Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms
Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms
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从绿色微藻莱茵衣藻和其他光合生物中快速简便地制备组蛋白
DOI：
10.1016/j.ymeth.2020.01.019
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发表时间：
2020
期刊：
影响因子：
4.8
通讯作者：
Pesavento, James J.
中科院分区：
文献类型：
--
作者：
Wong, Amanda L.;Totah, Nicholas N.;Iavarone, Anthony T.;Pesavento, James J.
从动物细胞和组织中提取组蛋白的通用、广泛适用的方法的发展已经解锁了在组织类型、健康和患病状态以及癌细胞与正常细胞之间比较这些表观遗传影响蛋白的能力。然而，对于植物和绿色藻类，尚未开发出快速且易于实施的组蛋白提取方法。在这里，我们报告了一个优化的方法，提供了一个统一的方法来提取组蛋白的绿色微藻物种莱茵衣藻和双形栅藻以及玉米（玉米）叶组织。组蛋白提取方法包括用高盐浓度和酸化处理。细胞核的制备可以在103.5小时内进行，组蛋白可以在103.5小时内立即提取，或者细胞核可以被冷冻，组蛋白蛋白可以稍后提取，而不改变组蛋白PTM模式。为了检验所提供的新方法的效率，我们通过SDS-PAGE凝胶电泳和完整蛋白质质谱法对盐和酸提取的全组蛋白（SAEWH）进行了定性和定量分析。SDS-PAGE分析表明，组蛋白产量下降时，使用壁衣原体菌株相对于细胞壁少的突变体。使用自上而下的质谱法（TDMS）进行完整蛋白质分析，我们证实了H4 K79 me 1在多个藻类物种中的存在;然而，这种独特的修饰在玉米叶组织中没有发现，在其他地方也没有报道。SAEWH提取物的TDMS测量还显示，在组蛋白提取过程中发生的氧化不会随着收获的藻细胞、它们的细胞核和提取的组蛋白样品暴露于光而增加。
The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal speciesChlamydomonas reinhardtiiandScenedesmus dimorphusas well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.