杜氏盐藻随机MAR文库的构建

核基质附着区(Matrixattachmentregion,MAR)是一段与核基质结合的DNA序列。为分离杜氏盐藻核基质附着区,我们首次构建了杜氏盐藻MAR文库。首先用0.5%TritonX-100裂解细胞,经30%和70%Percoll不连续梯度离心分离盐藻细胞核,再用二碘水杨酸锂(lithiumdiiodosalicy-late,LIS)去除组蛋白和限制酶消化除去结合松弛的DNA片段,最后通过蛋白酶K消化和乙醇沉淀提取盐藻核基质DNA。采用4种限制酶酶切,T_4DNA连接酶连至用相应限制酶酶切的pUC18载体上,转化E.coliJM109感受态细胞,挑选阳性克隆,构建MAR文库。部分测序分析表明,克隆的DNA片段具有明显的MAR特征。为进一步研究MAR对基因表达的调控作用及其作用机制提供了基础。     

核基质结合区提高报告基因在稳定转化的杜氏盐藻中的表达

前期的研究从杜氏盐藻（Dunaliella salina）中分离出一核基质结合区（matrix attachment region,MAR）片段——DSM1.实验证实,它在体外能与核基质结合且具有MAR的典型特征.为研究其在转基因盐藻中的作用,构建了RbcS启动子驱动、氯霉素乙酰转移酶（chloramp henicol acetyltransferase,CAT）基因为报告基因及表达盒两侧含DSM1 MAR的表达载体.电击法转化盐藻,随机挑选20株稳定转化的盐藻藻株,分析CAT酶活性.结果表明,在稳定转化的盐藻细胞中,MAR能使报告基因CAT的表达水平比对照藻株提高1-5倍,不同藻株之间个体表达的差异性也有所降低     

杜氏盐藻随机MAR文库的构建

核基质附着区(Matrix attachment region,MAR)是一段与核基质结合的DNA序列。为分离杜氏盐藻核基质附着区,我们首次构建了杜氏盐藻MAR文库。首先用0．5％ Triton X-100裂解细胞,经30％和70％ Percoll不连续梯度离心分离盐藻细胞核,再用二碘水杨酸锂(lithium diiodosalicylate,LIS)去除组蛋白和限制酶消化除去结合松弛的DNA片段,最后通过蛋白酶K消化和乙醇沉淀提取盐藻核基质DNA。采用4种限制酶酶切,T4DNA连接酶连至用相应限制酶酶切的pUC18载体上,转化E．coli JM109感受态细胞,挑选阳性克隆,构建MAR文库。部分测序分析表明,克隆的DNA片段具有明显的MAR特征.为进一步研究MAR对基因表达的调控作用及其作用机制提供了基础。     

杜氏盐藻核基质附着区的分离及特征性分析

采用0.5%TritonX 100破碎细胞,15%Percoll分离盐藻细胞核,25mM二碘水杨酸锂(lithiumdi iodosalicylate,LIS)抽提核蛋白,限制酶消化除去结合松弛的DNA,蛋白酶K SDS处理,酚/氯仿抽提,乙醇 沉淀提取核基质附着DNA,限制酶酶切连至pUC18载体上构建MARs文库。随机挑选6个克隆进行体外结 合实验筛选,筛选出一能与核基质结合的克隆,测序分析结果表明该序列具明显的MAR序列特征。     

核基质结合序列（MAR）与基因表达调控

核基质结合序列（MAR）是能在体外与核基质特异结合的DNA序列,广泛存在于染色质Loop结构的边界序列中。随着研究的深入,发现MAR序列不仅在染色质折叠中起到重要作用,影响邻近内源基因表达,而且将MAR序列构建到外源基因表达盒两侧转化动植物时,也影响外源基因的表达。因此,MAR序列是基因组中一种重要的基因间边界序列,为阐明非编码序列在基因表达中的作用和构建真核生物高效表达载体提供了新途径。     

核基质结合区对转基因表达的影响及其作用机制

核基质结合区(matrix attachment region,MAR)是一段在体外能与核基质结合的富含AT的DNA序列。研究发现MAR能使染色质形成环状结构;将其连到目的基因二侧构建载体并转至生物体中,发现它能增强基因转录表达水平及稳定性,在一定程度上降低转基因个体或细胞系之间转基因的表达水平的差异,这很可能是减低了基因沉默所致。现对MAR的序列特征、MAR对转基因表达的影响及对转基因效应的影响机制进行综述。     

牛乳腺核基质附着区多重顺式作用序列表明其功能的多样性

高等真核细胞的染色体DNA通过基质结合区(MAR)不时地与核基质特异性结合而组织成一种空间环状结构。为了研究以DNA套环形式附着于核基质上的DNA序列的特性,从处于泌乳期的乳腺组织中克隆了多个MAR DNA序列。体外结合实验表明,这些序列能够同核基质蛋白共结合成不溶性的复合物,这些复合物可较容易的通过离心去除。其中,两个MAR序列中包含有TL、CA—和GA—阻断以及ATTA基序。这两个序列中含有多个复制／转录因子的结合位点、增强子基序、多个完全的和非完全的反向重复序列以及潜在的DNA弯曲核心序列样结构。同一DNA序列中存在不同元件的组合可能说明在控制一系列细胞的发育过程中,它们可能发挥有正的或负的调控元件的功能。     

MAR结合蛋白及其调控功能

核基质结合区 （MAR)是真核生物中能与核基质结合的DNA片段.MAR通过与特异的MAR结合蛋白相互作用,在提高转基因表达水平、降低转基因个体之间表达水平差异以及染色体包装等方面具有重要的调控作用.目前,已在不同物种分离MAR结合蛋白,分别为核基质成分、核仁蛋白、组蛋白、叶绿体蛋白等,它们在调控基因表达、细胞发育、细胞凋亡、染色体包装等方面具有重要的功能.本文综述了目前分离出的MAR结合蛋白及其功能,并对MAR-结合蛋白研究作一展望.     

