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1.
The organisation of chromatin loops: characterization of a scaffold attachment site 总被引:36,自引:9,他引:27
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Previous experiments have identified a 657-bp restriction fragment in the non-transcribed region of the Drosophila histone gene cluster that is specifically associated with the histone-depleted nuclear scaffold. The remaining fragments of the 5-kb histone repeat were shown to be readily released from the scaffold; hence it was proposed that the tandemly repeated cluster of histone genes forms a series of 5-kb loops restrained by a nuclear substructure at the sites of attachment. Here we show that the attachment fragment is tightly associated with protease-sensitive material, whereas the solubilized fragments are relatively protein-free. Exonuclease III digestion has been used to map the location of protein complexes on the attachment fragment. We have defined two regions of ~200 bp whose borders provide kinetic barriers to exonuclease III degradation. They are separated by a nucleaseaccessible region of ~100 bp. The protected regions are sufficient to mediate association of the fragment with the histonedepleted nuclei. Sequence analysis reveals an enrichment for sequences closely related to the topoisomerase II cleavage consensus in these two domains. 相似文献
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Drosophila scaffold-attached regions bind nuclear scaffolds and can function as ARS elements in both budding and fission yeasts. 总被引:7,自引:2,他引:5
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Histone-depleted nuclei maintain sequence-specific interactions with genomic DNA at sites known as scaffold attachment regions (SARs) or matrix attachment regions. We have previously shown that in Saccharomyces cerevisiae, autonomously replicating sequence elements bind the nuclear scaffold. Here, we extend these observations to the fission yeast Schizosaccharomyces pombe. In addition, we show that four SARs previously mapped in the genomic DNA of Drosophila melanogaster bind in vitro to nuclear scaffolds from both yeast species. In view of these results, we have assayed the ability of the Drosophila SARs to promote autonomous replication of plasmids in the two yeast species. Two of the Drosophila SARs have autonomously replicating sequence activity in budding yeast, and three function in fission yeast, while four flanking non-SAR sequences are totally inactive in both. 相似文献
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We have mapped the DNA sequences bound to the nuclear scaffold along 320,000 base-pairs of a genetically well-defined region of the Drosophila chromosome. We have found that the domains delimited by the scaffold attachment regions are heterogeneous in size (ranging from 26,000 to 112,000 base-pairs in this interval), and that the attachment sites are within unique sequences as judged by blot hybridization. We also found that looped domains contain up to five, or even eight, unrelated genes including, in some cases, more than one transcribed gene. The loop organization unravelled here in cultured cells does not correspond to the banding pattern seen in salivary gland polytene chromosomes. 相似文献
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U Pauli J F Chiu P Ditullio P Kroeger V Shalhoub T Rowe G Stein J Stein 《Journal of cellular physiology》1989,139(2):320-328
Protein-DNA interactions within the promoter of a cell cycle-regulated human H4 histone gene were examined by binding of 5'-end-labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between -500 bp and -1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I-sensitive site in the center (-720 to -820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene-matrix interaction that are functionally related to gene expression. 相似文献
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Scaffold attachment of DNA loops in metaphase chromosomes 总被引:19,自引:0,他引:19
We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains. 相似文献
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Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. 总被引:22,自引:3,他引:19
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Using a low-salt extraction procedure, we isolated nuclear scaffolds from tobacco that bind specific plant DNA fragments in vitro. One of these fragments was characterized in more detail; this characterization showed that it contains sequences with structural properties analogous to animal scaffold attachment regions (SARs). We showed that scaffold attachment is evolutionarily conserved between plants and animals, although different SARs have different binding affinities. Furthermore, we demonstrated that flanking a chimeric transgene with the characterized SAR-containing fragment reduces significantly the variation in expression in series of transformants with an active insertion, whereas a SAR fragment from the human beta-globin locus does not. Moreover, the frequency distribution patterns of transgene activities showed that most of the transformants containing the plant SAR fragment had expression levels clustered around the mean. These data suggest that the particular plant DNA fragment can insulate the reporter gene from expression-influencing effects exerted from the host chromatin. 相似文献
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Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene. 总被引:30,自引:11,他引:19
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J P Linton J Y Yen E Selby Z Chen J M Chinsky K Liu R E Kellems G F Crouse 《Molecular and cellular biology》1989,9(7):3058-3072
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In an analysis of a 90-kb region around the human beta-globin gene complex we have identified at least eight sites of attachment to the nuclear scaffold (SARs). While these have many potential functions, there appears to be a particular association with sequences important in the regulation of the complex. Two SARs are close to the known enhancer-like elements of the beta-globin gene. SARs flanking the complex co-habit with the boundaries of the putative beta-like globin gene regulatory domain. In contrast, we have detected no SARs within a 140-kb region of the human alpha-globin gene complex. If SARs play a role in the regulation of gene expression then this structural difference would imply a difference in the regulation of the two complexes. 相似文献
13.
