欧洲黑杨MARs的分离克隆及序列分析

为获得林木植物核基质附着区(MARs)序列,诱导欧洲黑杨(Populus nigra)愈伤组织并制备悬浮细胞,裂解释放细胞核,除去非MARs的游离DNA片段,得到核基质。碱性缓冲液分离残留DNA与结合蛋白,回收并克隆残留DNA,测序后得到两个具有MARs序列特征的DNA片段A7和A23。序列特征分析结果表明它们都含有MARs的特征基元,通过与其他已发表的MARs序列比较,认为是从杨树中分离到的两个MARs片段,并预测了其功能。该研究是首次从木本植物获得MARs序列。     

烟草MARs的分离及其功能分析

从烟草基因组中克隆到两条新的MAR片段（M14和M17）,序列分析表明,它们具有90%AT-box,A-box,T-box,碱基非配对区域,拓扑异构酶Ⅱ识别位点,弯曲DNA序列,复制起始序列和ATATTT等典型的MAR序列特征,并与原有MAR序列的特征不同。将它们分别构建到植物表达载体pCAMBIA2301 GUS基因（uidA）表达盒一侧及两侧,通过农杆菌介导转化烟草。组织化学染色法定性检测GUS活性表明,带有M14和M17的uidA基因在转基因烟草中稳定表达。GUS活性的定量检测表明,表达载体上uidA基因一端或两端连接有MAR的转化烟草中,GUS的表达水平与对照相比都有了明显提高,而uidA基因两侧连有MAR的载体提高表达水平的效果优于一端连有MAR的载体,可使GUS活性增强3.14倍,但不同转化个体之间表达水平的差异仍然明显。上述结果表明,所得DNA序列为两条新的MAR片段,并且具有提高转基因表达水平的功能。     

AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs

Interstrand cross-links at T(A/T)4A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T)4A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200-1000 base pairs of approximately 85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (approximately 4.7-7.1 lesions/kilobase pair/microM drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (approximately 0.8-1.3 and approximately 0.9 lesions/kilobase pair/microM drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like AT-rich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.     

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.     

Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis.     

杜氏盐藻中的核基质与核基质结合区

真核生物细胞核DNA通过核基质结合区（Matrix attachment region,MAR）附着到核基质上。为了进一步探索染色体DNA与核基质之间的相互作用,从单细胞真核藻类-杜氏盐藻中克隆出了MAR片段。首先构建了杜氏盐藻的随机MAR文库,通过体外结合实验分离出能与核基质结合的MAR序列。从构建的MAR文库中,筛选出3个能与核基质结合的MAR,其中两个片段与核基质具有较强的结合力,测序分析表明具有MAR片段的一些典型特征性基序。     

Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

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。     

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 598–606.     

Protein:DNA interactions at chromosomal loop attachment sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.     

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)     

Molecular cloning and characterization of ARS elements from the mud loach (Misgurnus mizolepis)

Autonomously replicating sequences (ARSs) are thought to occur within, or adjacent to, the matrix attachment regions (MARs). To identify fish ARSs, MARs of the mud loach fish were obtained from nuclear matrices using a modified LIS method. These DNA fragments were screened for their ability to act as ARSs by being cloned into the ARS cloning vector, pURY19, and transformed into Saccharomyces cerevisiae. Sixteen ARSs were isolated, most of which were more efficient in transformation than the positive control vector, pURY19-2 microm, which contained the 2 microm circle origin of yeast. In particular, one clone, pURY19-ARS223, was 18 times more efficient in back-transforming E. coli than the positive control vector. Therefore, ARS223, which has strong ARS activity in yeast, could be a good candidate for inclusion in expression vehicles that are used to transfect fish cell lines or embryos. A DNA sequence analysis showed that the essential ARS elements contain potential ARS consensus sequences, and are predicted to have hairpin loop structures, or curved or kinked DNA. In addition, the MAR-Finder program suggested that ARSs also contain MAR motifs. These include AT tracts, ORI patterns, kinked DNA, ATC tracts, and Topoisomerase II consensus sequences. The in vitro matrix binding assay confirmed that all of the cloned ARSs could associate with the nuclear matrix. This indicates that ARSs elements may be located in or near the MARs. This is the first study that has identified and characterized ARSs in fish.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region

Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones.     

Identification and characterization of a silkgland-related matrix association region in Bombyx mori

From DNA fragments in vivo attached to the nuclear matrix in silkglands of Bombyx mori 5th instar larvae, we have screened a matrix association region (MAR), termed BmMAR1, by means of in vitro binding assay. BmMAR1 was identified to be specifically in vivo attached to the nuclear matrix only in the silkglands, neither in other tissues nor in the silkworm cell line Bm5, indicating its silkgland-relatedness. This 1983-bp DNA fragment contains a 1.1-kb core necessary for the effective in vitro binding although it is of relatively lower A/T composition (61%) compared to the 5' and 3' flanking regions (73 and 69%, respectively). Two degenerate sequences derived from Bm1 and L1Bm repetitive elements are located in the core region. BmMAR1 shares the widely considered typical MAR's features, DNA unwinding motif, A-box, T-box, H-box, replication origin, MAR recognition signature (MRS), the 90%AT box and Drosophila topoisomerase II consensus sequence. Furthermore we compared the occurrences of these patterns in BmMAR1 and some MARs from other organisms.