Functional analysis of cauliflower mosaic virus 35S promoter: re‐evaluation of the role of subdomains B5, B4 and B2 in promoter activity

Bhullar, Simran; Datta, Sudipta; Advani, Sonia; Chakravarthy, Suma; Gautam, Taru; Pental, Deepak; Burma, Pradeep Kumar

doi:10.1111/j.1467-7652.2007.00274.x

Cited by 35 publications

(27 citation statements)

References 34 publications

(75 reference statements)

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“…To investigate the methylation status, DNA samples from 7 each of the 4n and 2n T2 lines (indicated by asterisks in Figure 3A), composed of lowand high-copy number individuals, were treated with bisulphite-a process that converts unmethylated cytosines to uracils but does not affect methylated cytosines. A 442-bp sequence, from À108 to À549 of the 35S promoter including the B2-B5 domains important for the promoter activity ( Figure 1A and Figure S3A) (Benfey and Chua 1990;Bhullar et al 2007), was then PCR amplified with unbiased primers specific for the top strand. The PCR product was then digested with MseI or Bgl II/AccI and separated in an agarose gel.…”

Section: Resultsmentioning

confidence: 99%

Transgene Expression and Transgene-Induced Silencing in Diploid and Autotetraploid Arabidopsis

Finn¹,

Wang²,

Costa³

et al. 2011

Genetics

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Previous studies have suggested that transgene expression in plants can be affected by ploidy. Here we show that three different transgenes, a reporter transgene, an antisense transgene, and a hairpin RNA (hpRNA) transgene, are all expressed at a lower level in autotetraploid (4n) than in diploid (2n) Arabidopsis. RNA silencing of two endogenous genes was induced by the antisense and hpRNA transgenes and this silencing is significantly less effective in 4n than in 2n Arabidopsis; furthermore, the reduced silencing in 4n Arabidopsis correlated with reduced accumulation of silencing-inducer RNAs. Methylation analysis both of independent 2n and 4n transgenic lines and of 2n and 4n progeny derived from the same 3n transgenic parent, indicated that transgenes are more methylated in 4n than 2n Arabidopsis. These results suggest that transgenes are transcriptionally repressed in the 4n background, resulting in expression levels lower than in the 2n background. Transgenes designed to silence endogenous genes express lower concentrations of silencing-inducer RNAs in 4n Arabidopsis plants, resulting in less effective silencing of target genes than in 2n Arabidopsis plants.

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Section: Resultsmentioning

confidence: 99%

Transgene Expression and Transgene-Induced Silencing in Diploid and Autotetraploid Arabidopsis

Finn¹,

Wang²,

Costa³

et al. 2011

Genetics

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“…S1). We constructed individual expression vectors for 5 carotenogenic genes each under the control of a different endosperm-specific promoter to avoid potential gene silencing caused by promoter homology (20) and to ensure the coordinated, restricted expression of all transgenes, a feat that has not been achieved in previous reports of multigene transfer using marker genes to our knowledge (21). Direct DNA transfer with separate vectors usually results in transgene integration at a random single locus, in the form of a multigene array (22,23) containing any number of transgenes from 1 to n, with the distribution within the transgenic population tending to describe a normal curve as would be expected from random sampling (24).…”

Section: Discussionmentioning

confidence: 99%

“…Direct DNA transfer with separate vectors usually results in transgene integration at a random single locus, in the form of a multigene array (22,23) containing any number of transgenes from 1 to n, with the distribution within the transgenic population tending to describe a normal curve as would be expected from random sampling (24). Input transgenes once integrated remain linked and do not segregate in subsequent generation as is the norm for direct DNA transfer of multiple transgenes irrespective of whether these are on 1 cointegrate vector or independent plasmids (cotransformation) (19)(20)(21)(22)(23)(24). Plants carrying 1-5 transgenes in a roughly normal distribution could be identified from colored seed phenotypes arising from the accumulation of specific carotenoids, and these could be correlated with mRNA and metabolite profiles to identify and quantify the specific carotenoid molecules present in the endosperm.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Zhu

Naqvi

Breitenbach

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

332

263

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Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between ␤-carotene hydroxylase and bacterial ␤-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of ␤-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.induced mutation ͉ metabolic engineering ͉ transgenic plant ͉ provitamin A

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“…Furthermore, the 35S promoter consists of various subdomains. Cis-elements, important for promoter activity, have been identified in domains B1 (as-2 element), A1 (as-1 element), and the minimal promoter (TATA box) (Bhullar et al 2007).…”

Section: Introductionmentioning

confidence: 99%

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits

et al. 2009

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Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrub Thaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.

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Functional analysis of cauliflower mosaic virus 35S promoter: re‐evaluation of the role of subdomains B5, B4 and B2 in promoter activity

Cited by 35 publications

References 34 publications

Transgene Expression and Transgene-Induced Silencing in Diploid and Autotetraploid Arabidopsis

Transgene Expression and Transgene-Induced Silencing in Diploid and Autotetraploid Arabidopsis

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits

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