title: The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells.
abstract: We have subcloned the coding sequence for the Escherichia coli lysA gene coding for diaminopimelic acid decarboxylase (DAP decarboxylase) into a eukaryotic expression vector based on the SV40 early promoter. The activities of a series of constructs with different lengths of non-coding DNA at the 5' and 3' ends of the coding region have been compared by measuring the synthesis of lysine from diaminopimelic acid (DAP) in mouse 3T3 cells. A short non-coding sequence at the 3' end reduced the expression of enzyme activity. Stable lines of 3T3 cells have been produced by co-transfection of the chimeric gene with a plasmid coding for G-418 resistance. Cells were grown in medium containing G-418 and resistant clones were screened for an ability to synthesise lysine from DAP. [3H]Lysine produced from [3H]DAP was incorporated into cell proteins. An enzyme extract from a cell line which had incorporated two copies of the gene synthesised 0.082 nmol of lysine/min per mg protein. In the intact cell the rate of lysine synthesis is limited by the uptake of DAP which is taken up at only 5% of the rate of lysine. lysA has a potential as a reporter gene in studies of gene expression in mammalian cells.
authors.affiliation: Rowett Research Institute, Bucksburn, Aberdeen, UK.
authors.forename: K M
authors.initials: KM
authors.lastname: Saqib
authors.forename: S M
authors.initials: SM
authors.lastname: Hay
authors.forename: W D
authors.initials: WD
authors.lastname: Rees
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D005798:Genes, Bacterial; D017930:Genes, Reporter; D008239:Lysine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D002262:Carboxy-Lyases; C079912:LysA protein, Bacteria; C020031:diaminopimelic acid decarboxylase; D008239:Lysine