Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine‐free medium

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Abstract
The lysA gene of Escherichia coli encodes for a diaminopimelic acid (Dpm) decarboxylase which allows the conversion of Dpm into lysine in bacteria. It was cloned in a eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse .alpha.-globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) was introduced into several mammalian cell lines [FR3T3, TK-, CV1, CDS1, 3T6, BHK21, Hela] by cotransfection with a 2nd selectable marker i.e. the polyoma-transforming DNA. Selection for morphologically transformed rat cells which contained the intact lysA sequences, allowed the determination of the concentration of Dpm in the lysine-free medium that permitted cell growth. lysA-expressing clones were directly selected in a medium containing 10 mM Dpm after transfection with pSB99 alone. Southern blot analysis on selected clones have shown that they contain up to 30-50 integrated copies of the plasmid in tandem arrangement. lysA-expressing clones incorporate a significant amount of radiolabeled [3H] Dpm in acid-insoluble material. The recombinant plasmid can serve as a selectable marker, in growth medium in which lysine was replaced by its direct bacterial precursor.