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Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct  [PDF]
Jeff Velten, Cahid Cakir, Eunseog Youn, Junping Chen, Christopher I. Cazzonelli
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0030141
Abstract: Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.
Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian  [PDF]
Kei-ichiro Mishiba,Satoshi Yamasaki,Takashi Nakatsuka,Yoshiko Abe,Hiroyuki Daimon,Masayuki Oda,Masahiro Nishihara
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009670
Abstract: A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora × G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (?90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (?148 to ?85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the ?149 to ?124 and ?107 to ?83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.
Evaluation of Xanthophylls Extracted from Tagetes erectus (Marigold Flower) and Capsicum Sp. (Red Pepper Paprika) as a Pigment for Egg-yolks Compare with Synthetic Pigments  [PDF]
E. Santos-Bocanegra,X. Ospina-Osorio,E.O. Oviedo-Rondon
International Journal of Poultry Science , 2004,
Abstract: Three experiments were accomplished to compare the efficacy of yellow and red xanthophylls extracted from Tagetes erectus and Capsicum sp., with synthetic citranaxanthin, canthaxanthin, and ester of beta-apo-8-carotenoic pigments at different concentrations to enhance the yellow color of chicken egg yolk. In each trial brown laying hens were placed in cages, and after seven days of feeding a sorghum-soybean diet without pigments treatments were assigned. Yolk eggs were classified with the Roche yolk color fan (RYCF) or Minolta refractometer. In the first experiment, 168 hens were randomly distributed to seven treatments with one control group, synthetic pigments, and natural xanthophylls. In the second and third experiments, 100 hens were assigned to four different treatments consisting in the same basal diet with addition of natural xanthophylls, and three combinations of apo-ester and citranaxantin. In all experiments, after four weeks of feeding these experimental diets, the color of 10 egg yolks per treatment was evaluated 10 different times every other day or twice a week. Results indicated that live performance was not affected by treatments, but the egg yolk color changed significantly according to the pigment added. Hens fed with diets with 7.5 ppm of yellow xanthophylls extracted from Tagetes and 4.0 ppm of red xanthophylls from Capsicum had yolk eggs classified as color 11.7 ± 0.1. Capsicum extract levels had linear effect over RYCF. Synthetic carotenoids gave a yolk color that varies from 13 to 14 in the highest concentration, and from 12 (5%) to 14 (43%) at the lowest concentration.
Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues
Antonio AB Viana, Rodrigo R Fragoso, Luciane M Guimar?es, Naiara Pontes, Osmundo B Oliveira-Neto, Sinara Artico, Sarah M Nardeli, Marcio Alves-Ferreira, Jo?o AN Batista, Maria CM Silva, Maria F Grossi-de-Sa
BMC Biotechnology , 2011, DOI: 10.1186/1472-6750-11-115
Abstract: Nucleotide analysis revealed high identity with cotton E2 homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the E2 active site, and an intron that is spliced in E2 homologues, but not in GhGDRP85. The GhGDRP85 gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in Arabidopsis thaliana. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2.uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.Cotton plants (Gossypium spp.) produce the most widely used textile fiber in the world. Moreover, the cotton seed is used to feed livestock and its oil is important in biodiesel production and human consumption. In 2010, more than half world cotton area was planted with genetically modified (GM) cotton, which occupied 21.0 million hectares (14% global biotech area) [1]. Up to date, around 35 cotton events have been approved, in which insect-resistant trait is one of most used and spread, behind only of herbicide tolerance or both traits combined [1].Despite significant efforts towards the isolation and characterization of plant genes, only a reduced number of plant promoters have been isolated and functionally characterized [2-4]. The most widely used general-purpose promoter in GM plants is the Cauliflower Mosaic Virus (CaMV) 35S
35s标记重组植物钙调素的制备方法  [PDF]
崔素娟?,郭晓强?,毛国红?,赵军峰?,孙颖?,白娟?,马力耕?,孙大业?
生物化学与生物物理进展 , 2002,
Abstract: 利用35s标记的氨基酸混合物喂养工程菌,成功地制备了35s标记的拟南芥钙调素亚型2(35s-acam2),对其纯度、放射活度、电泳行为及其灵敏性等进行了检测.结果表明从工程菌中制备的35s-acam2纯度高、放射活度高、ca2+与egta存在时的电泳行为与未标记的acam2相同,可作为一种高灵敏性的探针用于检测钙调素结合蛋白.
