PMID: 6328427Apr 25, 1984Paper

Expression of the mouse dihydrofolate reductase cDNA in B. subtilis: a system to select mutant cDNAs coding for methotrexate resistant enzymes

Nucleic Acids Research
T GrangeR Pictet

Abstract

With the aim to obtain a cDNA coding for a mammalian methotrexate resistant dihydrofolate reductase (Dhfr) a plasmid ( pQS1 ) harboring the mouse wild type Dhfr cDNA was constructed and used to transform a methotrexate sensitive bacteria: B. subtilis. A plasmid, pQS4 , expressing large amount of Dhfr in both E. coli and B. subtilis was isolated through a two steps selection with two substrate analogues, trimethoprim followed by methotrexate. This new plasmid has a 54 bp duplication including the beta-lactamase promoter and a deletion of 564 bp removing the 5' end of the beta-lactamase coding region. These changes create a new -35 region TTGAAA and a potentially stronger binding site for both E. coli and B. subtilis 16S ribosomal RNA. pQS4 transformed B. subtilis were then grown in the presence of high level of methotrexate and resistant mutants isolated. One of them, pQS6 , which codes for an enzyme about 50 times more resistant to methotrexate than the wild type Dhfr was sequenced. It shows that a point mutation replaces the glutamine residue at position 35 by a proline.

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Citations

Sep 11, 2009·Journal of the American Chemical Society·Tracy M BloisJames U Bowie

Related Concepts

Natto Bacteria
DNA, Double-Stranded
DNA Restriction Enzymes
Drug Resistance
Alkalescens-Dispar Group
Cistron
Methotrexate, (DL)-Isomer
Episomes
Tetrahydrofolate Dehydrogenase

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