Transcription of a stem/loop region of a tumour-amplified sequence induces bacterial aggregation

A sequence of human DNA, amplified in a lung tumour, was shown to induce expression-dependent effects on Escherichia coli phenotype. Previous studies have demonstrated the induction of abnormal plasmid supercoiling and bacterial aggregation in host strains harbouring various constructs encoding this region. Subcloning identified a short stretch of 50 bp crucial for these effects. This study details further characterization of the active sequence. Computer analysis of the region predicted the formation of a stem/loop structure in transcribed RNA. Transcription, in the absence of translation, of a 22 bp sequence comprising this structure was sufficient to induce bacterial aggregation. Site-directed and random mutagenesis of the sub-region were carried out in order to identify critical factors in the induction of this phenotypic shift. It was found that changes in the loop sequence modulated activity, and mutations increasing predicted stem stability produced more active constructs. The wild-type sequence induced high level aggregation in only a limited number of strains but using the mutagenesis data, a sequence was synthesized that induced high level aggregation in most E. coli strains tested.