核基质结合区与转基因沉默

核基质结合区(matrix attachment region,MAR)又叫支架附着区(scaffold attachment region,SAIL)是染色质被限制酶消化后仍与核基质或核骨架结合的DNA序列。转基因沉默主要发生在两个水平上,一是转录水平,二是在转录后水平。位置效应是引起转录水平基因沉默的重要因素。研究发现,用MAR构建的载体能够提高转基因的表达水平,能在一定程度上克服基因沉默．降低转基因在不同转基因系间的表达差异,增强外源基因表达的稳定性。     

核基质附着区与转基因表达

核基质附着区 (matrixattachmentregions,MARs)是与核基质 (或核骨架 )特异结合的DNA序列 ,属于非编码序列 ,富含AT。通过与核基质的结合 ,它能使染色质形成独立的环状结构 ,调控基因的转录和表达 ,减少由于位置效应引起的转基因沉默。MARs在提高转基因表达水平、消除转基因个体间表达水平的差异、抑制转基因沉默等方面起着重要的作用。就MAR的一些结构功能特征及其在基因工程中的应用等方面进行了阐述。     

Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Chromosomal loop anchorage sites appear to be evolutionarily conserved

We have previously identified a class of DNA sequence elements, termed matrix association regions (MARs), which specifically bind to nuclear matrices in vitro and are believed to be at the bases of chromosomal loops in vivo. Here we demonstrate that nuclear matrices prepared from the yeast Saccharomyces cerevisiae will specifically bind an MAR sequence derived from the mouse kappa light chain immunoglobulin gene. This suggests that both MAR sequences and their binding sites have been strongly evolutionarily conserved.     

A short synthetic MAR positively affects transgene expression in rice and Arabidopsis

Matrix Attachment Regions (MARs) are DNA elements that are thought to influence gene expression by anchoring active chromatin domains to the nuclear matrix. When flanking a construct in transgenic plants, MARs could be useful for enhancing transgene expression. Naturally occurring MARs have a number of sequence features and DNA elements in common, and using different subsets of these sequence elements, three independent synthetic MARs were created. Although short, these MARs were able to bind nuclear scaffold preparations with an affinity equal to or greater than naturally occurring plant MARs. One synthetic MAR was extensively tested for its effect on transgene expression, using different MAR orientations, plant promoters, transformation methods and plant species. This MAR was able to increase average transgene expression and produced integration patterns of lower complexity. These data show the potential of making well defined synthetic MARs and using them to improve transgene expression.     

A matrix/scaffold attachment region binding protein: identification, purification, and mode of binding.

Matrix/scaffold attachment regions (MARs/SARs) partition chromatin into functional loop domains. Here we have identified a chicken protein that selectively binds to MARs from the chicken lysozyme locus and to MARs from Drosophila, mouse, and human genes. This protein, named ARBP (for attachment region binding protein), was purified to homogeneity and shown to bind to MARs in a cooperative fashion. ARBP is an abundant nuclear protein and a component of the internal nuclear network. Deletion mutants indicate that multiple AT-rich sequences, if contained in a minimal approximately 350 bp MAR fragment, can lead to efficient binding of ARBP. Furthermore, dimerization mutants show that, to bind ARBP efficiently, MAR sequences can act synergistically over large distances, apparently with the intervening DNA looping out. The binding characteristics of ARBP to MARs reproduce those of unfractionated matrix preparations, suggesting that ARBP is an important nuclear element for the generation of functional chromatin loops.     

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans ~3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed ‘MAR potential’ and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an ~10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.     

Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix

Scaffold/matrix-associated region (S/MAR) sequences are DNA regions that are attached to the nuclear matrix, and participate in many cellular processes. The nuclear matrix is a complex structure consisting of various elements. In this paper we compared frequencies of simple nucleotide motifs in S/MAR sequences and in sequences extracted directly from various nuclear matrix elements, such as nuclear lamina, cores of rosette-like structures, synaptonemal complex. Multivariate linear discriminant analysis revealed significant differences between these sequences. Based on this result we have developed a program, ChrClass (Win/NT version, ftp.bionet.nsc.ru/pub/biology/chrclass/chrclass.zip), for the prediction of the regions associated with various elements of the nuclear matrix in a query sequence. Subsequently, several test samples were analyzed by using two S/MAR prediction programs (a ChrClass and MAR-Finder) and a simple MRS criterion (S/MAR recognition signature) indicating the presence of S/MARs. Some overlap between the predictions of all MAR prediction tools has been found. Simultaneous use of the ChrClass, MRS criterion and MAR-Finder programs may help to obtain a more clearcut picture of S/MAR distribution in a query sequence. In general, our results suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder and MRS.