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Results for “Matrix Attachment Regions”

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PubMed2001

Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

Animals↗

PubMed1999

Activation of the N-myc2 oncogene by woodchuck hepatitis virus integration in the linked downstream b3n locus in woodchuck hepatocellular carcinoma.

In the woodchuck hepatitis virus (WHV)/woodchuck model for hepatitis B virus-induced hepatocellular carcinoma, frequent activation of N-myc oncogenes by WHV integration has been firmly established. N-myc2, the most frequently affected gene, was reported to be activated by WHV insertion either in the proximity of the gene or in a distant uncoding locus, win. We previously reported that a WHV integration cloned from a liver tumor was located in a chromosomal locus already described by others as the site of WHV integration in another hepatocellular carcinoma. On this basis, the locus, named b3n, was defined as a recurrent site of WHV integration. A scaffold or matrix attachment region (S/MAR) element was subsequently shown to be located in this locus approximately 1 kb from the WHV insertion sites. S/MARs are genetic elements involved both in structural and functional organization of chromosomal DNA and in stimulation of gene expression. In the present work, we investigated the possibility that an N-myc gene might be affected by integration in b3n. Analysis of a liver tumor harboring WHV integration in this locus showed N-myc2 overexpression. By restriction analysis, the b3n locus was shown to be located downstream of N-myc2, so the known sites of viral insertion in b3n were approximately 11 kb downstream of the N-myc2 promoter. Although these data support that WHV insertion in b3n activates N-myc2, the mechanisms previously described to be involved in N-myc2 activation do not appear to properly account for activation in this subset of WHV integrations. Available data suggest that activation of N-myc2 by WHV integration in b3n might be mediated by the S/MAR located near the WHV insertion.

Animals↗

PubMed1998

Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection against neonatal infections of the enteric tract.

Animals↗

PubMed1994

Approaches to maximizing stable expression of alpha 1-antitrypsin in transformed CHO cells.

A variety of approaches to maximizing the production of recombinant human alpha 1-antitrypsin (AAT) in Chinese hamster ovary (CHO) cells have been investigated. The highly active and inducible human cytomegalovirus immediate early (IE) promoter/enhancer was used to drive transcription of a recombinant AAT gene in transiently transfected and stably transformed CHO cells. The AAT gene was modified to incorporate highly efficient 3'RNA processing signals from the herpes simplex virus type 2 IE gene 5, and optimal translational initiation signals were created by site-directed mutagenesis. The effect of flanking the recombinant gene with matrix attachment regions was investigated. Combinations of these modifications allowed secretion of up to 44 micrograms AAT/ml per day by cell lines growing in serum-rich medium. This could be increased to up to 100 micrograms AAT/ml per day upon chemical induction of expression by propionate, butyrate or hexamethylene bisacetamide. Cell lines adapted to grow in protein-free medium produced less AAT but still responded to chemical induction to secrete up to 14 micrograms/ml per day of readily purified AAT.

Animals↗

PubMed2003

H2O2-induced higher order chromatin degradation: a novel mechanism of oxidative genotoxicity.

The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.

Animals↗

PubMed2002

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment.

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.

Journal Article↗

PubMed2004

Dissemination of the highly expressed Bx7 glutenin subunit (Glu-B1al allele) in wheat as revealed by novel PCR markers and RP-HPLC.

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called 'over-expressing' allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.

Base Sequence↗

PubMed2002

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants.

Segregating T(1), T(2) and T(3) transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected beta-glucuronidase ( gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed.

Journal Article↗

PubMed2006

Expression of active hBMP2 in transgenic tobacco plants.

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.

Animals↗

PubMed2002

Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.

Acetyl-CoA Carboxylase↗

PubMed2006

Nucleotides induce higher order chromatin degradation.

