Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene.

The trimethoprim-resistant dihydrofolate reductase associated with the R plasmid R388 was isolated from strains that over-produce the enzyme. It was purified to apparent homogeneity by affinity chromatography and two consecutive gel filtration steps under native and denaturing conditions. The purified enzyme is composed of four identical subunits with molecular weights of 8300. A 1100 bp long DNA segment which confers resistance to trimethoprim was sequenced. The structural gene was identified on the plasmid DNA by comparing the amino acid composition of the deduced proteins with that of the purified enzyme. The gene is 234 bp long and codes for 78 amino acids. No homology can be found between the deduced amino acid sequence of the R388 dihydrofolate reductase and those of other prokaryotic or eukaryotic dihydrofolate reductases. However, it differs in only 17 positions from the enzyme associated with the trimethoprim-resistance plasmid R67.

Isolation and characterization of DNA fragments containing the dihydrofolate-reductase gene of coliphage T4.

DNA of a mutant of the bacteriophage T4, which contains cytosine instead of glucosylated hydroxymethylcytosine, was shown to direct the synthesis of enzymatically active dihydrofolate reductase in a coupled in vitro transcription-translation system. The DNA-directed synthesis of the enzyme was used to localize the dihydrofolate-reductase gene frd on a 2300 bp long restriction-nuclease-generated DNA fragment. Fine structure mapping showed that the gene is encoded on a segment of less than 1850 bp but more than 700 bp length. The enzyme, which is synthesized in vitro from the DNA fragment, has a molecular weight of 18 500 to 19 500. A restriction map was constructed which extends about 10 kb to both sides of the reductase gene and which covers the T4 genome between the genes 55 and 63. The two genes which flank the frd gene, genes 32 and td (thymidylate synthetase), were mapped in detail. A correlation between the physical and genetic maps was established.

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.

Altered regulation of the rate of synthesis of dihydrofolate reductase in methotrexate-resistant hamster cells.

Dihydrofolate reductase was isolated from hamster cells sensitive to methotrexate and from a methotrexate-resistant subline which has an elevated enzyme level. The enzymes were compared by peptide map analysis, and no differences in primary structure could be detected. The rates of enzyme degradation and synthesis were determined in both cell lines by a novel approach based on the enzyme specific radioactivity (called radioaffinity labeling). Degradation of reductase was minimal in both cell lines, whereas the rates of synthesis were directly proportional to the steady state concentrations of enzyme. Thus the resistant cells synthesized reductase at a rate which was 140 times faster than that in sensitive cells. Therefore the high concentration of dihydrofolate reductase in the methotrexate-resistant cells is probably the result of an alteration of a cellular component which control the synthesis of the enzyme.