The role of scaffold attachment regions in the structural and functional organization of plant chromatin 总被引:5,自引:0,他引:5
Studies on nuclear scaffolds and scaffold attachment regions (SARs) have recently been extended to different plant species and indicate that SARs are involved in the structural and functional organization of the plant genome, as is the case for other eukaryotes. One type of SAR seems to delimit structural chromatin loops and may also border functional units of gene expression and DNA replication. Another group of SARs map close to regulatory elements and may be directly involved in gene expression. In this overview, we summarize the structural and functional properties of plant SARs in comparison with those of SARs from animals and yeast. 相似文献
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A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells. 总被引:5,自引:1,他引:4
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Integration of foreign genes into plant genomes by the Agrobacterium T-DNA transfer system has been considered to occur at random. It has been speculated that the chromosomal structure of the integration site might affect the expression pattern of the introduced genes. To gain insight into the molecular structure of T-DNA integration sites and its possible impact on gene expression, we have examined plant DNA sequences in the vicinity of T-DNA borders. Analysis of a transgenic petunia plant containing a chloramphenicol acetyltransferase (CAT) gene regulated by the hemoglobin promoter (PAR) from Parasponia andersonii revealed a scaffold attachment region (SAR) close to one T-DNA end. In addition to having strong binding affinities for both animal and plant nuclear scaffolds this petunia SAR element is as active in mammalian cells as the authentic elements from mammalian sources. 相似文献
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Interaction of DNA with nuclear scaffolds in vitro 总被引:25,自引:0,他引:25
We have previously identified a number of specific DNA fragments called SARs (scaffold-associated regions) that are associated with the nuclear scaffold and define the basis of DNA loops. We demonstrate that cloned DNA fragments containing SAR sequences bind to nuclear scaffolds in vitro with the same specificity as have genomic SAR fragments. This specific interaction is observed with the biochemically complex type I scaffolds. These scaffolds are composed of the nuclear lamina proteins and a set of other proteins that forms the internal network of these structures. So-called type II scaffolds, which are composed primarily of the lamina proteins and lack the proteins of the internal network, do not bind the SAR fragments at a detectable level. Competition experiments show that different SARs share common structural elements and can bind to the same sites on the nuclear scaffold, although with different affinities. Moreover, the SAR binding sites appear to be evolutionarily conserved, as all the Drosophila SARs also bind with identical specificity to nuclear scaffolds derived from rat liver nuclei. These Sar interaction studies were carried out with lithium 3,5-diiodosalicylate-extracted nuclei. Interestingly, scaffolds prepared by high-salt extraction also bind the genomic and exogenously added SAR fragments specifically. However, the endogenous transcribed sequences, as opposed to the same fragments added as purified DNA, associate randomly with these scaffolds. 相似文献
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In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene. 相似文献
19.
Nuclear scaffold attachment stimulates, but is not essential for ARS activity in Saccharomyces cerevisiae: analysis of the Drosophila ftz SAR 总被引:9,自引:1,他引:8
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Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix. 相似文献
20.
Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. 总被引:19,自引:2,他引:17
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G C Allen G E Hall Jr L C Childs A K Weissinger S Spiker W F Thompson 《The Plant cell》1993,5(6):603-613
The yeast ARS-1 element contains a scaffold attachment region (SAR) that we have previously shown can bind to plant nuclear scaffolds in vitro. To test effects on expression, constructs in which a chimeric beta-glucuronidase (GUS) gene was flanked by this element were delivered into tobacco suspension cells by microprojectile bombardment. In stably transformed cell lines, GUS activity averaged 12-fold higher (24-fold on a gene copy basis) for a construct containing two flanking SARs than for a control construct lacking SARs. Expression levels were not proportional to gene copy number, as would have been predicted if the element simply reduced position effect variation. Instead, the element appeared to reduce an inhibitory effect on expression in certain transformants containing multiple gene copies. The effect on expression appears to require chromosomal integration, because SAR constructs were only twofold more active than the controls in transient assays. 相似文献