Higher order chromatin degradation (HOCD) is a stepwise dismantling of the genome through the excision of chromatin loops and their oligomers at matrix attachment regions (MARs) during the early stages of programmed cell death. Although HOCD ultimately leads to the inactivation of the genome and cell death, a partial HOCD in cells receiving sublethal signals may result in the loss of genetic stability leading to neoplasia, degeneration, and aging. The present study was undertaken to determine the role of protein poly(ADP-ribosyl)ation in HOCD. Nuclei isolated from rat glioma C6 cells were able to carry poly(ADP-ribosyl)ation as assessed by the incorporation of (32)P-NAD(+) into TCA-insoluble fraction. Under the same experimental conditions, millimolar NAD(+) induced rapid HOCD in nuclei. However, while poly(ADP-ribosyl)ation was totally abrogated by specific inhibitor, benzamide, NAD(+)-induced HOCD was unaffected. Benzamide also failed to inhibit HOCD induced by H(2)O(2) exposure in intact cells. These results indicate that HOCD is not mediated through chromatin poly(ADP-ribosyl)ation, and that NAD(+) activates MAR-associated endonuclease or facilitates the access of the enzyme to DNA by other mechanisms. Furthermore, other nucleotides including NADP(+), ATP, UTP, GTP, and CTP were also found to induce HOCD in isolated nuclei indicating that HOCD is controlled by nucleotide-related ligands.

Animals↗

PubMed2006

DNA bending in the replication zone of the C3 DNA puff amplicon of Rhynchosciara americana (Diptera: Sciaridae).

Intrinsic bent DNA sites were identified in the 4289 bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides -6753, -5433, -5133 and -4757. Sequence analysis showed that these bends are composed of 2-6 bp dA.dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289 bp sequence revealed low helical stability (DeltaG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

Animals↗

PubMed2006

A Gateway-based platform for multigene plant transformation.

The post-genomic era offers unrivalled opportunities for genetic manipulation of polygenic traits, multiple traits, and multiple gene products. However, remaining technical hurdles make the manipulation of multiple genes in plants difficult. Here we describe a Gateway-based vector system to enable multiple transgenes to be directly linked or fused. The vector system consists of a destination vector and two special attL-flanked entry vectors each containing an attR cassette incompatible with the attL. By multiple rounds of LR recombination reactions, which we call MultiRound Gateway, multiple transgenes can be delivered sequentially and indefinitely into the Gateway-compatible destination vector through alternate use of the two special entry vectors. In our proof-of-principle experiments we have used this vector system to construct a plant transformation vector containing seven functional DNA fragments, including a screening marker gene, two reporter genes and four matrix attachment region sequences. This system provides a platform for fully realizing the potential of plant genetic manipulation.

Arabidopsis↗

PubMed2006

An inverted repeat transgene with a structure that cannot generate double-stranded RNA, suffers silencing independent of DNA methylation.

Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repeated T-DNAs. We have identified an Arabidopsis line (ex2-4 line 4) displaying silencing of the T-DNA-born nptII gene. This line contains a truncated copy of the T-DNA encompassing the nptII gene with its nos promoter adjacent to an intact T-DNA copy. The orientation of the intact and the truncated copies preclude the generation of a double-stranded nptII transcript. Therefore, we have investigated the genomic landscape surrounding T-DNA insertion in the silenced ex2-4 line 4 and five single-copy ex2-4 lines without silencing in search of features that might explain the silencing phenomenon. GC content, putative matrix-attachment regions and transcriptional interference from neighbouring genes could all be ruled out as major causes of silencing. Bisulphite sequencing revealed de novo methylation of the nos promoter both in non-silenced and silenced plants of this line, thus silencing was not correlated to DNA methylation level. Also, the methylation pattern deviated from that characteristic for RNA-mediated DNA methylation and silencing. Our data therefore suggest that ex2-4 line 4 represents a case where silencing is due to DNA-DNA pairing, i.e. pairing between the intact T-DNA and the adjacent truncated, inverted T-DNA copy.

Arabidopsis↗

PubMed1990

Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain.

We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.

Animals↗

PubMed1994

Production of pharmaceutical proteins from transgenic animals.

