Hautala J A, Conner B H, Jacobson J W, Patel G L, Giles N H
J Bacteriol. 1977 May;130(2):704-13. doi: 10.1128/jb.130.2.704-713.1977.
A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.
已开发出一种从粗糙脉孢菌的分生孢子期或萌发分生孢子生长阶段分离细胞核的方法。通过法国压力室使冷冻的分生孢子悬液裂解，然后在全能混合器中反复匀浆，使细胞核从破碎的细胞中释放出来。通过在Ludox梯度中进行密度梯度离心，从粗核组分中获得纯细胞核。最终的核产量为20%至30%。这些细胞核的脱氧核糖核酸（DNA）：核糖核酸（RNA）：蛋白质比例为1:3.5:7，并且在RNA合成中具有活性。用DNA染色剂4,6-二脒基-2-苯基吲哚染色后，在荧光显微镜下，细胞核呈现为直径1微米的亮蓝色球体，基本没有细胞质附着物。通过用2M氯化钠和5M尿素解离，以70%至75%的产率从细胞核中提取的染色质，其DNA:RNA:蛋白质比例为1:1.05:1.7。由此制备物重构的染色质表现出的RNA聚合酶模板活性水平低于纯粗糙脉孢菌DNA，但获得的最大重构水平仅为10%。在羟基磷灰石上对粗糙脉孢菌染色质进行分级分离，可将组蛋白与染色质酸性蛋白分开。在重构的和解离的染色质制备物中均存在正常的组蛋白补充。酸性蛋白组分在十二烷基硫酸钠凝胶电泳上呈现出多种条带，分子量范围从15,000到70,000。对于总解离染色质，凝胶图谱比重构染色质的要复杂得多。
