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。     

Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and downstream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which β-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccosvia Agrobacterium- mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.     

Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and downstream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which β-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and     

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.     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.     

The Influences of Two Plant Nuclear Matrix Attachment Regions (MARs) on Gene Expression in Transgenic Plants

Nuclear matrix attachment regions (MARs) are thought to influencegene expression by anchoring active chromatin to the proteinaceousnuclear matrix. In this study, two plant DNA fragments withstrong MAR activity were selected and tested for their effectson expression of a linked reporter gene in transgenic tobacco.One MAR was isolated from the 5' flanking region of a pea vicilingene previously reported to be expressed in a copy number-dependentmanner in transgenic tobacco. A second MAR was isolated fromthe genome of Arabidopsis thaliana by preselection for autonomouslyreplicating sequence (ARS) activity in yeast. Flanking copiesof the A. thaliana MAR stimulated median reporter gene expressionin transgenic plants by five to ten fold. Neither MAR significantlyreduced the variation in transgene expression between individualtransformants, or conferred copy number-dependence in gene expression. (Received July 24, 1997; Accepted November 10, 1997)     

Mapping long-range chromatin organization within the chicken alpha-globin gene domain using oligonucleotide DNA arrays

We have analyzed the organization of the chicken alpha-globin gene domain using DNA miniarrays and have found two novel chromatin loop attachment regions. We have found a 40-kb loop domain that includes all the alpha-globin genes in cells of erythroid origin. One of the domain borders colocalizes almost exactly with a strong MAR element and with a block of enhancer-blocking elements found earlier at the upstream end of the alpha-globin gene domain. The domain structure was found to be different in a lymphoid cell line DT40. We propose to use the technique of DNA arrays to map the nuclear matrix attachment sites that define the borders of chromosome loop domains. The technique of DNA arrays permits a large number of DNA sequences to be immobilized on a glass or nylon matrix. This may prove useful for mapping chromatin loop positions within the human genome by using a pool of chromatin loop attachment regions as a probe in a hybridization with a DNA chip containing a specific DNA region.     

MAR文库及其在真核基因组作图上的应用研究

基质结合区(matrix association regions,MAR) 是真核生物基因组中特有的一种参与基因表达调控和染色体动力学的顺式作用元件,也是真核染色体上的一种固有且各不相同的分子标记.通过体外(in vitro)或体内(in vivo)结合法可以得到与核基质特异性结合的MAR分子而构建MAR文库.体外MAR作为一种结构性的边界元件可用于染色体物理图谱的构建;而体内MAR作为某一组织中与核基质特异性结合的功能元件,则可用于研究已知基因的表达调控和染色体组织以及对新基因的分析.     

A non-curved chicken lysozyme 5'' matrix attachment site is 3'' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.     

A mini review of MAR-binding proteins

Genomic DNA encompasses several levels of organization, the nuclear matrix mediates the formation of DNA loop domains that are anchored to matrix attachment regions (MARs). By means of specific interaction with MAR binding proteins (MARBPs), MAR plays an important regulation role in enhancing transgene expression, decreasing expression variation among individuals of different transformants and serving as the replication origin. Through these years, some MARBPs have been identified and characterized from humans, plants, animals and algae so far and the list is growing. Most of MARBPs exist in a co-repressor/co-activator complex and involve in chromosome folding, regulation of gene expression, influencing cell development and inducing cell apoptosis. This review covers recent advances that have contributed to our understanding of MARBPs.     

S/MAR与基因表达

在真核生物的细胞核内,基因组是通过ＤＮＡ的核骨架附着（ＳＡＲ）或称核基质附着区（ＭＡＲ）（简记为Ｓ／ＭＡＲ）锚定在核骨架网状系统上的．Ｓ／ＭＡＲ既有一定的特征,又有多样性,研究认为它参与了ＤＮＡ复制调控和转录调控等多种核内生化过程,通过重组,在目的基因一侧或两侧带上Ｓ／ＭＡＲ后作基因转染或基因动植物,发现整合后的基因表达有时可增强几倍,甚至上万倍和／或显示位置独立效应,有些研究还报道,Ｓ／ＭＡＲ能     

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese‐hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human β‐globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 5′ or 3′ site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 5′ site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 3′ site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.     

Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach

Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.     

A matrix attachment region is located upstream from the high-molecular-weight glutenin gene Bx7 in wheat (Triticum aestivum L.).

A 2.2-kb nucleotide sequence rich in AT, located upstream from the Bx7 allele of the high-molecular-weight glutenin Glu-B1 locus in wheat (Triticum aestivum cv. Glenlea) was cloned following amplification by PCR. The 5' region of this sequence contains motifs typically found in matrix attachment regions (MARs) in other plants. We have shown that part of the 2.2-kb DNA binds to wheat nuclear matrix (NM) in vitro, at least as strongly as a known MAR (Adh1) from maize suggesting that there is a MAR upstream of Bx7. This MAR is approximately 800 bases in length running from -750 to -1560 bases, relative to the start codon. Although the MAR is associated with a tissue-specific gene and is beside a strong tissue-specific promoter, the MAR sequence did not lead to tissue-specific expression of the beta-glucuronidase marker gene under the control of the rice actin promoter in various tissues. Presence of the MAR was only slightly beneficial with respect to expression levels, which were not greatly altered in transient expression assays in various wheat tissues although a slight increase in the number of foci was observed in leaves, which have low transformation efficiencies.     

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.     

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.