28-Omp19 constructs using the native purified enzyme. The resulting
transcripts were analyzed by two independent methods; densitometry of radiolabeled transcripts and the Taq-Man probe-based, real-time RT-PCR. These analyses showed enhanced transcriptional activity in the presence of 4 μg of E. chaffeensis whole-cell lysate. Densitometric analysis revealed a 1.8-fold increase in transcriptional signal for the p28-Omp14 promoter construct and a 2.1-fold increase for p28-Omp19 construct
(Table 2). Addition of the same amount of protein yielded a Decitabine mouse similar fold increases when transcription was assessed with E. coli core enzyme saturated with E. chaffeensis recombinant σ70. No transcription occurred with the addition of whole-cell lysate alone in the absence of an enzyme, a potential source of E. chaffeensis RNAP. Similarly, the addition of boiled lysate did not cause any change in transcriptional signals. Quantitation by real-time RT-PCR for the calculation of fold increase in transcription in the presence of E. chaffeensis whole-cell protein lysate was carried out as described previously [30, 31]. Transcription of p28-Omp19 construct with purified E. chaffeensis RNAP, as quantified by real-time RT-PCR, showed a 2.24 fold enhancement in the presence of 4 μg of the protein lysate, whereas transcription of p28-Omp14 promoter construct resulted in a 1.81 fold-enhancement (Table 2), indicating a higher degree of agreement between the data generated by densitometric and real-time RT-PCR methods of quantitation (Table 2). Table 2 Effect of macrophage-culture grown E.