The gene 14 expression in E. chaffeensis also remained high for all time points analyzed post-inoculation in tick cells. In macrophage-derived E. chaffeensis, expression levels were reversed with significantly higher expression for gene 19 (Figure 2B). Figure 2 Quantitative RT-PCR analysis. TaqMan-based quantitative RT-PCR analysis was performed with RNA isolated from tick cell (A) and GSK2879552 nmr macrophage (B) cultures harvested at different high throughput screening assay times postinfection. Transcript numbers were estimated and presented per million E. chaffeensis organisms. Data are presented with SE

values calculated from three independent experiments (P ≤ 0.05). P28-Omp 14 and 19 promoter regions sequence analysis The entire non-coding sequences upstream to genes 14

and 19 were evaluated to identify sequences similar to the consensus E. coli RNA polymerase binding site sequences, -10 and -35, and ribosome binding site sequences (RBS) (Figure 3). Consensus -10 and -35 elements were identified and are located few bases upstream to the transcription start sites mapped by primer extension analysis (Figure 3). Similarly, putative RBS sequences [22] were identified 7 and 4 nucleotides upstream to the initiation codon of genes 14 and 19, respectively. Genes 14 and 19 sequences upstream to the predicted -10 and -35 sequences differed considerably in their lengths and homology Quinapyramine (Figure 3A and 3B). The gene 14 upstream sequence is 581 bp in length, which is 273 bp longer than the gene 19 upstream sequence (308 bp). The sequences included several MK 8931 gene-specific direct repeats and palindrome sequences. In addition, a unique 14 nucleotide-long ‘G’ rich sequence was detected in the gene 19 sequence. The consensus -35 sequence was identical for

both the genes, whereas the -10 and RBS sequences differed by one nucleotide each (Figure 3C). Relative distances of the consensus -10 and -35 sequences from transcription start sites also remained the same for both the genes (Figure 3C). Figure 3 P28-Omp genes 14 and 19 promoter region sequence analysis. Upstream sequences of genes 14 (panel A) and 19 (panel B) were evaluated for the presence of direct repeats (red text), palindromic sequences (pink text) and for the presence of unique sequences (G-rich region), consensus -35 and -10 regions (green text) and ribosome binding sites (blue text). Panel C shows the comparison of -10, -35 and ribosome binding sites of genes 14 and 19 with the E. coli consensus sequences. Transcription start sites for the genes mapped by primer extension analysis are identified with bold and grey color highlighted text or with an asterisk. Dashes were introduced in the p28-Omp gene 19 sequence to create alignment with the gene 14 sequence.