Different systems are being studied and used to prepare recombinant proteins for pharmaceutical use. The blood, and still more the milk, from transgenic animals appear a very attractive source of pharmaceuticals. The cells from these animals are expected to produce well-matured proteins in potentially huge amounts. Several problems remain before this process becomes used in a large scale. Gene transfer remains a difficult and costly task for farm animals. The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency. Improvement of these vectors includes the choice of efficient promoters, introns and transcription terminators, the addition of matrix attached regions (MAR) and specialized chromatin sequences (SCS) to enhance the expression of the transgenes and to insulate them from the chromatin environment. Mice are routinely used to evaluate the gene constructs to be transferred into larger animals. Mice can also be utilized to prepare amounts as high as a few hundred mg of recombinant proteins from their milk. Rabbit appears adequate for amounts not higher than 1 kg per year. For larger quantities, goat, sheep, pig and cow are required. No recombinant proteins extracted from the blood or milk of transgenic animals are yet on the market. The relatively slow but real progress to improving the efficiency of this process inclines to be reasonably optimistic. Predictive reports suggest that 10% of the recombinant proteins, corresponding to a 100 million dollars annual market, will be prepared from the milk of transgenic animals by the end of the century.

Animals↗

PubMed1995

Comparative study of induction of endogenous DNA degradation in rat liver nuclei and intact thymocytes.

A common strongly ordered multi-step-pattern of endogenous DNA degradation was induced in rat liver nuclei and intact thymocytes, prepared in the presence of chelating agents and incubated in the presence of CaCl2 and/or MgCl2. It consisted of sequential generation of 0.3 Mbp, then 0.05 Mbp DNA fragments and finally of oligo- and mononucleosomal DNA. Oligonucleosomal DNA was generated when the genome had already been disintegrated into 0.05 Mbp DNA fragments. ZnCl2 completely inhibited advanced genome cleavage to oligo- and mononucleosomal DNA without affecting the initial generation of large DNA fragments. Therefore, the endonucleolytic activity which produce large DNA fragments is different from Ca2+/Mg2+ endonuclease. The similar pattern of DNA degradation was observed in thymocytes treated with dexamethasone and with the topoisomerase II inhibitor VM-26, the agents known to induce apoptosis. The effect of VM-26 strongly suggests the involvement of topoisomerase II in generation of large DNA fragments. Multi-level organization and regulation of the chromatin structure determine the stepwise process of genome degradation. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation thus leading to cell death in terminal differentiation or stress-induced apoptosis.

Animals↗

PubMed2001

Higher order chromatin degradation in glial cells: the role of calcium.

Higher order chromatin degradation (HOCD), i.e. the scission of nuclear chromatin loops at the matrix attachment regions (MARs), is a hallmark of programmed cell death. We have previously demonstrated that hydrogen peroxide (H(2)O(2)) induces rapid HOCD in cultured oligodendrocytes generating two subpopulations of DNA fragments of >or=400 and 50-200 kb. In the present study, we examined the involvement of calcium in this process. HOCD was induced in primary rat oligodendrocytes by exposure to 1 mM H(2)O(2) and assessed by field inversion gel electrophoresis with and without S1 endonuclease digestion, to detect single and double stranded fragmentation, respectively. Chelating intracellular calcium with BAPTA/AM prior to H(2)O(2) exposure inhibited HOCD in a dose-dependent manner. Complete inhibition of HOCD was attained with 50 muM BAPTA/AM. The pretreatment of cells with desferroxamine mesylate, which may lower intracellular calcium levels, also resulted in a profound inhibition of HOCD, but the initial chromatin digestion into >or=400 kb single stranded DNA fragments was unaffected. Neither removing extracellular calcium nor blocking calcium release from intracellular stores with TMB-8 affected HOCD. Moreover, increasing intracellular calcium with A23187 calcium ionophore did not induce HOCD. Subsequent study in nuclei purified from C6 glioma cells revealed that the endonuclease responsible for HOCD is calcium-independent, but is magnesium-dependent. Magnesium-induced HOCD was not affected by the removal of calcium from nuclei with EGTA, but was practically abrogated in nuclei prepared from BAPTA/AM-pretreated cells. These results indicate that although H(2)O(2)-induced HOCD is not directly mediated by an increase of intracellular calcium concentration, normal resting levels of intracellular calcium are required for the maintenance of MAR-associated endonuclease in an active form.

Animals↗