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1.
Plant Cell Physiol ; 60(8): 1871-1879, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31135027

RESUMO

Wild-type plants of the Japanese morning glory (Ipomoea nil) produce blue flowers that accumulate anthocyanin pigments, whereas its mutant cultivars show wide range flower color such as red, magenta and white. However, I. nil lacks yellow color varieties even though yellow flowers were curiously described in words and woodblocks printed in the 19th century. Such yellow flowers have been regarded as 'phantom morning glories', and their production has not been achieved despite efforts by breeders of I. nil. The chalcone isomerase (CHI) mutants (including line 54Y) bloom very pale yellow or cream-colored flowers conferred by the accumulation of 2', 4', 6', 4-tetrahydoroxychalcone (THC) 2'-O-glucoside. To produce yellow phantom morning glories, we introduced two snapdragon (Antirrhinum majus) genes to the 54Y line by encoding aureusidin synthase (AmAS1) and chalcone 4'-O-glucosyltransferase (Am4'CGT), which are necessary for the accumulation of aureusidin 6-O-glucoside and yellow coloration in A. majus. The transgenic plants expressing both genes exhibit yellow flowers, a character sought for many years. The flower petals of the transgenic plants contained aureusidin 6-O-glucoside, as well as a reduced amount of THC 2'-O-glucoside. In addition, we identified a novel aurone compound, aureusidin 6-O-(6″-O-malonyl)-glucoside, in the yellow petals. A combination of the coexpression of AmAS1 and Am4'CGT and suppression of CHI is an effective strategy for generating yellow varieties in horticultural plants.


Assuntos
Benzofuranos/metabolismo , Flavonoides/metabolismo , Flores/metabolismo , Ipomoea nil/metabolismo , Engenharia Metabólica/métodos , Regulação da Expressão Gênica de Plantas , Transdução de Sinais/fisiologia
2.
Glycobiology ; 26(5): 482-92, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26747427

RESUMO

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the ß-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the ß-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The ß-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the ß-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The ß-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood.


Assuntos
Proteínas Antitrombina/metabolismo , Heparina/metabolismo , Manose/metabolismo , Oligossacarídeos/metabolismo , Proteínas Antitrombina/química , Glicosilação , Heparina/química , Humanos , Manose/química , Oligossacarídeos/química , Ligação Proteica
3.
Plant Biotechnol J ; 14(1): 354-63, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25923400

RESUMO

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical ß-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.


Assuntos
Produtos Agrícolas/genética , Embaralhamento de DNA/métodos , Flores/genética , Ipomoea nil/genética , Regiões Promotoras Genéticas , Arabidopsis/genética , Arabidopsis/ultraestrutura , Flores/anatomia & histologia , Flores/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Glucuronidase/metabolismo , Especificidade de Órgãos/genética , Fenótipo , Filogenia , Plantas Geneticamente Modificadas
4.
Proc Natl Acad Sci U S A ; 110(2): 767-72, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23267064

RESUMO

Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Inflorescência/anatomia & histologia , Meristema/crescimento & desenvolvimento , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Elementos de DNA Transponíveis/genética , Fluorescência , Perfilação da Expressão Gênica , Hibridização In Situ , Inflorescência/metabolismo , Proteínas de Domínio MADS/metabolismo , Meristema/metabolismo , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Plant J ; 78(2): 294-304, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24517863

RESUMO

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.


Assuntos
Flavonoides/metabolismo , Liases Intramoleculares/fisiologia , Proteínas de Plantas/fisiologia , Antocianinas/química , Antocianinas/metabolismo , Vias Biossintéticas , Flavonoides/química , Flores/anatomia & histologia , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Liases Intramoleculares/genética , Liases Intramoleculares/metabolismo , Ipomoea/anatomia & histologia , Ipomoea/genética , Ipomoea/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA
6.
Cancer Sci ; 106(1): 102-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25421609

RESUMO

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Gangliosídeo G(M2)/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Gangliosídeo G(M2)/metabolismo , Humanos , Masculino , Mesotelioma Maligno , Camundongos SCID , Pessoa de Meia-Idade , Engenharia de Proteínas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Planta ; 242(3): 575-87, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26007684

