orf43 specific mRNA levels were maximally up-regulated 7 minutes post exposure and elevated levels were sustained for over 30 minutes post exposure. Cytotoxic orf43 transcription is regulated through a region directly upstream of orf43 Based on previous observations with the Δ11 and ∆13 ICE R391 deletions, which deleted orfs40 to most of orf42 inclusive [8], the most likely location for an orf43 control site would be the last 36 bp specific

to orf42 directly in front of orf43. Comparative bioinformatic analysis of this region Foretinib price and the previously documented orfs90/91 regulated orf4 (jef) [14] uncovered a short 7 bp homologous DNA sequence (5’-AGAAGAT-3’) present in front of both genes. This conserved sequence was located 77 bp upstream of orf4 (jef) but directly in front

of orf43 where the last 2 base pairs of the sequence overlapped the first two base pairs of the predicted start codon of orf43. As no other recognisable promoter or operator region was predicted upstream of orf43, this 7 bp sequence may possibly represent a binding motif for the putative transcriptional enhancer (orfs90/91). However it is well known that transcriptional enhancer control sites can be difficult to predict [18] as they tend to be short DNA sequences see more lacking high sequence conservation even between enhancer types. To examine if the last 36 bp specific to orf42 and preceding orf43 did in fact contain a control site for orf43 transcription, orf43 specific mRNA expression was analysed in a number of specific deletion backgrounds spanning this putative control region [Table 1, Figure 4C]. Three directed ICE R391 deletion mutants were generated [Figure 4C] in an E. coli (AB1157 R391) background;

the KOA Branched chain aminotransferase deletion removed the genes orf32 to orf42 and placed the inserted ampicillin cassette on the reverse this website complement to ensure removal of all possible promoters of orf43 transcription except for the 36 bp directly in front of orf43, the KOB deletion removed the genes orf32 to orf42 similar to KOA but additionally removed the 36 bp directly in front of orf43 while the KOC deletion was identical to the KOA mutation, preserving the putative 36 bp control site but also contained an additional secondary zeocin resistant deletion which removed orfs90/91. These three deletion mutations were screened by both qualitative and quantitative UV survival assays to determine their effect on the cell-sensitising function [Figure 4A] and additionally were examined by RT-PCR to determine if orf43 specific mRNA transcription still occurred [Figure 4B]. The KOA mutant retained the UV-inducible sensitising function [Figure 4A] and orf43 mRNA transcription [Figure 4B], while the KOB and KOC mutations abolished the sensitising function as well as orf43 mRNA transcription.