Isolation of ribosomal gene chromatin.
Isolation of ribosomal gene chromatin.
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核糖体基因染色质的分离。
DOI：
10.1016/0012-1606(77)90180-4
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发表时间：
1977
影响因子：
2.7
通讯作者：
R. Reeder
中科院分区：
文献类型：
--
作者：
T. Higashinakagawa;H. Wahn;R. Reeder
本文介绍了从非洲爪蟾未成熟卵母细胞中分离扩增核仁的方法。通过用胶原酶-木瓜蛋白酶消化分离成年卵巢，通过一系列1g沉降步骤分离未成熟的无色素卵母细胞（Dumont阶段II和III），然后通过1.7M蔗糖离心。然后将未成熟的卵母细胞均质化，并通过在Metrizamide梯度中的浮力密度带分离核仁。通过光学显微镜和电子显微镜观察，通过该程序分离的核仁具有正常形态。它们含有完整的核糖体RNA前体分子和具有I型或核仁聚合酶特征的活性内源RNA聚合酶。核仁还含有一种酶活性，可以松弛闭合环状DNA中的超螺旋。该活性遵循甲泛葡胺梯度中的RNA聚合酶活性，表明该酶可能参与转录。总核仁蛋白的SDS-丙烯酰胺凝胶电泳显示，大部分的蛋白质迁移与蛋白质从细胞质核糖体。然而，一些非核糖体蛋白是清晰可见的，其中包括与组蛋白H2 a，H2 b，H3和H4共迁移的蛋白质。使用这种方法，可以分离出除扩增的核糖体DNA之外完全不含任何DNA组分的核仁。他们代表了第一个案例，其中有可能纯化一个单一的基因与其蛋白质仍然连接。
A procedure is described for isolating amplified nucleoli from immature oocytes ofXenopus laevis. Adult ovaries are dissociated by digestion with collagenase-papain and immature, pigment-free oocytes (Dumont stages II and III) are isolated by a series of 1gsedimentation steps followed by centrifugation through 1.7Msucrose. The immature oocytes are then homogenized and nucleoli are isolated by buoyant density banding in gradients of Metrizamide. Nucleoli isolated by this procedure have normal morphology by both light and electron microscopy. They contain intact ribosomal RNA precursor molecules and an active endogenous RNA polymerase which has the characteristics of the form I or nucleolar polymerase. The nucleoli also contain an enzymatic activity which relaxes superhelical turns in closed circular DNA. This activity follows RNA polymerase activity in a Metrizamide gradient suggesting that this enzyme may be involved in transcription. Electrophoresis of total nucleolar proteins on SDS-acrylamide gels shows most of the proteins migrating with protein from cytoplasmic ribosomes. Some nonribosomal proteins are clearly visible however, among which are proteins which comigrate with histones H2a, H2b, H3, and H4. Using this procedure nucleoli may be isolated completely free of any DNA component other than amplified ribosomal DNA. They represent the first case in which it has been possible to purify a single gene with its proteins still attached.