核基质结合区(MARs)与植物转基因的表达

基质结合区 (ｍａｔｒｉｘａｓｓｏｃｉａｔｉｏｎｒｅｇｉｏｎｓｏｒｍａｔｒｉｘａｔｔａｃｈｍｅｎｔｒｅ ｇｉｏｎｓ,ＭＡＲｓ)亦称核骨架结合区 (ｓｃａｆｆｏｌｄａｓｓｏｃｉａｔｉｏｎｒｅｇｉｏｎｓｏｒｓｃａｆｆｏｌｄａｔｔａｃｈｍｅｎｔｒｅｇｉｏｎｓ,ＳＡＲｓ) ,是真核基因组中能与核基质特异紧结合的ＤＮＡ序列。核基质与生物体内许多重要生命活动相联系 ,而结合的ＭＡＲｓ序列 ,参与ＤＮＡ复制 ,基因转录 ,基因表达调控[1 ] 和基因边界定位[2 ] 等过程。ＭＡＲｓ通过将染色质锚定在核基质上而使染色质形成拓扑学限制性Ｄ…     

烟草花粉母细胞细胞融合过程中细胞骨架的超微结构观察

应用普通电镜和DGD去包埋技术 ,研究了烟草花粉母细胞中的细胞融合现象及细胞融合过程中细胞骨架的变化。观察发现 ,处于凝线期的花粉母细胞 ,其内含物 ,包括细胞器和染色质 ,主要通过胞质通道向相邻细胞发生转移。DGD去包埋观察发现 ,花粉母细胞中核骨架与细胞质内及胞间连丝和胞质通道内胞质骨架连接成一个整体。在整个细胞融合过程中 ,均有核骨架纤维与染色质相连。本文讨论了细胞骨架在细胞融合过程中的作用     

染色质重塑复合物

人基因组伸展开来长约 1 .8米 ,而人细胞核的直径不到 6微米 ,ＤＮＡ在细胞核中以核小体的形式被高度压缩。这种结构在保证遗传物质稳定性的同时也阻碍了其它生物大分子接近ＤＮＡ双螺旋。许多重要生命过程的发生又都依赖功能大分子与ＤＮＡ的相互作用。机体内必然存在使核小体稳定性发生变化的调节机制以适应不同情况的需要。通常把染色质和单个核小体内发生的任何可检测到的变化称为染色质重塑。对核心组蛋白尾部的修饰 ,尤其是乙酰化修饰 ,与染色质结构变化密切相关。另有一类复合物也可以改变核小体稳定性 ,它们都含有一个ＤＮＡ依赖的…     

DNA的核骨架结合序列（MARs）研究进展

核骨架结合序列(Matrix-associated Reg-ions,MARs)是存在于真核细胞染色质中的一段与核骨架特异性结合的DNA序列。它常常位于基因的侧翼区内,与一些调控因子相邻;序列具有A-T丰富的特点,而且常常含有拓扑异构酶Ⅱ的酶切位点。近年来的研究表明MARs参与了细胞的许多重要的生命活动,包括染色质的组装,基因的复制和表达。因此,MARs被认为是一种决定染色质结构性和功能性区域的新的顺式调控因子。本文对MARs的特点,生物学功能以及研究方法等方面的最新进展进行了阐述。     

ScII类似蛋白是洋葱根端细胞核、染色体和染色体骨架的组成成分

采用机械破碎和蔗糖梯度离心方法从洋葱根端分生组织中分离出细胞核并制备出核骨架。细胞核SDS-PAGE谱带中135kD处有一多肽,免疫印迹实验结果表明,该多肽可被抗鸡ScⅡ抗体标记,核骨架中没有此多肽。经抗ScⅡ抗体和FITC偶联的二抗标记后,细胞核发出代表ScⅡ的特异性荧光,而核骨架中无荧光发出。经抗ScⅡ抗体和蛋白A胶体金处理后,金颗粒特异性地结合在核内染色质区域。说明ScⅡ类似蛋白是洋葱根端细胞核的组分,且位于核内染色质上,但该蛋白不是核骨架成分。免疫荧光和免疫电镜实验结果还说明ScⅡ类似蛋白是洋葱根端细胞染色体和染色体骨架的组成成分。     

