Application of Anhydrous Hydrogen Fluoride Extraction Method for the Cloning of Yeast and Fungal DNA

无水氟化氢提取法在酵母和真菌DNA克隆中的应用

• 批准号: 63570868
• 负责人: OSHIRO Satoru
• 金额: $ 1.34万
• 依托单位: Medical Research Institute, Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570868/
• 关键词: Hydrogen fluoride; DNA; RNA; Cell Wall; Fungi; Yeast; クローニング

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 … More 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

到目前为止，从真菌或酵母中提取DNA的最流行的方法是使用细胞壁裂解酶的原生质体裂解过程。这一过程涉及到对一系列物种的限制。已知无水氟化氢（HF）选择性地切割多糖的o -糖苷键。因此，我将这种化学方法应用于真菌和酵母细胞壁的破坏，以提取它们的DNA。此外，我检验了本方法是否适用于酵母和真菌DNA的克隆。为了研究HF提取工艺在酵母和真菌DNA分离中的有效性，选择了三种不同的菌株作为模型酵母。葡萄球菌F102携带两个线性杀伤质粒，pGKL1和pGKL2。第二株和第三株分别携带具有不同选择标记的质粒。在杀伤质粒的限制性内切酶分析中，限制性内切模式与原生质体裂解法获得的制剂相同。用这种方法获得的质粒的转化频率与用常规方法提取的DNA几乎相同。此外，高mr的酵母和真菌dna。DNA(约；得到的DNA片段用T4连接酶连接，对限制性内切酶敏感。为了检验甲骨文总RNA中mRNA的完整性，我们在兔网织红细胞裂解液无细胞系统中翻译了总RNA。mrna也是完全完整的：放射性氨基酸与蛋白质的结合取决于孵育时间和RNA浓度。此外，利用免疫印迹法检测生物合成蛋白中的葡聚糖酶。经HF处理后，还能从细柄草中纯化葡聚糖酶。从这些结果，我得出结论，用HF处理提取的酵母和真菌dna在生物学上是完整的，适用于酵母和真菌dna的克隆。该方法也可用于酵母或真菌中对细胞壁裂解酶不敏感的未知质粒载体的筛选。少

项目成果

Satoru Oshiro and Nobuhiko Katsura: "A Simple Method for Extraction of Nucleic Acids from the Fungus Chaetomium gracile with Anhydrous Hydrogen Fluoride." Analytical Biochemistry.

Satoru Oshiro 和 Nobuhiko Katsura：“用无水氟化氢从真菌毛壳菌中提取核酸的简单方法。”