RESUMO

MAIN CONCLUSION: UDP-glucose:flavonoid 3- O -glucosyltransferase is essential for maintaining proper production quantity, acylation, and glucosylation of anthocyanin, and defects cause pale and dull flower pigmentation in morning glories. The Japanese (Ipomoea nil) and the common (I. purpurea) morning glory display bright blue and dark purple flowers, respectively. These flowers contain acylated and glucosylated anthocyanin pigments, and a number of flower color mutants have been isolated in I. nil. Of these, the duskish mutants of I. nil produce pale- and dull-colored flowers. We found that the Duskish gene encodes UDP-glucose:flavonoid 3-O-glucosyltransferase (3GT). The duskish-1 mutation is a frameshift mutation caused by a 4-bp insertion, and duskish-2 is an insertion of a DNA transposon, Tpn10, at 1.3 kb upstream of the 3GT start codon. In the duskish-2 mutant, excision of Tpn10 is responsible for restoration of the expression of the 3GT gene. The recombinant 3GT protein displays expected 3GT enzymatic activities to catalyze 3-O-glucosylation of anthocyanidins in vitro. Anthocyanin analysis of a duskish-2 mutant and its germinal revertant showing pale and normal pigmented flowers, respectively, revealed that the mutation caused around 80 % reduction of anthocyanin accumulation. We further characterized two I. purpurea mutants showing pale brownish-red flowers, and found that they carry the same frameshift mutation in the 3GT gene. Most of the flower anthocyanins in the mutants were previously found to be anthocyanidin 3-O-glucosides lacking several caffeic acid and glucose moieties that are attached to the anthocyanins in the wild-type plants. These results indicated that 3GT is essential not only for production, but also for proper acylation and glucosylation, of anthocyanin in the morning glories.


Assuntos
Flores/metabolismo , Glucosiltransferases/metabolismo , Ipomoea/metabolismo , Uridina Difosfato Glucose/metabolismo , Antocianinas/metabolismo , Flores/enzimologia , Regulação da Expressão Gênica de Plantas , Ipomoea/enzimologia , Mutação
8.
Plant Mol Biol ; 85(3): 219-32, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24535433

RESUMO

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Arabidopsis , DNA (Citosina-5-)-Metiltransferases/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Mutação , Oryza/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo
9.
Plant Cell Physiol ; 55(1): 3-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24151203

RESUMO

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions. We have developed a specific method (nDart1-0-iPCR) for efficient detection of nDart1-0 insertions and successfully identified the SNOW-WHITE LEAF1 (SWL1) gene in a variegated albino (swl1-v) mutant obtained from the nDart1-promoted rice tagging line. The variegated albino phenotype was caused by insertion and excision of nDart1-0 in the 5'-untranslated region of the SWL1 gene predicted to encode an unknown protein with the N-terminal chloroplast transit peptide. SWL1 expression was detected in various rice tissues at different developmental stages. However, immunoblot analysis indicated that SWL1 protein accumulation was strictly regulated in a tissue-specific manner. In the swl1 mutant, formations of grana and stroma thylakoids and prolamellar bodies were inhibited. This study revealed that SWL1 is essential for the beginning of thylakoid membrane organization during chloroplast development. Furthermore, we provide a developmental perspective on the nDart1-promoted tagging line to characterize unidentified gene functions in rice.


Assuntos
Alelos , Genes de Plantas/genética , Mutação/genética , Oryza/genética , Proteínas de Plantas/genética , Tilacoides/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Oryza/ultraestrutura , Fenótipo , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Tilacoides/ultraestrutura
10.
Plant J ; 71(4): 564-74, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22448681

RESUMO

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.


Assuntos
Mutação , Oryza/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Cruzamentos Genéticos , Metilação de DNA , Técnicas de Introdução de Genes , Células Germinativas Vegetais , Germinação , Glucuronidase/genética , Recombinação Homóloga , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Pólen/genética , Regiões Promotoras Genéticas , Sementes/genética
11.
J Virol ; 86(11): 6189-96, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22457527

