The type IV plasmid-mediated dihydrofolate reductase (DHFR), from a clinical strain of isolated in South India, was prepared from a transconjugant containing the original clinical plasmid, J62-2 (pUK1123), and from C600 (pUK1150) containing a 2.6-kb dIII fragment of pUK1123 cloned into plasmid pBR322. Both preparations were purified by methotrexate affinity chromatography. Automatic amino-acid sequencing of the N-terminal of the purified type IV enzyme from both sources gave an identical sequence which was clearly distinct from other plasmid-mediated trimethoprim-resistant DHFRs. The type IV DHFR showed most homology with the endogenous, chromosomally-encoded enzyme. Amino-acid sequence analysis also showed that the type IV enzyme preparation from J62-2 harbouring the original clinical plasmid, pUK1123, also contained the DNA-binding protein NS1. Analysis by polyacrylamide gel electrophoresis suggested that the type IV enzyme, in its native form, consists of a DHFR of M 33 000 coupled to a DNA-binding protein.


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