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.     

Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar

We tested the value of a matrix attachment region (MAR) fragment derived from a tobacco gene for increasing the frequency of Agrobacterium-mediated transformation. A binary vector that carried a GUS reporter gene containing an intron and an nptII gene was modified to contain flanking MAR elements within the T-DNA borders. Vectors containing or lacking MARs were then used to transform tobacco, a readily transformabl e poplar clone (Populus tremula × P. alba), and a recalcitrant poplar clone (Populus trichocarpa × P. deltoides). MARs increased GUS gene expression approximately 10-fold in the two hybrid poplar clones and twofold in tobacco one month after cocultivation with Agrobacterium; MARs also increased the frequency of kanamycin-resistant poplar shoots recovered     

核基质结合序列（MAR）与基因表达调控

核基质结合序列（MAR）是能在体外与核基质特异结合的DNA序列,广泛存在于染色质Loop结构的边界序列中。随着研究的深入,发现MAR序列不仅在染色质折叠中起到重要作用,影响邻近内源基因表达,而且将MAR序列构建到外源基因表达盒两侧转化动植物时,也影响外源基因的表达。因此,MAR序列是基因组中一种重要的基因间边界序列,为阐明非编码序列在基因表达中的作用和构建真核生物高效表达载体提供了新途径。     

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.     

Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.S.-J. Oh, J.S. Jeong, E.-H. Kim, N.R. Yi and S.-I. Yi contributed equally to the paper     

Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize <Emphasis Type="Italic">adh-1</Emphasis> matrix attachment regions: tissue and developmental specificity in maize transgenic plants

Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms. In plants, contradictory data question the role of MAR sequences. To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region. The MARs have been cloned either 5 to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain. Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny. Furthermore, MARs systematically induced variegation. We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.Abbreviations adh-1 Alcohol dehydrogenase 1 - GUS -Glucuronidase - HSC80 Heat shock cognate 80 gene - MAR Matrix attachment regions - Rsyn-7 Root specific synthetic promoter     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

Summary We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5 flanking sequence and the total 5 untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and -glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.     

Expression of the blasticidin S deaminase gene (bsr) in tobacco: Fungicide tolerance and a new selective marker for transgenic plants

Summary Blasticidin S (BS), a fungicide of microbial origin, is used for the practical control of rice blast disease. It has broad antimicrobial activity but occasionally exhibits adverse phytotoxic effects on some dicot plants. An inactivating enzyme, BS deaminase, was discovered in the BS resistant strain, Bacillus cereus K55-S1, and the structural gene, bsr, for the enzyme has been cloned. We introduced the bsr gene into tobacco plants using the Ti plasmid vector system and demonstrated that the bsr gene conferred a BS resistant phenotype to the plants. Thus the bsr gene could be useful as a selective marker for plant transformation and provides an example for a new approach to the solution of phytotoxicity problems associated with the use of some types of fungicide.     

Increased availability of extrafloral nectar reduces herbivory in Lima bean plants (Phaseolus lunatus, Fabaceae)

Lima bean (Phaseolus lunatus) features two inducible indirect defences to protect itself against herbivores. Besides the emission of plant volatiles, extrafloral nectar is secreted to attract carnivorous arthropods to herbivore-damaged plants. The activation of both putative defences efficiently protects Lima beans from leaf damage. In a field experiment in Mexico, we studied whether extrafloral nectar alone can benefit the Lima bean under natural conditions. An artificial blend mimicking natural nectar both qualitatively and quantitatively was repeatedly applied to Lima bean tendrils. Ants, wasps and flies were significantly more abundant on treated tendrils than on untreated controls already after 1 week (i.e. after two treatment applications). Sticky traps were used to assess the functional groups of flying insects attracted to the Lima beans. After 24 h, 71% of all trapped flies and 98% of all wasps belonged to families comprising either parasitoid or predatory species. This observation suggests that also some of the flying visitors have played a role as putative defenders of Lima beans. Most of the trapped flies belonged to the families Dolichopodidae and Phoridae (each ca. one third of all individuals). Two thirds of the wasps belonged to Chalcidoidea (68%). All ant species that had been collected manually belonged to generalist genera with Camponotus novogranadensis and Cephalotes minutus being most regularly encountered on study tendrils. An additional experiment, where both ‘nectar’ and ‘control’ tendrils were treated with artificial nectar, revealed that ants responded with an increased abundance on tendrils that had experienced the ‘nectar’ treatment before.After 25 days, the treated tendrils showed a significantly reduced herbivory as compared to controls. The mere presence of increased amounts of extrafloral nectar thus can benefit the Lima bean under natural conditions.