RESUMO

Eliciting neutralizing antibodies is thought to be a key activity of a vaccine against human immunodeficiency virus (HIV). However, a number of studies have suggested that in addition to neutralization, interaction of IgG with Fc gamma receptors (FcγR) may play an important role in antibody-mediated protection. We have previously obtained evidence that the protective activity of the broadly neutralizing human IgG1 anti-HIV monoclonal antibody (MAb) b12 in macaques is diminished in the absence of FcγR binding capacity. To investigate antibody-dependent cellular cytotoxicity (ADCC) as a contributor to FcγR-associated protection, we developed a nonfucosylated variant of b12 (NFb12). We showed that, compared to fully fucosylated (referred to as wild-type in the text) b12, NFb12 had higher affinity for human and rhesus macaque FcγRIIIa and was more efficient in inhibiting viral replication and more effective in killing HIV-infected cells in an ADCC assay. Despite these more potent in vitro antiviral activities, NFb12 did not enhance protection in vivo against repeated low-dose vaginal challenge in the simian-human immunodeficiency virus (SHIV)/macaque model compared to wild-type b12. No difference in protection, viral load, or infection susceptibility was observed between animals given NFb12 and those given fully fucosylated b12, indicating that FcγR-mediated activities distinct from FcγRIIIa-mediated ADCC may be important in the observed protection against SHIV challenge.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Anti-HIV/administração & dosagem , HIV-1/imunologia , Receptores de IgG/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Células Cultivadas , Feminino , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/isolamento & purificação , Anticorpos Anti-HIV/metabolismo , Humanos , Macaca mulatta , Receptores de IgG/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Carga Viral
12.
Plant Cell Physiol ; 53(5): 857-68, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22514089

RESUMO

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.


Assuntos
Elementos de DNA Transponíveis/genética , DNA de Plantas/genética , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Azacitidina/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Ácidos Hidroxâmicos/farmacologia , Mutação/genética , Oryza/efeitos dos fármacos , Sementes/efeitos dos fármacos , Sementes/genética , Análise de Sequência de DNA , Transposases/genética , Transposases/metabolismo
13.
Genes Cells ; 16(11): 1071-80, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22023369

RESUMO

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Šstructure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Polissacarídeos/química , Receptores de IgG/química , Receptores de IgG/imunologia , Anticorpos/química , Anticorpos/imunologia , Anticorpos/uso terapêutico , Humanos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Polissacarídeos/imunologia , Conformação Proteica
14.
Breed Sci ; 62(1): 99-104, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23136520

RESUMO

Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.

15.
Plant Cell Physiol ; 52(4): 638-50, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21382978

RESUMO

GIGANTEA (GI) is a key regulator of flowering time, which is closely related to the circadian clock function in Arabidopsis. Mutations in the GI gene cause photoperiod-insensitive flowering and altered circadian rhythms. We isolated the GI ortholog PnGI from Pharbitis (Ipomoea) nil, an absolute short-day (SD) plant. PnGI mRNA expression showed diurnal rhythms that peaked at dusk under SD and long-day (LD) conditions, and also showed robust circadian rhythms under continuous dark (DD) and continuous light (LL) conditions. Short irradiation with red light during the flower-inductive dark period did not change PnGI expression levels, suggesting that such a night break does not abolish flowering by affecting the expression of PnGI. In Pharbitis, although a single dusk signal is sufficient to induce expression of the ortholog of FLOWERING LOCUS T (PnFT1), PnGI mRNA expression was not reset by single lights-off signals. Constitutive expression of PnGI (PnGI-OX) in transgenic plants altered period length in leaf-movement rhythms under LL and affected circadian rhythms of PnFT mRNA expression under DD. PnGI-OX plants formed fewer flower buds than the wild type when one-shot darkness was given. In PnGI-OX plants, expression of PnFT1 was down-regulated, suggesting that PnGI functions as a suppressor of flowering, possibly in part through down-regulation of PnFT1.


Assuntos
Ritmo Circadiano/genética , Flores/fisiologia , Ipomoea nil/fisiologia , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Ritmo Circadiano/efeitos da radiação , DNA Complementar/genética , DNA de Plantas/química , DNA de Plantas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escuridão , Regulação para Baixo/genética , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Ipomoea nil/genética , Ipomoea nil/crescimento & desenvolvimento , Ipomoea nil/efeitos da radiação , Luz , Dados de Sequência Molecular , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA de Plantas/genética , Análise de Sequência de DNA , Transdução de Sinais
16.
J Exp Bot ; 62(14): 5105-16, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21765172

RESUMO

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.


Assuntos
Antocianinas/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dahlia/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Vias Biossintéticas , Dahlia/química , Dahlia/classificação , Dahlia/genética , Flores/química , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alinhamento de Sequência
17.
J Plant Res ; 124(2): 299-304, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20680382

RESUMO

The mutable a(flaked (a(f)) allele at the A locus of the common morning glory (Ipomoea purpurea) confers incomplete dominance in flower pigmentation and is caused by insertion of the DNA transposon Tip100 into CHS-D, which encodes chalcone synthase and is required for anthocyanin biosynthesis. Levels of CHS-D transcripts, CHS-D protein, and anthocyanin pigment in heterozygous flowers were about half that in homozygous flowers, indicating that dosage-dependent expression of CHS-D is the primary cause of the observed incomplete dominance. This contrasts with the Nivea locus in snapdragon (Antirrhinum majus) in which incomplete dominance is caused by semi-dominant CHS alleles.