染色质的高级结构与调控

真核生物中,DNA作为遗传物质并不是裸露存在的,而是以染色质的形式存储在细胞核中。作为遗传信息的载体,长达数米的基因组DNA被压缩在微米级的细胞核中却能精确地控制生物高度有序的各种生理活动,因此,染色质的空间结构必定具有一定的规律性且高度可控。而30 nm染色质纤维作为从核小体串珠结构到高级染色质结构中间一个关键的结构阶段,在诸多DNA相关的生命活动中起重要调控功能,因此,对其结构及动态调控相关的研究具有重要的生物学意义。现总结近年来30 nm染色质纤维精细结构解析以及染色质结构动态调控方面的一些重要最新进展,同时,也对细胞核内染色质纤维结构的一些基因组学和电镜方面的最新研究进行了讨论和展望。     

百合花粉母细胞核骨架的超微结构观察

参照动物细胞核骨架的研究方法,用整装电镜技术和DGD包埋-去包埋技术研究了选择性抽提的和完整的百合(Lilium davidii var. willmottiae (Wilson) Roffill)花粉母细胞。结果表明,住减数分裂前期Ⅰ,百合花粉母细胞核内存在一个精细的非染色质纤维——核骨架。该网络由5～15nm的纤维交织而成,广泛地分布于细胞核内。这些核骨架有的分布于染色体间,有的分布于染色体周围,并与染色体和核仁相连。随着减数分裂时间的推移,染色体(质)间核骨架纤维逐渐减少,染色体(质)周围的核骨架纤维逐渐增多,并与染色体内部的纤维结构相连,表明核骨架一方面为染色体拓扑变化提供一个空间支架,另一方面也可能参与了染色体骨架的构建。     

ScⅡ类似蛋白是洋葱根端细胞核、染色体和染色体骨架的组成成分

采用机械破碎和蔗糖梯度离心方法从洋葱根端分生组织中分离出细胞核并制备出核 骨架。细胞核ＳＤＳ－ＰＡＧＥ谱带中１３５ｋＤ处有一多肽,免疫印迹实验结果表明,该多肽可被抗 鸡ＳｃⅡ抗体标记,核骨架中没有此多肽。经抗ＳｃⅡ抗体和ＦＩＴＣ偶联的二抗标记后,细胞核 发出代表ＳｃⅡ的特异性荧光,而核骨架中无荧光发出。经抗ＳｃⅡ抗体和蛋白Ａ胶体金处理 后,金颗粒特异性地结合在核内染色质区域。说明ＳｃⅡ类似蛋白是洋葱根端细胞核的组分, 且位于核内染色质上,但该蛋白不是核骨架成分。免疫荧光和免疫电镜实验结果还说明ＳｃⅡ 类似蛋白是洋葱根端细胞染色体和染色体骨架的组成成分。     

细胞辐射敏感性与核骨架系统

哺乳动物细胞辐射敏感性的分子学基础,是放射生物学及肿瘤生物学研究中的关键问题之一,本文以核骨架系统及其功能为基础,阐述了核骨架系统、染色质结构与细胞辐射敏感性三者间的重要相关性。     

染色质动态调整与基因转录

核小体是真核细胞染色质的基本单位。真核基因组ＤＮＡ在细胞核中储存在染色质结构中。核小体的核心结构是由１４６个碱基对的ＤＮＡ盘绕四对核心组蛋白的八聚体外面近两周所构成。核小体核心组蛋白Ｈ３／Ｈ４两对异源二聚体是核小体亚颗粒的核心,它能稳定地与ＤＮＡ结合。Ｈ２Ａ／Ｈ２Ｂ二聚体是核小体核心中较不稳定的成分。核小体组装是一个干扰ＤＮＡ复制、基因表达和细胞周期进展的过程,因此在细胞生命过程中极为重要。...     

MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix.     

Modular structural elements in the replication origin region of Tetrahymena rDNA.

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.     

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.