Zusammenfassung

Die Limabohne (Phaseolus lunatus) verfügt über zwei induzierbare, indirekte Verteidigungsformen zur Abwehr von Herbivoren. Neben der Emission volatiler Verbindungen ist die Limabohne zusätzlich dazu in der Lage, extrafloralen Nektar zu sezernieren. Beides dient der Anlockung von Fraßfeinden zu den von Herbivoren befallenen Pflanzen. In einem Freilandexperiment in Mexiko wurde untersucht, ob die Limabohne unter natürlichen Bedingungen von der Sekretion extrafloralen Nektars profitiert. Hierzu wurde ein künstliches Nektargemisch wiederholt auf Limabohnenranken aufgetragen, welches natürlichen Nektar quantitativ und qualitativ imitierte. Bereits nach einer Woche (d.h. nach zwei Behandlungen) war die Abundanz von Ameisen, Fliegen und Wespen auf behandelten Ranken signifikant höher als auf unbehandelten Kontrollranken. Zur Erfassung der zur Limabohne angelockten fliegender Insekten sowie deren Zugehörigkeit zu funktionellen Gruppen wurden die Versuchsranken mit Klebefallen bestückt. Mehr als zwei Drittel der nach 24 h gefangenen Fliegen und 98% aller Wespen gehörten parasitisch oder räuberisch lebenden Fliegen- bzw. Wespen-Familien an. Diese Beobachtung legt nahe, dass nicht nur Ameisen, sondern auch einige der gefangenen fliegenden Besucher eine Rolle als potentielle Verteidiger der Limabohne gespielt haben könnten. Von den gefangen Fliegen gehörten die meisten den Familien Dolichopodidae und Phoridae (je ca. ein Drittel aller gefangenen Individuen) an, wogegen die Chalcidoidea zwei Drittel (68%) der gefangenen Wespen ausmachten. Unter den durch Handaufsammlung gefangenen Ameisen gehörten Camponotus novogranadensis und Cephalotes minutus zu den am häufigsten auf behandelten Ranken angetroffen Arten. Ein zusätzliches Experiment, in dem das künstliche Nektargemisch sowohl auf ‘Nektar’- als auch auf ‘Kontroll’-Ranken aufgebracht wurde, ergab, dass die Ameisen mit einer erhöhten Abundanz auf solchen Ranken reagierten, die bereits vorher die, Nektar’-Behandlung erfahren hatten.Nach 25 Tagen zeigten behandelte Ranken signifikant weniger Blattfraß im Vergleich zu unbehandelten Kontrollranken. Die bloße Erhöhung der Menge an extrafloralem Nektar reichte offensichtlich dazu aus, unter natürlichen Bedingungen wachsenden Limabohnen einen Vorteil zu verschaffen.     

Common polymorphisms in interleukin genes (IL4, IL6, IL8 and IL12) are not associated with alcoholic liver disease or alcoholism in Spanish men

Background: Preliminary data suggest that polymorphisms in cytokine genes may be involved in the genetic predisposition to alcoholic liver cirrhosis or alcohol use disorders. We thus analyze the association between these diseases and the following polymorphisms: −33T > C IL4, −174 G > C IL6, −251 T > A IL8 and 1188 A > C IL12B. Methods: 258 male alcoholics (161 without liver disease and 97 with liver cirrhosis) and 101 healthy controls were genotyped for the above mentioned polymorphisms. We examined the relationship between genotype and allele frequencies and the presence of disease, as well as the correlation with combinations of putative pro-inflammatory genotypes. Haplotypes were inferred using the expectation–maximization algorithm and haplotype frequencies were compared. Results: We found no statistically significant association between any of these polymorphisms or the combinations of pro-inflammatory polymorphisms and the risk of alcoholic liver cirrhosis or alcohol abuse or dependence. Haplotype analysis of the IL4 and IL12B polymorphisms did not show any statistical relationship either. Conclusions: Our results do not support the hypothesis that the analyzed polymorphisms confer differences in alcoholic liver cirrhosis or alcohol use disorders susceptibility.     

Transgenic tobacco plants with strongly decreased expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase do not differ significantly from wild type in photosynthate partitioning,plant growth or their ability to cope with limiting phosphate,limiting nitrogen and suboptimal temperatures

Transformation of tobacco with the potato gene encoding the subunit of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) in the antisense orientation under the control of the constitutive CaMV 35S promoter, followed by selfing and crossing of the transformants, generated a line of tobacco (5–37) with up to an 85% reduction in PFP activity in the shoot. Transformants containing a sense construct (4-40-91) contained only 1–3% of wild-type PFP, presumably due to co-suppression. Rates of photosynthesis and partitioning between sucrose and starch in source leaves were identical in 4-40-91 transformants and the wild type. In the dark in sink leaves of 4-40-91 transformants, levels of hexose phosphates were up to 50% higher, glycerate-3-phosphate 30% lower and fructose-2,6-bisphosphate threefold higher than in the wild type; inorganic pyrophosphate, pyruvate and the ATP/ADP ratio were unaltered. Low -PFP and wild-type plants did not differ significantly in their rate of growth at 25° C and 200 mol quanta · m^–2 · s^–1 on full nutrient medium. Growth on limiting phosphate and limiting nitrogen was inhibited identically in the wild type and transformants, and transformants adjusted their shoot/root ratio in an identical manner to the wild type. Differences in fructose-2,6-bisphosphate and glycolytic metabolites between the wild type and transformants were no larger in these suboptimal nutrient conditions, than in optimal conditions. Growth of the wild type and 4-40-91 transformants was inhibited identically at 12° C compared to 25° C. Differences in fructose-2,6-bisphosphate were smaller when the genotypes were compared at 12° C than at 25° C. We conclude that PFP does not play an essential role in photosynthate partitioning in source leaves. During respiratory metabolism in sink leaves it catalyzes a net glycolytic flux, as in potato tubers. However, tobacco seedlings are able to compensate for a large decrease in expression of PFP without loss of growth, or the ability to cope with suboptimal phosphate, nitrogen or temperature.Abbreviations F2,6BP fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - PFK phosphofructokinase - PFP pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase - 3-PGA glycerate-3-phosphate - PPi inorganic pyrophosphate - PEP phosphoenolpyruvate This work was supported by the Bundesministerium für Forschung and Technologie (M.S, U.S.) and the Canadian Research Council (S.C., D.D). M.P. was supported by a Royal Society Fellowship.