Use of matrix attachment regions (MARs) to minimize transgene silencing

Matrix attachment regions (MARs) are operationally defined as DNA elements that bind specifically to the nuclear matrix in vitro. It is possible, although unproven, that they also mediate binding of chromatin to the nuclear matrix in vivo and alter the topology of the genome in interphase nuclei. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable expression in transgenic plants or cell lines, most likely by minimizing gene silencing. Our review explores current data and presents several plausible models to explain MAR effects on transgene expression.     

向烟草导入抗真菌病基因的研究

用花粉管通道法向烟草中导入几丁质酶基因,通过PCR检测的106株植株有4株表现出阳性,经点杂交检测得到3株阳性植株,用花粉管通道法向烟草导入几丁质酶基因具有可行性。     

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.     

Osmium tetroxide footprinting of a scaffold attachment region in the maizeAdh1 promoter

Osmium tetroxide (OsO₄) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO₄ hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO₄ will also identify other torsional perturbations of DNA. In this study, OsO₄ was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO₄ hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

The type of DNA attachment sites recovered from nuclear matrix depends on isolation procedure used

A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change.     

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.     

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken alpha-globin gene domain.

Summary The distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the -globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the -globin gene domain and the function of the replication origin is discussed.     

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var. capitata

INTRODUCTIONPlantshavedevelopedseveralbi0chemicaldefensemechanismsinresp0nsetopath0gensandabioticstress.Fo1l0wingpathogenattack,plantsynthesizephenyl-propaniodpr0ductssuchaslignin,l0wm0l.wt.antimicrobia1comp0undsknownasphyt0alexins,andseveraldefense-relatedproteins.Amongthesepr0teinsarepathogenesis-relatedproteinsincludingthefungalcellwalldegradingenzymeschitinaseandP-1,3-glucanase[1].Endochitinasefromhigherplantscatalyzethehydr0lysis0fchitin,aP-1,4-linkedhomop0lymerofN-acetyl-D-glucos…     

Computer prediction of sites associated with various elements of the nuclear matrix

Attachment regions of the eukaryotic chromosomal DNA to the nuclear scaffold/matrix (S/MARs) participate in various important cellular processes. However, no obvious characteristics common for these nucleotide sequences have been revealed, except that S/MARs are non-coding sites containing putative regulatory elements and binding sites of DNA-topoisomerase II. Heterogeneity among S/MARs can be caused by a variety of biological factors. In this paper, the accuracy of two S/MARs prediction programs, MAR-Finder (Singh, Kramer and Krawetz, 1997) and ChrClass (Glazkov, Rogozin and Glazko, 1998) are compared and it is concluded that both programs can be recommended for analysis of eukaryotic genomes. However, results of their prediction should be interpreted with caution since estimation of prediction accuracy of both programs needs further analysis. Problems of S/MARs prediction are illustrated on several examples of human protein-coding genes, repeated elements and the beta-globin locus from different mammalian species. Results of our analysis suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder (a default set of parameters).     

Sea urchin arylsulfatase insulator exerts its anti-silencing effect without interacting with the nuclear matrix

Chromatin insulators have been shown to stabilize transgene expression. Although insulators have been suggested to regulate the subcellular localization of chromosomes, it is still unclear whether this property is important for their anti-silencing activity. To investigate the underlying mechanisms governing the anti-silencing function of insulators, we studied the association of sea urchin arylsulfatase insulator (ArsI) with the nuclear matrix, which is a key component of the subnuclear localization of the genome. ArsI did not potentiate the nuclear matrix association with the transgene, even though it showed strong anti-silencing activity. This observation was in clear contrast to the results of the experiment using a human interferon-beta scaffold attachment region, in which the anti-silencing effect coincided with the enhanced matrix association. Chromatin immunoprecipitation analyses suggested that the absence of the matrix binding by ArsI was due to a lack of its binding to CCCTC-binding factor (CTCF), a protein known to be associated with matrix binding by chicken beta-globin insulator. Furthermore, ArsI maintained the nucleosome occupancy within the transgene at a constant level during long-term culture, although ArsI itself was not a nucleosome-excluding sequence. Taken together, these results suggest that this insulator exerts its anti-silencing activity by counteracting silencing-associated factors to maintain local chromatin environment, rather than by remodeling the subnuclear localization of the transgene locus.