| Project/Area Number |
04454192
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
寄生虫学(含医用動物学）
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| Research Institution | Juntendo University School of Medicine |
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Principal Investigator |
AOKI Takashi Juntendo University School of Medicine, Professor, 医学部, 教授 (20053283)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMADA Junko Juntendo University School of Medicine, Research Assoc., 医学部, 助手 (20211964)
SHIMOGAWARA Rieko Juntendo University School of Medicine, Research Assoc., 医学部, 助手 (50146776)
TAKAMIYA Shinzaburo Juntendo University School of Medicine, Assoc.Professor, 医学部, 助教授 (90138206)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1993: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1992: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Keywords | Trypanosoma / Glycosome / Pyrimidine biosynthesis / Orotate phosphoribo-syltransferase / Carbamoyl-phosphate synthetase II / Gene cloning / Localization / Cellular fractionation / オロト酸ホスホリボシルトランスフェラーセ / OMPデカルボキシラーゼ |
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| Research Abstract |
Pyrimidine and purine nucleotides are essential units for nucleic acid structure and function. Trypanosomes are incapable of synthesizing purines de novo ; in contrast, pyrimidine de novo biosynthetic pathway is operative, where the fifth and sixth enzymes (OPRT and OMP-DC) are thought to localize in the glycosome, a peculiar organelle of the single membrane vesicle containing glycolytic enzymes. Differential centrifugation of the homogenate of Trypanosoma cruzi epimastigotes yielded the 3,800g, 26,000g, and 225,000g precipitates and supernatant. The 26,000g fraction contained the majority of OPRT and OMP-DC activities and had glycosome-like vesicular structures.
The screening of Escherichia coli transformants resulted in the candidate six DNA fragments that may encode the T.cruzi OPRT, among which a 1.3 kb fragment was cut by a restriction enzyme into 2 fragments of 600 and 700 bp. The amino acid sequence deduced from the 600 bp fragment was compared with various OPRTs, showing no apparent homology between T.cruzi and other OPRTs. Other five candidate DNA fragments have been under the sequence analysis.
We have partially sequenced cDNA and gDNA fragments that encode T.cruzi CPS II, the first and key enzyme in de novo pyrimidine synthesis. The result indicated that the enzyme occurs as an independent protein similar to the E.coli CPS II.However, the T.cruzi enzyme is quite unique and differs completely from the E.coli and any other organisms' CPS II, since the parasite enzyme exists as a single polypeptide and does not exhibit a subunit structure. Reverse transcription (RT) and subsequent PCR of the purified T.cruzi mRNA yielded evidence that the trans-splicing mechanism does take place for the production of a mature CPS II mRNA.
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