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Identification of the type I trimethoprim-resistant dihydrofolate reductase specified by the Escherichia coli R-plasmid R483: comparison with procaryotic and eucaryotic dihydrofolate reductases.

By C C Simonsen, E Y Chen and A D Levinson


We have isolated and determined the nucleotide sequence of a 1,626-base-pair fragment from R-plasmid R483 which encodes a trimethoprim-resistant dihydrofolate reductase. Analysis of the nucleotide sequence of this fragment revealed the presence of two open reading frames, each sufficient to encode polypeptides of approximately 17,000 daltons. Both open regions are preceded by sequences conforming closely to the canonical description of procaryotic promoters. A 490-base-pair HpaI fragment spanning one of the potential coding regions was inserted into a plasmid vector under the transcriptional control of the trp promoter. Cells transformed with this plasmid were trimethoprim resistant and produced dihydrofolate reductase activity which in vitro was resistant to moderate levels of trimethoprim. Analysis of the predicted amino acid sequence of this protein indicated that the R483-encoded trimethoprim-resistant enzyme was distantly related to the trimethoprim-sensitive bacterial homologs. The conserved amino acids were localized primarily to the region of the enzyme previously shown to comprise the hydrophobic substrate binding pocket

Topics: Research Article
Year: 1983
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Provided by: PubMed Central
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