A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants

The RB7 matrix attachment region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60-fold in stably transformed tobacco suspension cell lines. We have now used the same co-transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co-transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MAR-containing lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC₁ generation. Sixty-three per cent of the 21 MAR-containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology-dependent gene silencing.     

High transformation frequency of tobacco and rice via Agrobacterium-mediated gene transfer by flanking a tobacco matrix attachment region

Nuclear matrix attachment regions (MARs) are suggested to regulate chromatin structure and influence the expression of flanking genes. Our previous study showed that TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro and increase transgene expression in vivo. Here, we investigated the role of TM2 MAR in improving transformation frequency of Agrobacterium -mediated transformation in tobacco and rice. The gusA reporter gene flanked by TM2 MAR in pBI121 and pCAMBIA-1301 vectors was controlled by a constitutive promoter or a photosynthetic tissue-specific promoter. The presence of TM2 MAR in different expression cassettes significantly increased the numbers of kanamycin-resistant tobacco shoots, hygromycin-resistant rice calli and shoots. Seeds from the independent transgenic lines with TM2 MAR can germinate normally on the medium containing 500 mg l⁻¹ kanamycin, whereas none of the seeds from the transgenic lines without TM2 MAR survived. Furthermore, RNA gel blot analysis revealed that nptII messenger RNA levels are more abundant in the independent transgenic lines with TM2 MAR than in the lines without TM2 MAR. Altogether, these data reveal a possible mechanism that TM2 MAR improves the transformation frequency by increasing nptII gene expression.     

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven -glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies 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     

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.     

Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants

We have isolated and analyzed a pre-ferredoxin gene from Arabidopsis thaliana. This gene encodes a 148 amino acid precursor protein including a chloroplast transit peptide of 52 residues. Southern analysis shows the presence of a single copy of this ferredoxin (Fd) gene in the A. thaliana genome. Its expression is tissue-specific and positively affected by light. Response times, both to dark and light conditions, are remarkably rapid.A chimeric gene consisting of a 1.2 kb Fd promoter fragment fused to the -glucuronidase reporter gene was transferred to tobacco. This fusion gene is expressed in a tissue-specific way; it shows high levels of expression in green leaves, as compared to root tissue.     

Expression of the human granulocyte–macrophage colony stimulating factor (hGM-CSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice

Expression of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected “protective or enhancer effect” from the MAR element on the hGM-CSF gene expression was not observed.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Tobacco matrix attachment region sequence increased transgene expression levels in rice plants

In this study, six plasmids were constructed to study the effects of the tobacco Rb7 matrix attachment region (MAR) sequence on rice transgene expression. Among them, each of four plasmids contained two identical copies of the MAR sequence flanking two different reporter genes, which encode the green fluorescent protein and -glucuronidase. Two control plasmids contained no MAR sequences. Microprojectile bombardment was used to separately introduce these six plasmids into rice calli. Transgenic rice plants were regenerated, and gene expression was measured in rice leaf extracts of two-month-old transgenic plants. By comparing transgenic plants with the corresponding control plants, transgenic plants harboring each of four plasmids that included the MAR sequence resulted in average gene expression levels enhanced by 3.3-fold, 18-fold, 376-fold and 650-fold. These results suggest that the same MAR sequence can affect the expression of different genes to different extents. One goal of plant scientists and breeders is to maximize expression levels of transgenes, especially in the production of transgene-encoded protein. Therefore, inclusion of the Rb7 MAR sequence would be beneficial.     

Isolation and Characterization of cDNA Clones with Enhanced Expression in Tobacco Plants Expressing the Rice Homeobox Gene, OSH1

We have used subtractive hybridization to isolate cDNA cloneswhose expression were up-regulated in transgenic tobacco ectopicallyexpressing the rice homeobox gene, OSH1. Thirty-nine distinctcDNA clones, which we term HRGs (Homeobox Regulated Genes),were identified. Some of them were specifically expressed intransformants, indicating that their expression was possiblyregulated by transgene. (Received January 9, 1997; Accepted March 8, 1997)     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Effect of nuclear matrix attachment regions on transgene expression in tobacco plants

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.