RESUMEN

With the increasing prevalence of allergies, accurate identification of allergens is a major priority for allergists, scientists, the food industry, and food regulators. Knowledge of allergens is essential for risk assessment of novel genetically modified (GM) foods, and to develop recombinant proteins for the treatment and diagnosis of allergies. This Opinion Paper considers the lack of standardization for the clinical and scientific assessment of proteins before they are labelled as allergens. Food allergens are being reported and recorded in allergen databases, with minimal or in some cases apparently no published justification. IgE binding, rather than clinically relevant reactivity, is inappropriately used to confirm allergenicity. Using kiwifruit as an example, the lack of rigor in identifying allergenic proteins is considered.

Asunto(s)

Actinidia/inmunología , Hipersensibilidad a los Alimentos/diagnóstico , Alérgenos/inmunología , Bases de Datos de Proteínas , Hipersensibilidad a los Alimentos/inmunología , Alimentos Modificados Genéticamente , Humanos , Inmunoglobulina E/sangre

RESUMEN

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

Asunto(s)

Actinidia/inmunología , Antígenos de Plantas/clasificación , Antígenos de Plantas/inmunología , Cisteína Endopeptidasas/inmunología , Hipersensibilidad/diagnóstico , Adolescente , Adulto , Western Blotting , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Frutas/inmunología , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Pruebas Cutáneas , Reino Unido/epidemiología

RESUMEN

In kiwifruit, much of the softening process occurs prior to the respiratory climacteric and production of ethylene. This fruit therefore represents an excellent model system for dissecting the process of softening in the absence of endogenous ethylene production. We have characterized the expression of three polygalacturonase (PG) cDNA clones (CkPGA, B and C) isolated from fruit of Actinidia chinensis. Expression of CkPGA and B was detected by northern analysis only in fruit producing endogenous ethylene, and by RT-PCR in other tissues including flower buds, petals at anthesis, and senescent petals. CkPGA promoter fragments of 1296, 860 and 467 bp fused to the beta-glucuronidase (uidA) reporter gene directed fruit-specific gene expression during the climacteric in transgenic tomato. CkPGC gene expression was observed in softening fruit, and reached maximum levels (50-fold higher than for CkPGA and B) as fruit passed through the climacteric. However, expression of this gene was also readily detected during fruit development and in fruit harvested prior to the onset of softening. Using RT-PCR, expression of CkPGC was also detected at low levels in root tips and in senescent petals. These results suggest that PG expression is required not only during periods of cell wall degeneration, but also during periods of cell wall turnover and expansion.

Asunto(s)

Frutas/genética , Poligalacturonasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/genética , Etilenos/biosíntesis , Frutas/enzimología , Frutas/crecimiento & desarrollo , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Glucuronidasa/metabolismo , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución Tisular

RESUMEN

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.

Asunto(s)

Frutas/genética , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Frutas/crecimiento & desarrollo , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Homeobox , Hibridación in Situ , Datos de Secuencia Molecular , Fenotipo , Plantas Modificadas Genéticamente , Polen/genética , Polen/crecimiento & desarrollo , ARN Mensajero/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ADN , Distribución Tisular , Factores de Transcripción/genética

RESUMEN

Levels of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and polygalacturonase (PG) mRNAs were characterized during ripening of Royal Gala, Braeburn and Granny Smith apples. Both ACC-oxidase and PG mRNAs were up-regulated in ripening fruit of all three cultivars. Expression in Royal Gala was detected earlier than in Braeburn and Granny Smith, relative to internal ethylene concentration. Genomic clones corresponding to the ACC-oxidase and PG mRNAs expressed in ripe apple fruit were isolated and ca. 2 kb of each promoter was sequenced. The start point of transcription in each gene was mapped by primer extension, and sequences homologous to elements in other ethylene-responsive or PG promoters were identified. The fruit specificity of the apple ACC-oxidase and PG promoters was investigated in transgenic tomato plants using a nested set of promoter fragments fused to the beta-glucuronidase (gusA) reporter gene. For the ACC-oxidase gene, 450 bp of 5' promoter sequence was sufficient to drive GUS expression, although this expression was not specific to ripening fruit. Larger fragments of 1966 and 1159 bp showed both fruit and ripening specificity. For the PG gene, promoter fragments of 1460 and 532 bp conferred ripening-specific expression in transgenic tomato fruit. However GUS expression was down-regulated by 2356 bp of promoter, suggesting the presence of a negative regulatory element between positions -1460 and -2356.

Asunto(s)

Aminoácido Oxidorreductasas/genética , Poligalacturonasa/genética , Rosales/enzimología , Rosales/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reporteros , Glucuronidasa/genética , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Rosales/crecimiento & desarrollo

Asunto(s)

Plantas Modificadas Genéticamente/genética , Plantas/genética , ARN de Planta/genética , Supresión Genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/virología , Proteínas de Plantas/metabolismo , Virus de Plantas/genética , Virus de Plantas/fisiología , Plantas/metabolismo , Plantas/virología , Plantas Modificadas Genéticamente/metabolismo , ARN Complementario/genética , ARN Complementario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de Señal , Moldes Genéticos , Transgenes

RESUMEN

Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) was purified 3000-fold to homogeneity. The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8. The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for [3H]XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan. Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of [3H]XXXG-ol by hydrolysis, and in the presence of [3H]XXXG-ol by hydrolysis and endotransglycosylation. Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA. The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes. Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6. Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft. A full-length cDNA clone (AdXET5) was overexpressed in E. coli to produce a recombinant protein that showed endotransglycosylase activity when refolded.

Asunto(s)

Frutas/enzimología , Glucanos , Glicosiltransferasas/metabolismo , Xilanos , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cartilla de ADN , ADN Complementario , ADN de Plantas , Escherichia coli/metabolismo , Expresión Génica , Genes de Plantas , Glicosiltransferasas/biosíntesis , Glicosiltransferasas/genética , Glicosiltransferasas/aislamiento & purificación , Marcaje Isotópico , Datos de Secuencia Molecular , Oxidación-Reducción , Polisacáridos/metabolismo , Polisacáridos/farmacología , Especificidad por Sustrato , Tritio

RESUMEN

We have constructed a binary vector containing elements of the monopartite geminivirus, tobacco yellow dwarf virus (TYDV). The vector is designed to be stably integrated into the plant genome, via Agrobacterium-mediated transfer. Upon expression of the viral replication-associated protein the TYDV elements are released from the T-DNA and then replicate episomally. We refer to these released forms as multicopy plant episomes (MPE). Tobacco plants (Nicotiana tabacum cv `Samsun') transformed with the vector showed MPE release and subsequent episomal replication in 6 of 11 of these plants. An MPE vector containing the uidA gene faithfully replicated and maintained the reporter gene, even though the construct was considerably larger than the wild-type TYDV genome. Histochemical staining revealed a speckled GUS expression phenotype which could be correlated with MPE replication.

RESUMEN

Single PCR primers complementary to microsatellite repeats were used to amplify genomic DNA samples from various plant species, as well as from human, yeast, and Escherichia coli DNA. Most primers generated distinct amplification products, resulting in fingerprint-like banding patterns after agarose gel electrophoresis and ethidium bromide staining. These fingerprints allowed distinction among different plant taxa at an interspecific as well as intraspecific level. Unexpectedly, some of the primers produced bands with the E. coli template DNA as well. A detailed examination of the influence of PCR conditions, especially the annealing temperature, on the quality of banding patterns suggested that the majority of bands were generated by mismatch priming in a way similar to random amplified polymorphic DNAs (RAPDs).

Asunto(s)

Dermatoglifia del ADN , ADN Satélite/genética , Genoma , Reacción en Cadena de la Polimerasa/métodos , Clonación Molecular , Colorantes , Cartilla de ADN , ADN Bacteriano/genética , ADN de Hongos/genética , ADN de Plantas/genética , Electroforesis en Gel de Agar/métodos , Escherichia coli/genética , Etidio , Humanos , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Moldes Genéticos

Asunto(s)

ADN de Plantas/genética , Bases de Datos Factuales , Frutas/genética , Poligalacturonasa/genética , ADN Complementario , Frutas/enzimología , Datos de Secuencia Molecular

Asunto(s)

Frutas/genética , Genes de Plantas , Poligalacturonasa/genética , Secuencia de Bases , Secuencia Conservada , Bases de Datos Factuales , Frutas/enzimología , Datos de Secuencia Molecular

RESUMEN

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the ß D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.

RESUMEN

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (ß-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.