Assuntos
Aciltransferases/genética , Antocianinas/metabolismo , Ipomoea/genética , Pigmentação/genética , Alelos , Antocianinas/genética , Sequência de Bases , Elementos de DNA Transponíveis , DNA de Plantas/genética , Flores/anatomia & histologia , Flores/química , Flores/genética , Dosagem de Genes , Genes de Plantas/genética , Ipomoea/enzimologia , Dados de Sequência Molecular , Mutagênese Insercional , Oxirredutases/genética , Análise de Sequência de DNA
18.
Plant J ; 60(2): 386-96, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19519802

RESUMO

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive-negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding beta-glucuronidase fused with the endogenous promoter of MET1a, one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T(0)) transgenic knock-in plants obtained were found to carry only one copy of GUS, with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter-fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive-negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Dosagem de Genes , Técnicas de Introdução de Genes/métodos , Oryza/enzimologia , Recombinação Genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genoma de Planta , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Seleção Genética , Transgenes
19.
Mol Genet Genomics ; 284(5): 343-55, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20830488

RESUMO

The nonautonomous nDart1 element in the hAT superfamily is one of a few active DNA transposons in rice. Its transposition can be induced by crossing with a line containing an active autonomous element, aDart1, and stabilized by segregating aDart1. No somaclonal variation should occur in nDart1-promoted gene tagging because no tissue culture is involved in nDart1 activation. By transposon display analysis, we examined the activities of nDart1-related elements in the selfed progeny of a mutable virescent pyl-v plant containing aDart1. Although various nDart1-related elements are present in the rice genome, only nDart1-3 subgroup elements, nDart1-0 and nDart1-3 in particular, were found to be transposed frequently and integrated into various sites almost all over the genome, and a fraction of the transposed elements were found to be transmitted to the next generation. More than half of the newly integrated elements were identified as nDart1-0. Analysis of the newly inserted sites revealed that the nDart1-3 subgroup elements were predominantly integrated into single-copy regions. More than 60% of the transposed elements were inserted into the genic regions that comprise putative coding regions and their 0.5-kb flanking segments, and approximately two-thirds of them were within the 0.5-kb area in front of the putative initiation codons, i.e., promoter-proximal genic regions. These characteristic features of nDart1-3 subgroup elements seem to be suitable for developing an efficient and somaclonal variation-free gene tagging system for rice functional genomics.


Assuntos
Elementos de DNA Transponíveis , DNA de Plantas/genética , Oryza/genética , Cromossomos de Plantas , Genoma de Planta
20.
Nucleic Acids Res ; 36(14): 4727-35, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18632759

RESUMO

Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination. Through a gene-targeting procedure with positive-negative selection, we previously reported the generation of fertile transgenic rice plants with a positive marker inserted into the Adh2 gene by using an Agrobacterium-mediated transformation vector containing the positive marker flanked by two 6-kb homologous segments for recombination. We describe here that base changes within the homologous segments in the vector could be efficiently transferred into the corresponding genomic sequences of rice recombinants. Interestingly, a few sequences from the host genome were flanked by the changed sequences derived from the vector in most of the recombinants. Because a single-stranded T-DNA molecule in Agrobacterium-mediated transformation is imported into the plant nucleus and becomes double-stranded, both single-stranded and double-stranded T-DNA intermediates can serve in gene-targeting processes. Several alternative models, including the occurrence of the mismatch correction of heteroduplex molecules formed between the genomic DNA and either a single-stranded or double-stranded T-DNA intermediate, are compared to explain the observation, and implications for the modification of endogenous genes for functional genomic analysis by gene targeting are discussed.


Assuntos
Marcação de Genes , Vetores Genéticos , Genoma de Planta , Oryza/genética , Recombinação Genética , Quebras de DNA de Cadeia Dupla , Reparo de Erro de Pareamento de DNA , DNA Bacteriano/metabolismo , Vetores Genéticos/química , Ácidos Nucleicos Heteroduplexes/química , Mutação Puntual
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