Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens
Section snippets
Materials and methods
Materials. Bodo saliens (ATCC 50358) was cultured at 25 °C in 500-cm2 flasks (No. 132867, Nalge Nunc International, Denmark), each containing 250 ml of artificial seawater. They were fed with Klebsiella pneumoniae subsp. (ATCC 27889) every other day, and charged with mixed gas (5% O2 and 5% CO2 in N2) in an anaerobic chamber every day. They were harvested and washed as described [8].
Northern blotting. Total RNA was prepared from B. saliens using TRIZOL reagent (Invitrogen, San Diego, CA)
Results
Northern blot analysis of B. saliens total RNA, using DIG-labeled DNA probes specific for full-length ACT–DHOD, ACT, and DHOD showed that all three probes bound strongly to a 2.6-kb band, probably representing full-length ACT–DHOD mRNA (Fig. 1A). The open reading frame of the ACT–DHOD gene consists of 1944 bp [8], and thus, the other region, the sum of the spliced leader sequence and 3′ UTR may become approximately 650 bp. Prolonged development did not show any smaller bands, indicating the
Discussion
We have shown here that, in the kinetoplastid protist B. saliens, the ACT–DHOD gene is transcribed to a single ACT–DHOD mRNA, that its primary translation product is ACT–DHOD, that 35-kDa DHOD arose from ACT–DHOD in an in vitro processing assay, and finally that post-translational processing results in the production of N-terminal blocked mature DHOD. To our knowledge, the ACT–DHOD fused gene product and its maturation process is reported here for the first time, but the final product of B.
Acknowledgments
This work was supported in part by Grants-in-Aid for scientific research (Nos. 18890188, 17390123, and 17590377) from the Ministry of Education, Sports, Culture, Science, and Technology of Japan. T. Annoura and T. Aoki were supported by a Grant-in-Aid for 21st Century COE Research from the Ministry of Education, Sports, Culture, Science, and Technology of Japan.
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