Cytosine deaminase as a negative selective marker for Arabidopsis.
Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.
Perera
R J
RJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139-4307.
Linard
C G
CG
Signer
E R
ER
D017360:Arabidopsis / Q000235:genetics*; D015415:Biomarkers; D043525:Cytosine Deaminase; D029968:Escherichia coli Proteins; D005437:Flucytosine / Q000633:toxicity*; D005472:Fluorouracil / Q000633:toxicity; D015870:Gene Expression; D009700:Nucleoside Deaminases / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics
D015415:Biomarkers; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; C468827:codA protein, E coli; D005472:Fluorouracil