Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor.
DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate  reductase. The enzyme was found to be stable at 100 degrees. Some aspects of the synthesis of dihydrofolate reductase are discussed.
D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D011815:R Factors; D011995:Recombination, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014169:Transformation, Bacterial; D014295:Trimethoprim / Q000494:pharmacology
D004269:DNA, Bacterial; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase