RESUMO
The amounts of specific transcripts that accumulate in chloroplasts are determined by the rates of synthesis and degradation of the transcripts. The 3' untranslated region of transcripts is a major determinant of the stability of transcripts in chloroplasts. The half-lives of specific transcripts can be determined by northern blot analysis of a time course of transcripts in detached tobacco leaves incubated with actinomycin D, a potent transcription inhibitor. This analysis may be applied to transcripts of endogenous genes or of transgenes introduced into the chloroplast genome in transplastomic plants. Sequence determinants of transcript stability can be identified by analysis of transplastomic plants containing constructs of the green fluorescent protein (gfp) reporter gene fused to the sequences of interest.
Assuntos
Cloroplastos/genética , Nicotiana/genética , Estabilidade de RNA/genética , RNA de Cloroplastos/genética , Regiões 3' não Traduzidas/genética , Sequência de Bases , Dactinomicina/farmacologia , Genes Reporter , Genoma de Cloroplastos/genética , Proteínas de Fluorescência Verde/genética , Meia-Vida , Inibidores da Síntese de Ácido Nucleico/farmacologia , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , RNA Ribossômico 16S/genética , Ribulose-Bifosfato Carboxilase/genética , Análise de Sequência de RNA , Nicotiana/citologiaRESUMO
Rotavirus is the main cause of gastroenteritis in children worldwide, and the World Health Organisation has recommended that a rotavirus vaccine should be included in all infant immunization programmes. VP6 is the most immunogenic rotavirus subunit and is a potential target for an oral subunit vaccine. VP6 accumulated at up to 3% of total soluble protein in the young leaves of transplastomic tobacco plants, but the protein was unstable and was lost as the leaves aged. The aim of this study was to alter the 5'-untranslated region (5'-UTR) and the 5' end of the coding region of VP6 cDNA in an attempt to increase the expression and stability of VP6 protein in tobacco chloroplasts. The inclusion of the 5'-UTR from gene 10 of bacteriophage T7 (T7g10) and the addition of 15 nucleotides, encoding five additional amino acid residues, at the 5' end of the coding region increased the expression to >15% of total leaf protein and stabilized the protein in ageing leaves. Plants containing VP6 expression constructs with the rbcL 5'-UTR and with the native VP6 5' end of the coding region produced VP6 protein at only 1.9% of total leaf protein. Both the T7g10 5'-UTR and the additional 15 nucleotides increased transcript accumulation and translational efficiency compared with VP6 constructs containing the rbcL 5'-UTR. The VP6 protein produced from all gene constructs appeared to be susceptible to proteolytic processing at its N-terminal region. However, in all transplastomic lines, VP6 proteins assembled into the trimeric form found in the rotavirus capsid.
Assuntos
Regiões 5' não Traduzidas/genética , Antígenos Virais/metabolismo , Proteínas do Capsídeo/metabolismo , Cloroplastos/metabolismo , Nicotiana/metabolismo , Fases de Leitura Aberta/genética , Cloroplastos/genética , DNA de Cloroplastos/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/genética , Immunoblotting , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Multimerização Proteica , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Plântula/metabolismo , Fatores de Tempo , Nicotiana/genética , Transformação GenéticaRESUMO
Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.
Assuntos
Arabidopsis/citologia , DNA de Plantas/metabolismo , Nicotiana/citologia , Plastídeos/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Transporte Biológico , DNA de Cloroplastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Nicotiana/genética , Transformação GenéticaRESUMO
Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.
Assuntos
Ácido Abscísico/farmacologia , Nicotiana/citologia , Células Vegetais/fisiologia , Plastídeos/metabolismo , Triticum/citologia , Aminoácidos Cíclicos/farmacologia , Secas , Luz , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas/citologia , Cloreto de Potássio/farmacologia , Plântula/fisiologia , Cloreto de Sódio/farmacologia , Estresse Fisiológico , TemperaturaRESUMO
High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to â¼4% and â¼40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24. Oral administration of a partially purified preparation of chloroplast-derived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Cloroplastos/genética , Proteína do Núcleo p24 do HIV/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Cloroplastos/imunologia , Feminino , Proteína do Núcleo p24 do HIV/administração & dosagem , Proteína do Núcleo p24 do HIV/genética , Imunidade Humoral/imunologia , Imunização Secundária , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nicotina/toxicidade , Plantas Geneticamente Modificadas/genética , Nicotiana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/administração & dosagem , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Stromules are stroma-filled tubules that extend from the plastids in all multicellular plants examined to date. To facilitate the visualisation of stromules on different plastid types in various tissues of bread wheat (Triticum aestivum L.), a chimeric gene construct encoding enhanced yellow fluorescent protein (EYFP) targeted to plastids with the transit peptide of wheat granule-bound starch synthase I was introduced by Agrobacterium-mediated transformation. The gene construct was under the control of the rice Actin1 promoter, and EYFP fluorescence was detected in plastids in all cell types throughout the transgenic plants. Stromules were observed on all plastid types, although the stromule length and abundance varied markedly in different tissues. The longest stromules (up to 40 µm) were observed in epidermal cells of leaves, whereas only short beak-like stromules were observed on chloroplasts in mesophyll cells. Epidermal cells in leaves and roots contained the highest proportion of plastids with stromules, and stromules were also abundant on amyloplasts in the endosperm tissue of developing seeds. The general features of stromule morphology and distribution were similar to those shown previously for tobacco (Nicotiana tabacum L.) and arabidopsis (Arabidopsis thaliana (L.) Heynh.).
Assuntos
Proteínas de Fluorescência Verde/análise , Triticum/metabolismo , Triticum/ultraestrutura , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células do Mesofilo/metabolismo , Células do Mesofilo/ultraestrutura , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos/genética , Sementes/genética , Sementes/metabolismo , Sementes/ultraestrutura , Triticum/genéticaRESUMO
Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5'-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5'-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5'-UTR construct, described above, and another containing the same terminator, but with the promoter and 5'-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.
Assuntos
Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Plastídeos/genética , Solanum tuberosum/genética , Transformação Genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Biotecnologia/métodos , Cloroplastos/genética , Cloroplastos/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folhas de Planta/genética , Raízes de Plantas/genética , Plastídeos/metabolismo , Recombinação Genética , Sequências Reguladoras de Ácido Nucleico , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , TransgenesRESUMO
The 3' untranslated region (3' UTR) of transcripts is a major determinant of transcript stability in plastids and plays an important role in regulating gene expression. In order to compare the effect of different 3' UTRs on transgene expression in tobacco chloroplasts, the 3' UTRs from the tobacco chloroplast rbcL, psbA, petD and rpoA genes and the terminator region of the Escherichia coli rrnB operon were inserted downstream of the gfp reporter gene under the control of the psbA promoter, and the constructs were introduced into the plastid genome by particle bombardment. RNA-gel blot analysis of homoplasmic transplastomic plants identified gfp transcripts of ~1.0 and ~1.4 kb from all constructs and showed that plants expressing gfp with the rrnB terminator contained 4 times more gfp transcripts than plants expressing gfp with the rbcL and rpoA 3' UTRs. The amounts of transcripts accumulated roughly correlated with the half-life of the transcripts, determined by RNA-gel blot analysis of transcripts present in leaves treated with actinomycin D to prevent continued transcription of the chimeric gfp genes. Transcripts containing the 3' region of rrnB were most stable, with half-lives of ~43 h, considerably longer than the half-lives of the other ~1.0 kb gfp transcripts (13-26 h). Immunoblot analysis with antibodies to GFP indicated that all plants contained about the same amount of GFP (~0.2% total soluble protein), suggesting either that translation was limited by something other than the amount of transcript or that the 3' UTR was affecting translation.
Assuntos
Regiões 3' não Traduzidas , Cloroplastos/genética , Proteínas de Fluorescência Verde/genética , Nicotiana/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , Transgenes , Sequência de Bases , Southern Blotting , Primers do DNA , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of approximately 600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins.
Assuntos
Imunoprecipitação da Cromatina/métodos , Genoma de Cloroplastos , Repressores Lac/metabolismo , Nicotiana/genética , Regiões Operadoras Genéticas , Sítios de Ligação , Linhagem Celular , DNA de Cloroplastos/química , DNA de Cloroplastos/metabolismo , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Óperon Lac , Repressores Lac/genética , Proteínas Recombinantes de Fusão/análise , Nicotiana/citologiaRESUMO
Stromules are highly dynamic stroma-filled tubules extending from the surface of plastids and occasionally interconnecting individual plastids, allowing the movement of complex biological molecules between the interconnected plastids. Experiments with inhibitors of cytoskeleton assembly have indicated the involvement of an actin-based system in stromule movement. However, the motor protein associated with the system had not been identified. Here, we present direct evidence that myosin XI is involved in the formation and movement of stromules in tobacco leaves. Application of 2,3-butanedione 2-monoxime, an inhibitor of myosin ATPase activity, resulted in the loss of stromules from tobacco leaf epidermal cells. Transient RNA interference of myosin XI in leaves of Nicotiana benthamiana also resulted in the loss of stromules from epidermal cells, without any effect on transcripts for actin or myosin VIII. Transient expression of a GFP-tagged myosin XI tail domain in tobacco leaf epidermal cells showed that the fusion protein localized to the chloroplast envelope, as well as to mitochondria and other organelles. Our findings identify myosin XI as a key protein involved in the formation and movement of stromules.
Assuntos
Actinas/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Nicotiana/metabolismo , Plastídeos/metabolismo , Cloroplastos/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Miosinas/antagonistas & inibidores , Organelas/metabolismo , Folhas de Planta/enzimologia , Nicotiana/enzimologiaRESUMO
We have completed a single ascending dose clinical study of the proposed chemopreventive agent 3,3'-diindolylmethane (DIM). The study agent was nutritional-grade, absorption-enhanced BioResponse 3,3'-diindolylmethane (BR-DIM). We determined the safety, tolerability, and pharmacokinetics of single doses of BR-DIM in drug-free, non-smoking, healthy men and women. Groups of four subjects were enrolled for each dose level. After randomization, one subject in each group received placebo whereas three received active BR-DIM. The doses administered were 50, 100, 150, 200, and 300 mg, with the 300-mg dose repeated in an additional group. No BR-DIM-related adverse effects were reported at doses up to 200 mg. At the 300-mg dose, one of six subjects reported mild nausea and headache and one also reported vomiting. Only the latter effect was judged as probably related to the study agent. Analysis of serial plasma samples showed that only one subject at the 50-mg dose had detectable concentrations of DIM. The single 100-mg dose of BR-DIM resulted in a mean maximum plasma concentration (C(max)) of 32 ng/mL and a mean area under the curve (AUC) of 128 h ng/mL, and a single 200-mg dose produced a mean C(max) of 104 ng/mL and a mean AUC of 553 h ng/mL. The single 300-mg dose of BR-DIM resulted in a mean C(max) of 108 ng/mL and a mean AUC of 532 h ng/mL. We conclude that BR-DIM is well tolerated at single doses of up to 200 mg, and that increasing the dose to 300 mg did not result in an increase in C(max).
Assuntos
Indóis/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Relação Dose-Resposta a Droga , Feminino , Humanos , Indóis/administração & dosagem , Indóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , PlacebosRESUMO
We describe a novel, typically prokaryotic, sensor kinase in chloroplasts of green plants. The gene for this chloroplast sensor kinase (CSK) is found in cyanobacteria, prokaryotes from which chloroplasts evolved. The CSK gene has moved, during evolution, from the ancestral chloroplast to the nuclear genomes of eukaryotic algae and green plants. The CSK protein is now synthesised in the cytosol of photosynthetic eukaryotes and imported into their chloroplasts as a protein precursor. In the model higher plant Arabidopsis thaliana, CSK is autophosphorylated and required for control of transcription of chloroplast genes by the redox state of an electron carrier connecting photosystems I and II. CSK therefore provides a redox regulatory mechanism that couples photosynthesis to gene expression. This mechanism is inherited directly from the cyanobacterial ancestor of chloroplasts, is intrinsic to chloroplasts, and is targeted to chloroplast genes.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Histidina Quinase/genética , Histidina Quinase/metabolismo , Fotossíntese/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , Cianobactérias/enzimologia , Cianobactérias/genética , Primers do DNA/genética , Evolução Molecular , Expressão Gênica , Genes de Plantas , Dados de Sequência Molecular , Mutação , Oxirredução , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Simbiose , Nicotiana/enzimologia , Nicotiana/genética , Transcrição GênicaRESUMO
Genes encoding plastid ribosomal proteins are distributed between the nuclear and plastid genomes in higher plants, and coordination of their expression is likely to be required for functional plastid protein synthesis. A custom microarray has been used to examine the patterns of accumulation of transcripts from plastid and nuclear genes encoding plastid ribosomal proteins during seedling development in tobacco and Arabidopsis. The transcripts accumulate coordinately during early seedling development and show similar responses to light and to inhibitors, such as norflurazon and lincomycin, affecting plastid signaling. Computational analysis of the promoters of these genes revealed a shared initiator motif and common cis-elements characteristic of photosynthesis genes, specifically the GT-1 element, and the I-box. Analysis of the RPL27 gene of Arabidopsis thaliana indicated that transcription initiates from an initiator-like region. Deletion analysis of the RPL27 promoter in transgenic plants revealed that the identified shared cis-elements were not all required for wild-type expression patterns, and full developmental, light- and plastid-regulation can be conveyed by a region of the promoter from -235 to +1 relative to the transcription start site.
Assuntos
Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Nicotiana/metabolismo , Plastídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Plântula/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Luz , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Ribossômicas/genética , Plântula/genética , Plântula/crescimento & desenvolvimento , Alinhamento de Sequência , Transdução de Sinais , Fatores de Tempo , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T-cell-mediated immune responses in HIV-positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non-food crop, and tomato, a food crop with an edible fruit. Optimized p24-Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.
Assuntos
Genomas de Plastídeos , Antígenos HIV/genética , HIV/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Solanum lycopersicum/genética , Sequência de Bases , Expressão Gênica , Marcadores Genéticos/genética , Vetores Genéticos , Íntrons/genética , Dados de Sequência Molecular , Recombinação Genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Chloroplast transformation of the high-biomass tobacco variety Maryland Mammoth has been assessed as a production platform for the human immunodeficiency virus type 1 (HIV-1) p24 antigen. Maryland Mammoth offers the prospect of higher yields of intact functional protein per unit floor area of contained glasshouse per unit time prior to flowering. Two different transformation constructs, pZSJH1p24 (for the insertion of a native p24 cDNA between the rbcL and accD genes) and pZF5 (for the insertion of a chloroplast-codon-optimized p24 gene between trnfM and trnG) were examined for the production of p24. Plants generated with construct pZSJH1p24 exhibited a normal green phenotype, but p24 protein accumulated only in the youngest leaves (up to approximately 350 microg/g fresh weight or approximately 2.5% total soluble protein) and was undetectable in mature leaves. In contrast, some of the plants generated with pZF5 exhibited a yellow phenotype (pZF5-yellow) with detectable p24 accumulation (up to approximately 450 microg/g fresh weight or approximately 4.5% total soluble protein) in all leaves, regardless of age. Total protein in pZF5-yellow leaves was reduced by approximately 40%. The pZF5-yellow phenotype was associated with recombination between native and introduced direct repeat sequences of the rbcL 3' untransformed region in the plastid genome. Chloroplast-expressed p24 was recognized by a conformation-dependent monoclonal antibody to p24, and p24 protein could be purified from pZF5-yellow leaves using a simple procedure, involving ammonium sulphate precipitation and ion-exchange chromatography, without the use of an affinity tag. The purified p24 was shown to be full length with no modifications, such as glycosylation or phosphorylation, using N-terminal sequencing and mass spectrometry.
Assuntos
Proteína do Núcleo p24 do HIV/genética , HIV-1/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Southern Blotting , Cloroplastos/genética , Cloroplastos/virologia , Primers do DNA , DNA de Plantas/genética , DNA Viral/genética , Flores/genética , Genoma de Planta , Folhas de Planta/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Nicotiana/crescimento & desenvolvimento , Transformação GenéticaRESUMO
Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.
Assuntos
RNA Polimerases Dirigidas por DNA/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Plastídeos/genética , Plântula/crescimento & desenvolvimento , Transcrição Gênica , Transgenes/fisiologia , Proteínas Virais/farmacologia , Bacteriófago T7/enzimologia , Núcleo Celular/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/metabolismo , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
We have completed a phase I trial in women of the proposed chemopreventive natural product indole-3-carbinol (I3C). Women received oral doses of 400, 600, 800, 1,000, and 1,200 mg I3C. Serial plasma samples were analyzed by high-performance liquid chromatography-mass spectrometry for I3C and several of its condensation products. I3C itself was not detectable in plasma. The only detectable I3C-derived product was 3,3'-diindolylmethane (DIM). Mean Cmax for DIM increased from 61 ng/mL at the 400-mg I3C dose to 607 ng/mL following a 1,000-mg dose. No further increase was observed following a 1,200-mg dose. A similar result was obtained for the area under the curve, which increased from 329 h ng/mL at the 400-mg dose to 3,376 h ng/mL after a 1,000-mg dose of I3C. Significant interindividual quantitative variation was seen in plasma DIM values within each dosing group, but the overall profiles were qualitatively similar, with no quantifiable DIM before dosing, tmax at approximately 2 h, and DIM levels near or below 15 ng/mL (the limit of quantitation), by 24 h. Different results were obtained for 14 subjects who received a 400-mg dose of I3C after 8 weeks of twice-daily I3C dosing. Although the predose sampling occurred at least 12 h after the last known ingestion of I3C, 6 of 14 subjects exhibited Cmax for DIM in their predose plasma. Despite this high initial value, plasma DIM for all subjects decreased to near or below the limit of quantitation within the 12-h sampling period. Possible reasons for this disparity between apparent t1/2 of DIM and the high predose values are discussed.
Assuntos
Anticarcinógenos/farmacocinética , Indóis/metabolismo , Indóis/farmacocinética , Administração Oral , Adulto , Área Sob a Curva , Cromatografia Líquida de Alta Pressão , Esquema de Medicação , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Tomato, Solanum lycopersicum (formerly Lycopersicon esculentum), has long been one of the classical model species of plant genetics. More recently, solanaceous species have become a model of evolutionary genomics, with several EST projects and a tomato genome project having been initiated. As a first contribution toward deciphering the genetic information of tomato, we present here the complete sequence of the tomato chloroplast genome (plastome). The size of this circular genome is 155,461 base pairs (bp), with an average AT content of 62.14%. It contains 114 genes and conserved open reading frames (ycfs). Comparison with the previously sequenced plastid DNAs of Nicotiana tabacum and Atropa belladonna reveals patterns of plastid genome evolution in the Solanaceae family and identifies varying degrees of conservation of individual plastid genes. In addition, we discovered several new sites of RNA editing by cytidine-to-uridine conversion. A detailed comparison of editing patterns in the three solanaceous species highlights the dynamics of RNA editing site evolution in chloroplasts. To assess the level of intraspecific plastome variation in tomato, the plastome of a second tomato cultivar was sequenced. Comparison of the two genotypes (IPA-6, bred in South America, and Ailsa Craig, bred in Europe) revealed no nucleotide differences, suggesting that the plastomes of modern tomato cultivars display very little, if any, sequence variation.
Assuntos
DNA de Cloroplastos/genética , Evolução Molecular , Plastídeos/genética , Solanaceae/genética , Solanum lycopersicum/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA de Cloroplastos/química , DNA de Plantas/química , DNA de Plantas/genética , Genes de Plantas/genética , Genoma de Planta/genética , Dados de Sequência Molecular , Filogenia , Edição de RNA/genética , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas Ribossômicas/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T(1) plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.
Assuntos
Engenharia Genética/métodos , Lactuca/citologia , Lactuca/genética , Plastídeos/genética , Transformação Genética/genética , Cruzamentos Genéticos , Resistência a Medicamentos/genética , Vetores Genéticos/genética , Lactuca/efeitos dos fármacos , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polietilenoglicóis , Plântula/efeitos dos fármacos , Plântula/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Espectinomicina/farmacologia , Transgenes/genéticaRESUMO
We completed a phase I trial of indole-3-carbinol (I3C) in 17 women (1 postmenopausal and 16 premenopausal) from a high-risk breast cancer cohort. After a 4-week placebo run-in period, subjects ingested 400 mg I3C daily for 4 weeks followed by a 4-week period of 800 mg I3C daily. These chronic doses were tolerated well by all subjects. Hormonal variables were measured near the end of the placebo and dosing periods, including determination of the urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio. Measurements were made during the follicular phase for premenopausal women. Serum estradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, and sex hormone binding globulin showed no significant changes in response to I3C. Caffeine was used to probe for cytochrome P450 1A2 (CYP1A2), N-acetyltransferase-2 (NAT-2), and xanthine oxidase. Comparing the results from the placebo and the 800 mg daily dose period, CYP1A2 was elevated by I3C in 94% of the subjects, with a mean increase of 4.1-fold. In subjects with high NAT-2 activities, these were decreased to 11% by I3C administration but not altered if NAT-2 activity was initially low. Xanthine oxidase was not affected. Lymphocyte glutathione S-transferase activity was increased by 69% in response to I3C. The apparent induction of CYP1A2 was mirrored by a 66% increase in the urinary 2-hydroxyestrone/16alpha-hydroxyestrone ratio in response to I3C. The maximal increase was observed with the 400 mg daily dose of I3C, with no further increase found at 800 mg daily. If the ratio of hydroxylated estrone metabolites is a biomarker for chemoprevention, as suggested, then 400 mg I3C daily will elicit a maximal protective effect.