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

到目前为止，最流行的从真菌或酵母中提取DNA的方法是使用细胞壁裂解酶的原生质体裂解物过程。这一程序涉及对一系列物种的限制。已知无水氟化氢(HF)选择性地裂解多糖的O-糖苷键。因此，我应用了这种化学方法来破坏真菌和酵母的细胞壁，以提取它们的DNA。此外，我还检验了该方法是否适用于酵母和真菌DNA的克隆。为了考察HF提取方法在酵母和真菌DNA分离中的有效性，我们选择了三种不同的酵母菌株作为模型酵母。酿酒酵母F102携带两个线性杀伤性质粒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。在对杀人质粒的限制性内切酶分析中，限制性内切酶图谱与…制剂的相同。更多的是通过原生质体裂解程序获得的。用本方法获得的质粒转化频率与用常规方法提取的DNA几乎相同。此外，还获得了高Mr DNA的酵母和真菌DNA。获得了对限制性内切酶敏感的20-40kb的DNA片段，并将其与T4连接，在兔网织红细胞裂解液无细胞体系中进行翻译，以检测甲藻总RNA中mRNA的完整性。MRNAs也是完全完整的：蛋白质中放射性氨基酸的掺入依赖于孵育时间和RNA浓度。此外，免疫印迹法检测到生物合成蛋白中存在葡聚糖酶。从这些结果中，我得出结论，用HF处理提取的酵母和真菌DNA具有生物完整性，适用于酵母和真菌DNA的克隆。这种新发展的HF方法也可用于从酵母或真菌中筛选对细胞壁裂解酶不敏感的未知质粒载体。较少

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