共查询到20条相似文献,搜索用时 31 毫秒
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Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter
when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays
ADH1 5′ MAR, Nicotiana tabacum
Rb7 3′ MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer–promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed
between the cauliflower mosaic virus
35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::β-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated.
Accession numbers: TBS from Petunia hybrida cultivar V26, GenBank accession number EU864306. 相似文献
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There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of
the enhancer–promoter interaction in yellow. In this paper, we have found that the yellow sequence from −100 to −69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the
same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4)
u1
background. These finding suggest that proteins bound to the −100 to −69 sequence are essential for communication between
the yellow promoter and upstream regulatory elements. 相似文献
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Evidences for insulator activity of the 5′UTR of the Drosophila melanogaster LTR-retrotransposon ZAM
Crescenzio Francesco Minervini Simona Ruggieri Michele Traversa Leonardo D’Aiuto Rene’ Massimiliano Marsano Daniela Leronni Isabella Centomani Claudio De Giovanni Luigi Viggiano 《Molecular genetics and genomics : MGG》2010,283(5):503-509
Insulators or chromatin boundary are DNA elements that organize the genome into
discrete regulatory domains by limiting the actions of enhancers and silencers through a
“positional-blocking mechanism”. The role of these sequences, both in modulation of the
enhancers range of action (enhancer–promoter selectivity) and in the organization of the
chromatin in functional domains, is emerging strongly in these last years. There is a great
interest in identifying new insulators because deeper knowledge of these elements can
help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation
and genome functioning, only a few have been reported so far.
Here, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila
retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on
the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in
the human cell line HEK293.
These results suggest the possibility of employing the ZAM insulator in gene transfer
protocols from insects to mammals in order to counteract the transgene positional and
genotoxic effects. 相似文献
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M. V. Kostyuchenko E. E. Savitskaya M. N. Krivega P. G. Georgiev 《Russian Journal of Genetics》2008,44(12):1468-1471
Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers
and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine
proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role
in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and
promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer
and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated
promoter. The best studied insulator of Drosophila melanogaster was found in the 5′ regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated
that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression,
induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines,
two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter
and functionally opposite elements, responsible for either gene activation, or repression.
Original Russian Text ? M.V. Kostyuchenko, E.E. Savitskaya, M.N. Krivega, P.G. Georgiev, 2008, published in Genetika, 2008,
Vol. 44, No. 12, pp. 1693–1697. 相似文献
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The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower
mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic
virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 –
15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between
independent transgenic lines for all the constructs tested. 相似文献
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Jinjin Zhang Xiping Wang Stacy Singer Zhongchi Liu Yingjun Yang Guohua Yan Zongrang Liu 《Plant biotechnology journal》2014,12(7):951-962
Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed. 相似文献
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Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this
study we isolated two similar, but distinct, alleles of the Citrus sinensis
sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis
thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements
required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression.
Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and
separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development
of transgenic plants resistant to a wide variety of target pests. 相似文献
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Xiaoping Chen Zhangying Wang Jianhua Wang Maoyan Wang Li Zhao Guoying Wang 《Plant Cell, Tissue and Organ Culture》2007,88(1):11-20
ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region
preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied
via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb
fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking
fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous
system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression
in embryos, which is different from similar promoters tested in maize. 相似文献
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The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis
AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether
a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented
chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the β-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results
indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity
in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers
exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been
observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of
targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for
the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions. 相似文献