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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Amplification and modification of dihydrofolate reductase in Escherichia coli. Nucleotide sequence of fol genes from mutationally altered plasmids.

Recombinant plasmids carrying the structural gene for Escherichia coli dihydrofolate reductase (fol) were mutagenized in vitro and in vivo and were used to transform a suitable recipient strain. Twenty-three transformants were isolated that were able to grow in the presence of high levels of the folate analog trimethoprim, and, in each strain, the resistance determinant was shown to be carried on the plasmid. Three of the strains produced dihydrofolate reductase with an increased Ki value for trimethoprim. DNA sequence analysis showed that the plasmids in these strains had mutations in fol which altered a conserved region of the polypeptide that forms part of the dihydrofolate-binding site. Two other strains had approximately 3-fold elevated dihydrofolate reductase levels, apparently resulting from plasmid copy number mutations. The remaining 18 strains had dihydrofolate reductase levels that were 10-30 times higher than those of the starting strain. Surprisingly, three of these strains had no discernible changes either in plasmid copy number or in the nucleotide sequence of the plasmid fol gene. Sequence analysis of the plasmids in 12 more of the strains revealed mutations in the promoter region adjacent to the fol gene. Most of these mutations occurred in the conserved sequences known as the Pribnow box and the -35 region and increased the homology of these sequences with the consensus E. coli promoter sequence. Strains carrying these plasmids produced a significant fraction of their total cell protein as wild type dihydrofolate reductase and should therefore be useful as sources of the purified enzyme.[1]

References

  1. Amplification and modification of dihydrofolate reductase in Escherichia coli. Nucleotide sequence of fol genes from mutationally altered plasmids. Smith, D.R., Rood, J.I., Bird, P.I., Sneddon, M.K., Calvo, J.M., Morrison, J.F. J. Biol. Chem. (1982) [Pubmed]
 
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