The expression of Escherichia coli diaminopimelate decarboxylase in mouse 3T3 cells.
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.
Saqib
K M
KM
Rowett Research Institute, Bucksburn, Aberdeen, UK.
Hay
S M
SM
Rees
W D
WD
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
D001426:Bacterial Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D002262:Carboxy-Lyases; C079912:LysA protein, Bacteria; C020031:diaminopimelic acid decarboxylase; D008239:Lysine