非洲爪蟾卵提取物中组装的染色质的原子力显微镜成像。
Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.
作者信息
Fu Hongxia, Freedman Benjamin S, Lim Chwee Teck, Heald Rebecca, Yan Jie
机构信息
Mechanobiology Institute, National University of Singapore.
出版信息
Chromosoma. 2011 Jun;120(3):245-54. doi: 10.1007/s00412-010-0307-4. Epub 2011 Jan 11.
Abstract
Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.
摘要
我们对染色质低级和高级结构的理解仍存在差距。非洲爪蟾卵提取物提供了一种研究在活细胞中难以操作的重要染色质成分的方法，但对提取物中组装的染色质进行纳米级成像具有挑战性。我们描述了一种利用限制性酶切消化，然后转移到云母表面，制备用于原子力显微镜（AFM）的提取物中组装染色质的方法。使用这种方法，我们发现染色质组装提取物的缓冲液稀释或染色质在低离子强度溶液中的孵育会导致染色质纤维松散压缩，容易解旋成裸DNA。我们还描述了一种不使用限制性酶的染色质直接AFM成像方法，该方法揭示了不同宽度的高级纤维。由于能够控制染色质组装条件，我们认为这些方法在研究生理相关染色质结构方面具有广泛的潜力。
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