DNA-蛋白质相互作用研究的方法及其新进展

DNA与蛋白质的相互作用参与生命体内的许多生物学过程,关于二者相互作用的研究是人们了解基因表达机制、揭开生命奥秘的关键所在.该文简要阐述了传统研究DNA-蛋白质相互作用的常用方法及其优缺点,并综述了近年来该领域所采用的新技术及其新进展.     

染色质免疫沉淀技术在研究DNA与蛋白质相互作用中的应用

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域。与其他方法相比,染色质免疫沉淀技术（chromatin immunoprecipitation assay, ChIP）是一种在体内研究DNA-蛋白质相互作用的理想的方法。近年来这种方法与DNA芯片和分子克隆技术相结合,可用于高通量的筛选已知蛋白因子的未知DNA靶点和研究反式作用因子在整个基因组上的分布情况,这将有助于深入理解DNA-蛋白质相互作用的调控网络。总结了染色质免疫沉淀技术的方法,特别介绍了使用这些方法取得的最新进展。     

用碱洗脱法检测DNA-蛋白质交联和DNA链间交联

本方法以DNA单链断裂的检测为基础,在背景γ射线照射下进行DNA交联检测。所建方法与Kohn氏原法相比,洗脱时间大为缩短,实验所用主要材料都能立足国内。本文引入“交联度”这个参数,能同时相对定量地表示DNA总交联、DNA-蛋白质交联和DNA链间交联。此外还从DNA、蛋白质两方面确证了DNA-蛋白质交联的存在。     

运用染色质免疫沉淀技术筛选出AP-2α的靶基因GALK1

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.与其他方法相比,染色质免疫沉淀技术 (chromatin immunoprecipitation assay, ChIP ) 是一种近来研究体内 DNA 与蛋白质相互作用的最好方法之一.本研究利用 ChIP 克隆方法, 找出了 AP-2α所调控的新的下游靶基因 GALK1,并应用 Luciferase assay和 RT-PCR 实验进行了初步的验证.这一新发现,有利于我们进一步研究转录因子AP-2α的功能.     

运用染色质免疫沉淀技术筛选AP-2α的靶基因

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.染色质免疫沉淀技术(chromatin immunoprecipitation assay,简称CHIP)是目前唯一研究体内DNA与蛋白质相互作用的方法.对与ChIP有关的实验条件进行了优化,获得了较优的实验条件,并运用ChIP实验筛选出了转录因子activator protein-2 alpha (AP-2a)的未知靶基因,对于进一步研究AP-2a的功能和调控网络打下了基础.     

介绍一种脱嘌呤干扰足纹法

报道了一种鉴定蛋白质与DNA的相互作用位点的新方法——脱嘌呤干扰足纹法,并用该方法鉴定大鼠脂酰-CoA氧化酶基因的表达调控部位。此方法基于用甲酸使DNA脱嘌呤后,再与核蛋白相互作用,然后通过凝胶电泳迁移率的改变,将游离DNA和与蛋白质结合的DNA片段分开,再经六氢吡啶降解DNA中无嘌呤部位的磷酸酯键和进行电泳分析。此方法具有分辨率高、重复性好、干扰少等优点,适用于对结合位点中缺乏鸟苷酸的基因进行分析。     

检测DNA-蛋白质交联的新方法

将彗星实验进行改进以用于DNA-蛋白质交联作用的检测。利用甲醛对受试动物肝细胞的影响来判定此法是否适用于检测DNA-蛋白质交联。由于在实验中添加一定量的蛋白酶K, 可使单细胞在电泳时产生更大的迁移, 因此可以利用添加蛋白酶K前后的彗星尾距比来判断外来化合物对生物机体产生DNA损伤效应的时候是否有出现DNA-蛋白质交联作用。结果表明, 该方法快速、经济、灵敏度较高, 可以在单细胞水平对甲醛等强交联剂引发的不同组织的DNA-蛋白质交联效应进行检测, 希望该方法能成为指示DNA交联能力的有用工具。     

