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Nucleic Acids Research, 1984, Vol. 12, No. 8 3585-3601
© 1984

ENZYMOLOGY

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

T. Grange, F. Kunst, J. Thillet, B. Ribadeau-Dumas1, S. Mousseron and A. Hung

Institut Jacques Monod du CNRS, Unité INSERM 257 — Université Paris VII Tour 43,2 Place Jussieu, Paris 75251, Cedex 05, France

Received March 5, 1984. Accepted March 30, 1984.

With the aim to obtain a cDNA coding for a mammalian methotrexate reaistant dihydrofolete reductase (Dhfr)a plasmid (pQSl) harboring the mouse wild type Dhfr cl was constructed and used to tranaform 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, trlimethoprim followed by methotrexate. This new plesmid 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 165 ribosomal RNA. pQS4 tranaformed 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 glutamins residue at position 35 by a prolins.

1Present address: INRA CNRZ 78350 Jouy-en-Josas, France


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