Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine-free medium.
The lysA gene of Escherichia coli encodes for a diaminopimelic acid decarboxylase (EC 4.1.1.20) which allows the conversion of diaminopimelic acid into lysine in bacteria. It has been cloned in an eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse alpha-globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) has been introduced into several mammalian cell lines by cotransfection with a second 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 diaminopimelic acid in the lysine-free medium that permitted cell growth. lysA-expressing clones were directly selected in a medium containing 10 mM diaminopimelic acid, 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. Finally, we demonstrated that lysA-expressing clones incorporate a significant amount of radiolabelled [3H]diaminopimelic acid 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.
D000818:Animals; D001426:Bacterial Proteins; D002262:Carboxy-Lyases / Q000235:genetics / Q000502:physiology*; D002455:Cell Division; D003001:Cloning, Molecular; D003470:Culture Media; D003960:Diaminopimelic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D051381:Rats; D014162:Transfection
D001426:Bacterial Proteins; D003470:Culture Media; D003960:Diaminopimelic Acid; D002262:Carboxy-Lyases; C079912:LysA protein, Bacteria; C020031:diaminopimelic acid decarboxylase; D008239:Lysine