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Publisher Summary This chapter investigates pyrimidine biosynthesis in Saccharomyces cerevisiae ( S. cerevisiae )by gas chromatography–mass spectrometry (GC–MS) analysis, using a stable isotope-labeled tracer. The amide nitrogen atom of glutamine is incorporated into N-3 and amino nitrogen (amino-N) at C-4 of the pyrimidine moiety of thiamin in S. cerevisiae . However, addition of casamino acids to the medium decreases incorporation of the amide nitrogen atom of glutamine into the pyrimidine. This suggests that another amino acid in casamino acids is the direct precursor. To determine the direct precursor, the competitive effects of 14 N-labeled amino acids on the incorporation of 15 NH 4 Cl into pyrimidine are investigated. To confirm the direct incorporation of histidine, S. cerevisiae is grown in the presence of L -[1,3- 15 N 2 ]histidine. The two nitrogen atoms of the imidazole ring in histidine are incorporated directly into pyrimidine. The base peak m / z 122 is shifted to m / z 124. The shift of the fragment ion at m / z 54 to 55 shows that one of the nitrogen atoms of imidazole in histidine is incorporated into the amino-N of pyrimidine. The fragment ion at m / z 81 contains the N-1 and amino-N atoms of pyrimidine. The one nitrogen of histidine is incorporated into this fragment. These results show that the nitrogen atom of histidine is incorporated into the amino-N atom but not the N-1 of pyrimidine. Therefore, the other nitrogen of histidine is incorporated into the N-3 of pyrimidine. |
