| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Until now, the most popular method for the extraction of DNA from fungi or yeasts has been the protoplast lysate procedure using cell wall lytic enzymes. This procedure involves limitations in a range of species. It has been known that anhydrous hydrogen fluoride ( HF ) selectively cleaves the O-glycosidic linkages of polysaccharides. I therefore applied this chemical method to the disruption of fungal and yeast cell walls for their DNA extraction. Moreover, I examined whether the present method is applicable to the cloning of yeast and fungal DNA.
To investigate the effectiveness of the HF extraction procedure in the isolation of yeast and fungal DNA, three different strains were chosen as model yeasts. S.cerevesiae F102 carries two linear killer plasmids, pGKL1 and pGKL2. The second and the third strains bear plasmids with different selective markers, respectively. In restriction enzyme analysis of the killer plasmids, the restriction patterns were identical to those of preparations o
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btained by the protoplast lysate procedure. The transformation frequencies of the plasmids obtained by the present method were almost the same as that of DNA extracted using the conventional procedure.
In addition, the yeast and fungal DNAs with high-Mr.DNA ( approx. 20-40 Kb )obtained were susceptible for restriction endonucleases, and their DNA fragments were ligated with T4 ligase.
In order to examine the integrity of mRNA among total RNA prepared from C.Oracile, the total RNA was translated in a rabbit reticulocyte lysate cell-free system. The mRNAs was also fully intact: the incorporation of radioactive amino acids to proteins was dependent on the incubation time and RNA concentration. Moreover, dextranase was detected in biosynthetic proteins by using immune-blotting. I could also purify dextranase from C.gracile after HF treatment.
From these results, I conclude that yeast and fungal DNAs extracted by employing HF treatment are biologically intact and applicable to the cloning of yeast and fungal DNAs. This newly developed HF method should be also useful for the screening of unknown plasmid vector from yeast or fungi, which is insensible to cell wall lytic enzymes. Less
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