Finally the influence of the host background was also explored. These experiments revealed that the two ICEs harbor closely related core regions, differ in their transcriptional organization and regulation. They provide further evidence of ICE replication. Our MEK inhibition results also pointed
out an impact of host cell on the ICE behavior. Results Transcriptional organization and promoter analyses of the ICESt1 and ICESt3 core region Previous sequences analyses suggested that the thirteen ORFs belonging to the conjugation module and the genes encoding the excisionase and integrase (recombination module) of ICESt1/3 could be transcribed as a unique polycistronic mRNA while the regulation module could LY3009104 order have a two-operon organization . Gene organization, position of predicted promoters and rho-independent transcription terminators of the ICESt1/3 core region are schematically presented in the Figure 1. As some ICE activities were reported to be affected by growth phase and/or cell density [17, RG7112 molecular weight 18], CNRZ368 and CNRZ385, strains carrying ICESt1 and ICESt3 respectively, were harvested in exponential growth phase as well as in stationary phase for total RNA extraction and subsequent transcriptional organization studies. Figure 1
Comparison of ICE St1 and ICE St3 regulation, conjugation and recombination modules. Location and orientation of ORFs and a truncated IS are indicated by arrowed boxes and a rectangle, respectively. ORF names beginning with “”orf”" are abbreviated with the corresponding letters or numbers. The pattern of the arrowed boxes depicts the relationships of each ORF deduced from functional analyses or from BLAST comparisons. White arrowed boxes correspond to unrelated ORFs of the two elements. Black arrowed box is the chromosomal fda gene. The grey areas indicate closely related sequences with the nucleotide identity
percentage value. The angled arrows and the lollipops indicate the experimentally demonstrated promoters and rho-independent transcription terminators predicted from in silico analysis (black) or unpredicted (grey). The star corresponds to the putative transfer origin. Horizontal lines delimitate functional modules with their names above. Dashed lines indicate the A, B and Nutlin3 C intergenic regions of both ICEs; their nucleotide sequence alignments are detailed below. (A) Region upstream from the orfQ gene, (B) Region upstream from the arp2 gene, (C) Parp2s region. The position of the ribosome binding sites (RBS), initiation and stop codons are annotated in bold. Coding regions are boxed. The -10 and -35 boxes of the promoters and transcriptional start sites (+1) determined by 5′RACE PCR are in boldface and underlined. Numbers indicate the nucleotide position on the ICE sequence [GenBank:AJ278471 for ICESt1 and GenBank:AJ586568 for ICESt3].