Neovascularisation by tattoo pigment
Abdul Razack E,Ravichandran G,Somasundaram V,Umaselvam M
Indian Journal of Dermatology, Venereology and Leprology , 1991,
Abstract: Split skin grafting for the removal of a tattoo resulted in the appearance of pigmented papules in the periphery of the grafted skin as well as distal to it on the normal skin. Histologically they showed large vascular laminae containing red blood corpuscles and pigment deposits, a hitherto not documented complication of tattoo pigment.
The Heredity of Flower Colors and the Discovery of Flower Color Chimera in Chrysanthemum Species
菊花花色遗传及花色嵌合体发现

LI Mao-Teng,YU Long-Jiang,WANG Li-Mei,LIU Jian-Min,LEI Cheng,
栗茂腾
,余龙江,王丽梅,刘建民,雷 呈

遗传 , 2005,
Abstract: The reciprocal crosses of yellow colored chrysanthemum x red colored chrysanthemum and white colored chrysanthemum x red colored chrysanthemum were conducted in order to analyze the heredity of flower colors. The results revealed that the heredity of flower colors was very complicated, and mainly exhibited matroclinous characteristics when red colored materials was used as maternal parent but not in the combinations when the yellow or white colored materials were used as maternal parents. The incomplete dominance and mosaic dominance also existed in the heredity of chrysanthemum flower colors. The flower-color chimeras with two kinds of flower buds were discovered in the cross of 3501 x 3509, i.e. one side of the flower buds was completely in red color, which was same as the parental material of 3509, and another side was generally in yellow color with red spots on them. Cytological analysis showed that two sides were both with 36 chromosomes, indicating that the formation of chimera was not resulted from the changes of chromosome numbers, but from the destruction of pigment synthesis genes by the insert of transposable element.
A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants  [cached]
Kanoria Shaveta,Burma Pradeep Kumar
BMC Biotechnology , 2012, DOI: 10.1186/1472-6750-12-85
Abstract: Background A high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ), which enhances gene expression in tobacco and cotton. Results The influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants. Conclusions synJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.
Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period
Ewa Borejsza-Wysocka, John L Norelli, Herb S Aldwinckle, Mickael Malnoy
BMC Biotechnology , 2010, DOI: 10.1186/1472-6750-10-41
Abstract: Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control.Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.Genetic transformation technology has facilitated studies of gene regulation in several plant species including trees [1,2]. Some of the most problematic barriers to genetic improvement of trees, such as their large size and long breeding cycles, can be circumvented by the application of these techniques. Because trees have a long lifespan, knowledge of the genetic regulation of mature tissues is of major importance. The successful introduction of transgenic trees depends on improving the horticultural performance of the modified plants and on the stable expression of the transgene [3]. There is no need to consider the inheritance pattern to successive generations in trees, since grafting is the normal method of propagating fruit trees and unlimited numbers of T0 transgenic lines can be selected for evaluation. The study of transgene expression is of vital importance whenever transgenic plants are produced. Transgene expression levels are influenced by many factors, in particular the site of integration of the transgene within the plant genome, gene silencing,
Regulation of transgene expression in genetic immunization
Harms, J.S.;Oliveira, S.C.;Splitter, G.A.;
Brazilian Journal of Medical and Biological Research , 1999, DOI: 10.1590/S0100-879X1999000200003
Abstract: the use of mammalian gene expression vectors has become increasingly important for genetic immunization and gene therapy as well as basic research. essential for the success of these vectors in genetic immunization is the proper choice of a promoter linked to the antigen of interest. many genetic immunization vectors use promoter elements from pathogenic viruses including sv40 and cmv. lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. our objective was to determine the effect of ifn-g on transgene expression driven by viral sv40 or cmv promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class i (mhc i) gene. we transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. luciferase assays of transfected cells untreated or treated with ifn-g indicated that although the viral promoters could drive luciferase production in all cell lines tested to higher or lower levels than the mhc i promoter, treatment with ifn-g inhibited transgene expression in most of the cell lines and amplification of the mhc i promoter-driven transgene expression in all cell lines. these data indicate that the sv40 and cmv promoter/enhancers may not be a suitable choice for gene delivery especially for genetic immunization or cancer cytokine gene therapy. the mhc i promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.
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