By plotting the mean arbitrary fluorescent units from untreated and canavanine treated pools, we could clearly determine the can1 can1 deletion strain as extremely enriched inside the population following canavanine treat ment. In the robot assisted experiments, four replicates of a deletant strain for each from the recognized yeast genes encod ing transporter proteins have been spotted onto strong med ium. Growth on a plate containing canavanine identified only the identified canavanine resistant strain can1 can1, in full agreement with published data and with our results from the competi tion experiment described above. We validated the outcomes from both higher throughput experiments by performing development experiments in a BioScreen C instrument, which generates robust development curves under a lot more strictly controlled situations.
We calculated the maximum growth rate from the WT and can1 can1 strains inside the presence of canavanine, and confirmed that, unlike the wild variety, can1 can1 mutants are insensitive to canavanine. In addition, a competition experiment in between canava nine as well as the ON-01910 ic50 native Can1p substrate, arginine, illustrates the fully protective impact of arginine. Both of those benefits suggest that the cellular import of canavanine happens exclusively via Can1p, as reported previously. Drugs with a single protein carrier The two screening procedures identified several transporters which clearly represented the sole transporter responsible for the uptake of a specific drug into yeast cells. The first example is comparable to that of Can1p trans porter and canavanine.
We screened for transporters the full report responsible for the uptake on the anticancer drugs five fluor ocytosine and 5 fluorouracil and, as could happen to be expected, identified that the fcy2 fcy2 mutant was the most resistant strain. Fcy2p is usually a recognized cytosine transporter and is so named due to the fluorocytosine resistant phenotype of its mutant alleles. The analysis of data from pool competitors experiments with diphenyleneiodonium chloride, by plotting the imply arbitrary fluorescence of untreated and treated pools, identified the nrt1 nrt1 deletant as very enriched in the population following DPI treatment. Robot assisted experiments using individual transporter deletants spotted onto agar also identified Nrt1p as the probably DPI transporter. We next performed growth assays on WT and nrt1 nrt1 strains within the presence of increasing DPI concentrations and verified the resistance conferred by the deletion of the candidate transporter. DPI is an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase and related enzymes, and bears some structural similarity to nicotina mide riboside. Furthermore, each nicotinamide riboside and thiamine are known to become transported by Nrt1p.