DNA足纹步移作图法

一种以PCR介导的、可对任意长度靶DNA片段上的核蛋白结合位点进行DNA足纹作图分析的新方法.原理是：采用被随机降解靶DNA分子作为模板,用标记的跨越整个模板的足够多条特异性引物进行单链扩增.首先,利用某种化学试剂或酶如DNaseⅠ对已与蛋白质结合或未结合的双链靶DNA进行随机降解,在一定条件下使每一个DNA分子恰好只有一个位点被切割.然后,这些被随机降解DNA分子即可作为模板,用同位素标记的跨越整个模板的足够多条特异性引物（正向或反向）进行单链扩增.最后,扩增的单链产物通过变性聚丙烯酰胺凝胶电泳和放射自显影形成DNA片段梯队,而被蛋白质保护的位点则在DNA片段梯队中形成位置缺口,从而确定DNA与蛋白质相结合的精确位点.该方法被应用对人干细胞因子基因5′旁侧－1190～－273区域的DNA足纹部分作图.     

蛋白质点阵/芯片技术的新进展

蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具.     

从七肽噬菌体展示库中筛选内毒素结合蛋白质配基

目的:研究从噬茵体展示库中筛选内毒素结合蛋白质配基,为其在内毒素致病作用机理及在内毒素血症防治研究中的应用奠定基础.方法:以内毒素为靶分子从随机七肽噬菌体展示库中筛选内毒素的高亲和力噬菌体配体,通过ELISA鉴定,DNA测序及相关软件分析.结果:所筛选的亲和力最高的噬菌体的ELISA检测值A405nm可达1.965通过比较亲和性噬菌体外源插入肽的DNA序列,认为FHENWPS肽段中包含有与内毒素分子发生亲和结合的一个共有序列.该序列展示肽的等电点为5.36,具有双嗜性,这有利于肽与LPS分子表面的位点相互作用从而产生亲和吸附.结论:运用亲和筛选方法从肽库中筛选内毒素结合蛋白质配基是可行的.     

染色质免疫共沉淀实验方法

染色质免疫共沉淀(ChIP)技术是一种检测蛋白质与DNA结合的实验技术。该方法可以先进行样品交联, 然后将蛋白质与DNA复合物进行随机DNA切断, 再借助免疫学方法特异性富集与目的蛋白相结合的DNA片段, 从而检测转录因子等目的蛋白质与DNA的结合情况, 鉴定基因启动子或其它DNA结合位点。该方法同时也可应用于研究基因组特定位点的组蛋白修饰情况。该文介绍了依赖交联固定的常规免疫共沉淀(X-ChIP), 以及适用于10³细胞级别微量实验材料的基于微球菌核酸酶非交联免疫共沉淀(ULI-NChIP)具体操作过程和注意事项。     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

Characterization of a DNA-protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis-N,N''-tetraacetic acid and dithiothreitol.

Treatment of polyoma virions with ethyleneglycol-bil-N,N'-tetraacetic acid (EGTA) and dithiothreitol (DTT) at pH 8.5 resulted in the dissociation of the virions into a DNA-protein complex and individual structural capsomere subunits. The sedimentation value of the DNA-protein complex in sucrose gradients was approximately 48S, and it had a density of 1.45 g/cm3 in equilibrium CsCl gradients. Alkaline sucrose analysis of the DNA within this DNA-protein complex demonstrated that approximately 75% of the DNA is component 1. The proteins associated with the DNA were dissociated by treatment with either NaCl or the anionic detergent Sarkosyl. VP1 and the histone proteins VP 4--7 were the major proteins associated with the DNA. Treatment of the DNA-protein complex with alkaline pH resulted in the specific removal of FP1. Electron microscopy of the 48S DNA-protein complex demonstrated that it is a very tightly coiled structure that is slightly larger than the intact virion. Treatment of the complex with either NaCl or with pH 10.5 buffer resulted in the loss of protein and subsequent loosening of the DNA-protein complex such that the DNA could be visualized. The capsomere subunits released as a result of the EGTA-DTT treatment sedimented as 18S, 12S, and 5S subunits in sucrose gradients. Electrophoretic analysis of the isolated capsomeres demonstrated that VP1, VP2, and VP3 were present in each species, although the ratios of the proteins varied. In addition to the structural proteins, histones VP 4--7 were found to be predominantly associated with the 5S capsomere subunit.