Purification of low-abundance mRNAs by polysome immunoprecipitation and its use for cDNA cloning
| Project/Area Number |
59870009
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Chiba University |
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Principal Investigator |
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| Project Period (FY) |
1984 – 1985
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| Project Status |
Completed (Fiscal Year 1985)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1985: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1984: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | Free and membrane-bound polysomes / polysome immunoprecipitation / cDNA clonong / ornithine transcarbamylase / 3-oxoacyl-CoA thiolase / 3-ケトアシル-CoAチオラーゼ |
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| Research Abstract |
cDNA cloning of low abundance mRNA species is one of the most critical steps in recombinant DNA studies. Polysome immunoprecipitation is one of the most powerful means to purify low-abundance mRNAs and the purified mRNAs should facilitate cDNA cloning. We developed a simple and rapid procedure for isolation of polysomes from rat liver and applied the procedure for cDNA cloning.
1. A procedure was developed for preparation of free and membrane-bound polysomes from rat liver. The procedure involves separation of the two classes of polysomes by differential centrifugation of liver homogenate and magnesium precipitation of both classes of polysomes. The polysomes were essentially undegraded and highly active in cell-free synthesis.
2. A polysome class synthesizing ornithine transcarbamylase was purified about 50-fold from the free polysomes by immunoprecipitation. A cDNA clone with a 1600base insert was isolated from the enriched mRNA and the entire primary structure of the transcarbamylase precursor was determined.
3. 3-Oxoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase mRNAs were purified 25-40-fold and cDNA clones for the two enzymes were isolated. The longest clones containing 1400- and 1200-base insert, respectively, are expected to carry more than 80% of the thiolase and dehydrogenase mRNAs.
4. The mRNA for carbamyl phosphate synthetase (subunit molecular weight, 160,000) could not be effectively enriched by the polysome immunoprecipitation procedure. Thus, the present procedure may not be suitable for purification of very large mRNAs.
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Research Products
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