The difference in enzyme activity is much higher than the difference in mRNA LY3023414 chemical structure levels as known in other cases [20–22]. Figure 4 Quantitative PCR analysis of LacZ reporter gene. Fold difference in transcript level in pPr591 over that of pPrRv in log phase and stationary phase cultures are shown. The fold difference observed is the average of three independent experiments. Error bars represent the standard deviation. Mapping the transcription start site in M.tuberculosis We identified transcription

start site of Rv0166 and Rv0167 in vivo in M.tuberculosis H37Rv and VPCI591 using fluorescence tagged primers in primer extension assay using RNA templates. The absence of DNA contamination in BMN 673 datasheet RNA preparation was confirmed by PCR for Rv0166 and Rv0167 in absence of reverse transcriptase (data not shown). The sizing of the products was carried out by genescan analysis and the TSS was detected at -65 position from the Selleckchem LCZ696 translation initiation site of Rv0166 and at -56 position from the translation initiation site of Rv0167 (Figure 5B-E), suggesting that there are two potential promoters for mce1 operon generating two transcripts, one including Rv0166 and the other without it (Figure 5A). Further, this demonstrated that both promoters are active in the genomic context of M.tuberculosis. Considering

the translation initiation site of Rv0167 as +1, we map the transcription start site within IGPr at -56 position and the mutation in VPCI591 at -61 position. Figure 5 Mapping of selleck inhibitor transcription start site (TSS) in mce1 operon. A -Line diagram indicating the position of

primers used for mapping TSS by primer extension. The numbers in parenthesis indicate the map position on the reference sequence of M.tubersulosis H37Rv. Filled boxes indicate non-coding regions, filled arrowheads indicate translation start site, tsp1 is HEX-labeled primer beginning at 195092, tsp2 is FAM-labeled primer beginning at 196960. P1 and P2 represent the TSS detected. B-E show Genescan analysis of the products of primer extension reactions on mRNA from M.tuberculosis H37Rv (B, D) and VPCI591 (C, E) with fluorescence labeled primers is shown in A. The peak at 165 bp position is transcript from P1 promoter and the peak at 156 position transcript from P2 promoter. Estimation of mce1 operon transcript levels in M.tuberculosis The transcript level of Rv0167, Rv0170 and Rv0174 of mce1 operon downstream to IGPr in M.tuberculosis and VPCI591 was analyzed by quantitative PCR with rpoB as the endogenous control (Figure 6A). The data reveals 1.5 fold upregulation of the mce1 operon genes in VPCI591 as compared to M.tuberculosis H37Rv (Figure 6B). The difference at protein level is considerably higher than at the transcript levels in case of β-galactosidase, similar enhancement in Mce1 protein levels could also be anticipated.