| Project/Area Number |
08044215
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Applied animal science
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| Research Institution | Miyazaki University |
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Principal Investigator |
ONODERA Ryoji Miyazaki University, Faculty of Agriculture, Professor, 農学部, 教授 (60040862)
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| Co-Investigator(Kenkyū-buntansha) |
KARRER Kathl マルケット大学生物学科, 助教授
WHITE Brvan イリノイ大学動物科学科, 教授
TOMITA Yoshifumi Miyazaki University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (70113230)
KARRER Kathleen M. Marquette University, Department of Biology, Associate Professor
WHITE Bryan A. Illinois University, Department of Animal Science, Professor
WHITE Bryan イリノイ大学, 動物科学科, 教授
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| Project Period (FY) |
1996 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Rumen protozoa / Entodinium caudatum / cDNA library / Lysine-requiring mutant of Escherichia coli / lysA screening / Diamionopimelate / Transmission electron micrograph / Endosymbiont / エンドシンピオント / ジゴキシゲニン / Entodinium caudatuin / ジアミノピメリン酸脱炭酸酵素遺伝子 / LysAスクリーニング |
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| Research Abstract |
In connection with the fact that the head investigator had discovered an ability of the rumen protozoa to synthesize lysine from diaminopirnelate (DAP), this project was conducted to screen a gene for coding DAPdecarboxylase to produce lysine (lysA) from a cDNA library of Entodinium caudsturn, one of the popular ruinen protozoa, and to determine its DNAsequence, in order to clarify whether or not the enzyme could be produced by the protozoa themselves. At first, mono-faunated goats which contain only E.caudatum as protozoa in the rinnen were prepared. Then the cDNA library was made from the protozoa collected from the goats and was used for screening lysA using lysine-requiring mutant of Escherichia coli. However, the result was negative, In the next step, the library was again used for screening lysA with digoxigenin using a probe made from the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria. As a result a positive plaque was found and its DNA seque
… More
nce was determined. The result of the analysis of the DNA sequence, however, revealed high homologies not with lysA but with sperm-tail specific proteins and surface antigen of Paramecium. Then the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria was again used as primers to examine PCR products using the cDNA library as a template, but no products were obtained. Since every trial resulted in failure as mentioned above, lysA seemed not to exist in the cDNA library. Finally we marked endosymbionts in the cells of E.caudatwn and examined the distribution of DAP decarboxylase activity in the cells. it was shown that the precipitates after centrifugation at 8,000 and 37,000 x g had high activity of the enzyme. Observations of the precipitates by a transmission electron microscope revealed that a lot of oval bacterium-like particles were included in both precipitates and the particles also existed in vesicles in the intact cells. DNA (20,000 bp) has been extracted from the particles. We already started screening lysA again from the DNA and expect to attain the initial objective. Less
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