The location of TnlO genes encoding tetracycline resistance and its regulation was determined by analyzing the properties of recombinant plasmids carrying partial HpaI digestion products of A::TnlO transducing phage deoxyribonucleic acid. Within a 2,700-base pair region are encoded tetracycline resistance, the structural gene (tet) for a tetracycline-inducible polypeptide, and the regulatory elements for the induction of both the resistance phenotype and the polypeptide. Fusion of different sequences to an HpaI site in the tet gene alters the molecular weight and stability of the polypeptide as well as the tetracycline resistance phenotype of strains producing fusion polypeptides. These results indicate the orientation of the tet gene and support the conclusion that the tet polypeptide is required for tetracycline resistance. A Hincd cleavage site immediately upstream from the tet gene is protected by ribonucleic acid polymerase, but only in the absence of ribonucleotide triphosphates. The possibility that tet transcription is initiated at this site is discussed.
The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.
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