The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7

Abstract

The complete nucleotide sequence of the type I dihydrofolate reductase gene from Tn7 was determined. The structural gene coded for a polypeptide of 157 amino acid residues. The polypeptide deduced from the DNA sequence had a molecular weight of 17,577 which was in good agreement with that estimated by mobility in SDSpolyacrylamide gels. Sequences were identified proximal to the coding region which were similar to those found in the consensus E. coli promoter region and for the initiation of protein synthesis. Features consistent with the termination of RNA transcription were present distal to the structural gene. No homology was apparent when the DNA sequence of the type I gene was compared to the sequence of the type II plasmid DHFR genes, but sequence homology was evident when the type I and E. coli chromosomal enzymes were compared. Homology was greatest in the regions coding for amino acids which in the E. coli chromosomal enzyme are associated with substrate, cofactor and inhibitor binding.

Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

“…1A, lanes 3 and 4; B, lanes 1 and 3 (is also apparent. Although we have not pursued the identity of this band, its size coupled with its ability to be bound by MTX are consistent with the properties of bacterial DHFR (12,18). Its apparent failure to bind to MTX-affinity resin when isolated from Tp-resistant cells that are grown in the presence of high concentrations of Tp and presumably, therefore, contain elevated intercellular levels of this drug, may reflect the site-specific nature of DHFR-antifolate binding in general and is consistent with the possibility that Tp-bound bacterial DHFR may not be capable of efficiently binding to MTX-affinity resin.…”

Detection of novel trimethoprim resistance determinants in the United Kingdom using biotin–labelled DNA probes

“…This increased complexity, coupled with the difficulties of performing biochemical analysis on an epidemiological scale, means that DNA hybridization procedures probably offer the best approach for monitoring the evolution and distribution of these medically important enzymes. The type I DHFR gene probe consists almost entirely of the structural gene [17] and, when used in combination 68 Trimethoprim resistance genes in the UK 69 with high stringency wash conditions, forms an extremely useful and highly specific probe for initial screening purpose [11]. The work described in this paper confirmed that the type I group remains the predominant plasmid-encoded group conferring trimethoprim resistance among strains of E. coli from the Nottingham area of the UK.…”

“…DNA probes specific for known DHFR genes comprised the following: type I 499 bp HIpaI fragment of pFE872 [17]; type II, 275 bp Sau3A/EcoRI fragment of pWZ820 [18]: type Illa, 700 bp Pst I/EcoRI fragment of pUN972 [19] or 855 bp EcoRI/HindlIl fragment of pFE1242 [11]; type IV, 1 7 kb Cla I fragment of pUK1 148 [16]; type V, 500 bp HincIl fragment of pLK09 [16]; type VII, 300 bp EcoRV fragment of pUN1042 [20]. There is no currently available probe for the type VI DHFR.…”

Prevalence and genetic location of non-transferable trimethoprim resistant dihydrofolate reductase genes in South African commensal faecal isolates

“…Fifteen of these trimethoprim resistant enzymes have been identified in Enterobacteria and have been characterized according to their biochemistry and DNA sequence. They are the Ia [3], lb [4], Ila [5], Ilb [6], Ile [7], Illa [8], IIIb [9], IIIc [10] renamed the type VIII [11], IV [12], V [13], VI [14], VII [15], IX [16], X [17] and XII [18].…”