The inability of many higher eukaryotes to convert 5-fluorocytosine to
cytotoxic 5-fluorouracil presents the possibility of using the bacter
ial cytosine deaminase codA gene for negative selection. In transforme
d plant callus, expression of codA results in cell death on 5-fluorocy
tosine. In transgenic tobacco and Lotus japonicus plants the substrate
-dependent negative marker segregates as a single dominant gene, and o
n 5-fluorocytosine CodA+ seedlings stop growing at the early seedling
stage. Positive selection of CodA+ tobacco on the pyrimidine biosynthe
tic inhibitor N-(phosphonacetyl)-L-aspartate was obtained, by pyrimidi
ne salvage from external cytosine. Activity of cytosine deaminase was
determined by conversion of labelled cytosine to uracil followed by se
paration in thin layer chromatography. The codA marker therefore provi
des substrate-dependent negative and positive selection, together with
cytosine deaminase reporter activity.