Figure 1.
Drug resistance of yeast colonies containing different plasmids.
The drugs tested were 5-FU, 5-FC, 6-azauracil, 5-FOA, and methotrexate in combination with sulfanilamide. Yeast cells were serially diluted and spotted onto uracil-less synthetic media with or without drugs at indicated concentrations. Growth was scored after incubation at 30°C for four days.
Figure 2.
Sensitivity of pyrimidine biosynthesis mutants to 5-FU.
All mutants tested were kanMX gene disruptions in the BY4742 genetic background. Since the BY4742 background is ura3, all strains except the ura3 strain were first transformed with the HIS3 URA3 plasmid pHGZ252 in order to restore URA3 function. The ura3 strain is BY4742 transformed with the HIS3 plasmid pRS413. URA3 stands for BY4742 with a restored wild type URA3 locus transformed with with pHGZ252, included as a control. URA3* is BY4742 transformed with pHGZ252, included as an additional control. Cells grown in histidine-less media were diluted 10-fold in water and spotted onto synthetic complete plates in the presence or in the absence of 80 µg/ml of 5-FU. Growth was scored after incubation at 30°C for three days.
Figure 3.
Genetic interactions affecting 5-FU sensitivity.
(A) Dependency of the 5-FU resistance conferred by different plasmids on URA2 function. A ura2 strain containing different plasmids was grown to late exponential phase in synthetic uracil-less medium supplemented with 1 g/l of orotic acid, serially diluted, and spotted onto plates with or without 80 µg/ml of 5-FU. (B) Effect of arginine biosynthesis mutants on 5-FU sensitivity. Since the deletion mutants were made in BY4742, which is ura3, all strains were transformed with the URA3 plasmid pHR81 in order to restore a functional pyrimidine biosynthesis. Transformants were grown in liquid medium to late exponential phase, serially diluted, and spotted onto synthetic uracil-less plates with or without 10 µg/ml of 5-FU. Growth was scored after incubation at 30°C for four days.
Figure 4.
Effect of excess arginine on 5-FU resistance conferred by different plasmids.
Ten-fold serial dilutions of yeast strains containing different plasmids were plated onto proline-based synthetic complete media containing 20 µg/ml 5-FU and either the normal amount of arginine (0.1 mM), or a 100-fold excess of arginine (10 mM).
Figure 5.
Effect of plasmids on the metabolism of 5-FU and uracil.
(A) Uptake of 14C-labelled uracil or 5-FU. (B) Incorporation of 14C-labelled uracil or 5-FU into tRNA. Yeast cells containing different plasmids, as indicated in the figure, were incubated with 14C-labelled uracil or 5-FU. Uptake of the 14C-labelled compounds and their incorporation into tRNA was quantified as described in Materials and Methods. The error bars show standard errors of the mean in experiments performed in quadruplicate. Bars with two asterisks differ significantly from the pHR81 value at p<0.01 and bars with one asterisk at p<0.05.
Figure 6.
Schematic overview of the pyrimidine biosynthetic pathway and its effect on the sensitivity of yeast cells to 5-FU under different conditions.
(A) Wild type cells. 5-FU is taken up and converted to 5-FUTP, which competes with freshly synthesized UTP for incorporation into RNA. (B) In an arginine biosynthesis mutant, more carbamoyl-P is shunted into the pyrimidine biosynthesis, and the resulting increased in UTP reduces the amount of 5-FUTP that is incorporated into RNA. (C) In cells overexpressing CPA1, CPA2 or URA2, more carbamoyl-P is made, which also results in an increase in UTP that reduces the incorporation of 5-FUTP into RNA. (D) In cells overexpressing HAM1, the amount of 5-FU that is incorporated into RNA is reduced by dephosphorylation of 5-FUMP back to 5-FUTP. Genetic interactions suggest that YJL055W also may affect this process.