Abstract
Uridine-5'-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment. A mutagenesis using an alkylation agent, N-ethyl-N-nitrosourea, was carried out to the marine diatom Phaeodactylum tricornutum. Cells were treated with 1.0 mg mL(-1) N-ethyl-N-nitrosourea and were screened on agar plates containing 100 to 300 mg L(-1) 5-fluoroorotidic acid (5-FOA). Two clones survived the selection and were designated as Requiring Uracil and Resistant to FOA (RURF) 1 and 2. The 50% effective concentration of 5-FOA on growth of RURF1 was about 5 mm, whereas that in wild-type cells was 30 μm. The ability to grow in the absence of uracil was restored by a P. tricornutum gene that potentially encoded UMPS or the human umps gene, HUMPS. Because the P. tricornutum gene was able to restore growth in the absence of uracil, it was designated as ptumps, encoding a major functional UMPS in P. tricornutum. RNA interference to the ptumps targeting the 5' region of ptumps resulted in the occurrence of a clear RURF phenotype in P. tricornutum. This RNA interference phenotype was reverted to the wild type by the insertion of HUMPS, confirming that the ptumps encodes UMPS. These results showed direct evidence of the occurrence of novel-type UMPS in a marine diatom and also revealed the potential usage of this gene silencing and complementation system for molecular tools for this organism.
Highlights
Uridine-5#-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment
To determine the minimum inhibitory concentration of 5-fluoroorotidic acid (5-FOA) on the growth of wild-type P. tricornutum, cells were spotted as plaques on artificial seawater supplemented with half-strength Guillard’s f solution (F/2ASW)-agar plates and cultured for 2 to 3 weeks. 5-FOA showed a strong growth inhibition at 100 mg L21 (Fig. 2B), and the inhibitory effect became stronger when cells were exposed to 200 and 300 mg L21 5-FOA (Fig. 2B)
About 4 3 106 cells of wild-type P. tricornutum were mutagenized with 1 mg mL21 ENU for 30 min at room temperature, plated on agar plates containing 100 mg mL21 5-FOA and 50 mg mL21 uracil at the density of about 104 cells per plate, and allowed to form colonies for 2 to 3 weeks
Summary
Uridine-5#-monophosphate synthase (UMPS), the critical step of the de novo pyrimidine biosynthesis pathway, which is a housekeeping plastid process in higher plants, was investigated in a marine diatom, the most crucial primary producer in the marine environment. This RNA interference phenotype was reverted to the wild type by the insertion of HUMPS, confirming that the ptumps encodes UMPS These results showed direct evidence of the occurrence of novel-type UMPS in a marine diatom and revealed the potential usage of this gene silencing and complementation system for molecular tools for this organism. The de novo pyrimidine biosynthesis pathway consists of six emzymatic reactions that react from relatively simple substrates, HCO32, Asp, ATP, and Gln, to make UMP This pathway functions as a housekeeping process that is essential for the production of uracil and conserved in all species of living organisms so far examined (Zrenner et al, 2006). When the pathway from orotate to 5#-UMP operates, cells synthesize the lethal inhibitor for thymidylate synthetase, 5-fluorouracil, in the presence of 5-FOA as a precursor (Fig. 1), whereas cells can survive on
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