Nucleic Acids Research, 1989, Vol. 17, No. 17 6893-6901
© 1989
MOLECULAR BIOLOGY |
Unexpected effects on bacterial phenotype induced by expression of a tumour-amplified human sequence
Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute Manchester M20 9BX, UK
Received June 13, 1989. Accepted July 28, 1989.
An amplified human sequence was isolated from a metastatic human lung carcinoma. A fragment of this sequence situated behind the lac promoter of pUC19 appeared to affect plasmid DNA supercoiing in certain E.coli strains. Subclones were constructed to identity the smallest region of the human insert that conferred the activity and a 369 bp fragment was identified, expression of which appeared to result in abnormal plasmnid supercoiling. In order to study this phenotype, various constructs were introduced into the minicell-producing strain E.coli DS410 and an unexpected effect was observed. The bacteria, after normal early growth clumped together in late log phase, leaving virtually clear medium. Other E.coli strains were also shown to be affected to a lesser degree. The sequence producing this effect was also mapped to the 369 bp fragment and a critical region of approximately 50 bp was identified using site-directed in vitro mutagenesis and Bal31 deletion analysis. The plasniid encoded product responsible for this phenotypic alteration did not appear to be a peptide and clumping was observed when the human DNA was expressed from several bacterial promoters. It would seem likely that this sequence encodes a biologically active RNA which affects gene expression in the host cell.