Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.
We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
D
D
Santoso
R
R
Thornburg
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004080:Dihydroorotase / Q000378:metabolism; D000090722:Dihydroorotate Dehydrogenase; D018506:Gene Expression Regulation, Plant; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010641:Phenotype; D010947:Plants, Toxic; D011743:Pyrimidines / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000032:analysis; D013941:Thymine / Q000378:metabolism* / Q000494:pharmacology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D000090722:Dihydroorotate Dehydrogenase; D009097:Multienzyme Complexes; D011743:Pyrimidines; D012333:RNA, Messenger; D009963:Orotic Acid; C027157:uridine 5'-monophosphate synthase; C001242:5-fluoroorotic acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D001221:Aspartate Carbamoyltransferase; D009962:Orotate Phosphoribosyltransferase; D004080:Dihydroorotase; D009964:Orotidine-5'-Phosphate Decarboxylase; C030986:pyrimidine; D013941:Thymine